Practical approaches to

the analyses for pesticide residues

in essential oils

A report for the Rural Industries Research and Development Corporation by Sandra M. Garland Prof. Robert C. Menary NW Davies and Garth S. Oliver

July 2004 RIRDC Publication No 04/109 RIRDC Project No UT-36A

 2004 Rural Industries Research and Development Corporation. All rights reserved. ISBN 1 74151 018 X ISSN 1440-6845 Practical approaches to the analyses for pesticide residues in essential oils Publication No. 04/109 Project No. UT-36A The views expressed and the conclusions reached in this publication are those of the author and not necessarily those of persons consulted. RIRDC shall not be responsible in any way whatsoever to any person who relies in whole or in part on the contents of this report. This publication is copyright. However, RIRDC encourages wide dissemination of its research, providing the Corporation is clearly acknowledged. For any other enquiries concerning reproduction, contact the Publications Manager on phone 02 6272 3186.

Researcher Contact Details Prof. R. C. Menary Tasmanian Institute of Agricultural Research (TIAR) University of Tasmania GPO Box 252-54 HOBART Tas 7001 Phone: Fax:( Email: (03) 6226 2723 (03) 6226 7609 R.Menary@utas.edu.au

In submitting this report, the researcher has agreed to RIRDC publishing this material in its edited form.

RIRDC Contact Details Rural Industries Research and Development Corporation Level 1, AMA House 42 Macquarie Street BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: Fax: Email: Website: 02 6272 4539 02 6272 5877 rirdc@rirdc.gov.au http://www.rirdc.gov.au

Published in July 2004 Printed on environmentally friendly paper by Canprint

ii

Contents
Acknowledgments...................................................................................................................... iv

Introduction....................................................................................................................... 1
Pesticides commonly used in the essential oil industry. ............................................................. 2 Abbreviations .............................................................................................................................. 3 Chemical Properties of Essential Oils......................................................................................... 4 Practical considerations for laboratory procedures in pesticide analyses ................................... 7

Categorisation of Pesticides ............................................................................................. 8

Chlorinated chemicals................................................................................................................. 8 Experiments undertaken for equipment assessment.................................................................. 10 Organophosphates ..................................................................................................................... 11 Urea Chemicals ......................................................................................................................... 14 Carbamate Derivatives .............................................................................................................. 16 Dithiocarbamates ...................................................................................................................... 17 Pesticides with Acidic Moieties ................................................................................................ 20 Quaternary Nitrogen Herbicides ............................................................................................... 22

Analytical Equipment..................................................................................................... 25
Gas Chromatography ................................................................................................................ 25 Liquid Chromatography ............................................................................................................ 57 Assessment of the application of ICP OES to the screening of essential oils........................... 84

Preliminary clean-up methodology ............................................................................... 87

Liquid / liquid extraction of pesticides from solvent extracted oils .......................................... 87 Preliminary Development of Solid Phase Extraction of Pesticides from Essential Oils........... 88 The application of ion exchange chromatography to the clean-up of acidic pesticides with detection by GC ECD................................................................................................................ 95

Literature Cited ............................................................................................................ 100 Appendix........................................................................................................................ 103

iii

Acknowledgments
Our appreciation is extended to Dr Ashley Townsend whose expertise in ICP OES was instrumental in developing the methodology for the screening of essential oils for mancozeb contamination.

iv

Introduction
Many papers have been published detailing methods for the analysis of pesticide residues in matrices such as water and vegetables. The detection of analytes in essential oils, however, has specific problems associated. The chemical properties of the active ingredients of many pesticides, such as polarity and retention behaviour in chromatography media, are often very similar to those observed for components of essential oils.

Clean-up, or rather the limited applicability of standard pre-concentration steps, presents as the greatest limitation. The greater majority of commercially produced, solid phase extraction columns are designed to trap low levels of pesticide residues from large quantities of water. Even in the analysis of vegetables, an aqueous phase is the predominant matrix from which pesticides are absorbed. Essential oils are usually a complex mixture of medium polarity and non-polar extracts of plant material concentrated to as little as 5% of the source material. Development of an analytical methodology for any one contaminant can be achieved, but the pre-concentration of a number of pesticide residues within one screen is problematic.

For the great majority of pesticides, the structure and chemical properties of the active ingredient confer physical properties, such as polarity, solubility and elution characteristics which can be used as a predictive indicator for their behaviour in clean-up methodology involving chromatography. The components which present as the most likely to co-extract with essential oils are, by the nature of their extractability, the most difficult to separate from the matrix and the most likely to interfere with the analysis, having similar behaviour in liquid partition and chromatographic methodologies. This manual is designed to provide an overview of the applicability of the analytical technology generally available, to the detection of analytes. Methodologies are designed based on the chemical type of the active ingredient.

The Manual can be read in conjunction with reports on four RIRDC projects detailing the development of analytical techniques for the determination of pesticide minimum residue limits in essential oils: UT-8A, UT-13A Publication No 98/123, UT23A Publication No 04/023 and UT36A Publication No 04/104.

Pesticides commonly used in the essential oil industry.

Active ingredient acephate asulam bromacil & diuron carbaryl carbendazim chloropropham chlorpyrifos clopyralid dicamba difenoconazole dimethenamid dimethoate diuron ethofumesate fluazifop fluroxypyr glyphosate haloxyfop isoxaben linuron mancozeb MCPA mecoprop monocrotophos norflurazon oryzalin oxyflurofen paraquat & diquat pendimethalin pirimicarb procymidone prometryn propazine propiconazole sethoxydim simazine sulfosulfuron tebuconazole terbacil trichlopyr Key : Commercial preparation Orthene Asulox Krovar Carbaryl Bavistan Allicide Lorsban Lontrel Dicamba Score Frontier Rogor Diuron Tramat Fusilade Starane Roundup Verdict Flexidor Linuron Dithane MCPA mecoprop Nuvacron Solicam Snapshot Goal Gramoxone Stomp Pirimor Sumisclex Geasaguard Gesamil Tilt Sertin Gesatop Maverick Folicur Sinbar Garlon I - insecticide H - herbicide F - fungicide Function I H H I F H I H H F H I H H H H H H H H F H H I H H H H H I F H H F H H H F H H

Abbreviations ai amu APCI BAP C.E. DCM DETA DMED ECD ESI ESP FID FPD GC HPLC HR ICP OES LC MRL MS MSD MSDs MSMS PDA. R.P. r.s.d. RVE. SIM SPE SPI TBAS TIC TLC TSP active ingredient Atomic Mass Units Atmospheric pressure chemical ionisation Best Agricultural Practices collision energy Dichloromethane Diethylene triamine
Dimethyl ethylenebis(dithiocarbamate)

Electron Capture Detector Electrospray ionisation Electrospray Flame Ionisation Detector Flame Photometric Detector Gas Chromatography High Pressure Liquid Chromatography High Resolution Inductively Coupled Plasma Optical Emission Spectrophotometer Liquid chromatography Maximum Residue Limit Mass Spectrometry Mass Selective Detector Material Safety Data sheets Daughter Mass Spectra Generated by Fragmentation of a Parent Ion Photo Diode Array Detector Reverse Phase Relative Standard Deviation Rotary Vacuum Evaporation Single Ion Monitoring Solid Phase Extraction Septum Equipped Programmable Injector Tetrabutylammonium hydrogen sulfate Total Ion Chromatogram Thin Layer Chromatography Thermospray

Chemical Properties of Essential Oils

Two of the most widely used methods for the production of essential oils include solvent extraction and steam distillation. Concretes are produced by the steeping of raw plant material in organic solvents such as petroleum ether, ethanol or acetone, with or without maceration and agitation. The solvents are normally removed from the extracts by rotary vacuum evaporation (RVE) at low temperatures (usually below 60C). Absolutes are prepared by cold filtration of chilled ethanol solutions of concretes, whereby waxes and other high molecular weight, non-polar components are removed to produce a less viscous, more concentrated product. Steam distilled oils, or essential oils, are those obtained by steam distillation. The distillation is conducted at atmospheric pressure or at elevated pressures for products such as sandalwood oil, patchouli oil, vetiver oil and several other oils composed mainly of sesquiterpenoid alcohols, ketones and diterpenoids. All methods of essential oil production have implications for the likelihood of pesticide residue contamination and confer associated chemical characteristics to be considered when clean-up and analytical techniques are applied. Whatever the method of essential oil production, one aspect is common for all pesticide residues which do co-extract. The level of pesticide residues present in plant material is extracted into a significantly smaller volume of oil, such that essential oil production, can in effect, concentrate pesticide residues. With essential oil yields from plant material often in the vicinity of 3 to 5% by weight, contamination of, for example, 1 mgkg-1 in harvested crops has the potential to result in 20 a to 33 mgkg-1 level of contamination in the oil, assuming 100% recovery of the residues.

Solvent extracts
Concretes Many concretes are produced by the extraction of flowers, leaves or buds using low polarity organic solvents such as hexane and petroleum ether. Although the plant components targeted by this production method are usually aromatic volatile chemicals, high amount of waxes and non-volatiles are co-extracted. In addition, the co-extraction of any pesticide contaminant present in the plant material is also more likely occur in the production of concretes, as opposed to steam distilled oils. In most of the concretes produced in Australia, fresh or frozen material is used. Water content is often quite high, as much as 50% of the weight of plant material. For highly polar pesticides this increases the likelihood that much of residues present will remain in the vegetative

material. Highly water soluble pesticides, such as the quaternary ammonium salts, should not coextract with the oil components. However, a great number of commonly used pesticides are readily soluble in organic solvents, having similar polarities and solubilities to many of the desirable essential oil components such as hydrocarbons and oxygenated hydrocarbons. Concretes are also the most chemically complex of the oils produced using the various production methods. As such, clean-up techniques are almost essential, as solvent extracts often contain non-volatile components not amenable to gas chromatography (GC) or liquid chromatography (LC).

Absolutes Many of the considerations presented for concretes also apply to absolutes. However, the cold ethanol extraction process usually removes many of the waxes and lipids common to concretes which would otherwise present difficulties for volatilisation in GC or contaminate columns in liquid chromatography. However, the likelihood of co-extraction of moderately polar pesticide residue remains. The removal of contaminants which precipitate out with the waxy components during the production of absolutes, is only relevant to lipophilic chemicals.

Distilled Oils
Distilled oils, by the nature of their production, have few contaminants that are non-volatile. This has implications, not only for the degree of clean-up required, but also implies that nonvolatile residues should not be present in the oil. However, even non-volatile contaminants may be carried over in the distillation process when water droplets or particulate matter, splashed to the head of the distillation vessel, are washed through to the distillate. Whatever the process, pesticide residues are commonly detected in distilled oil. (Gould, 1960; Starr et al., 1963; Ballee et al., 1970, Garland et al., 1999). With sufficiently low detector sensitivity and specificity, analysis by GC may be conducted with no clean-up and little risk of accelerated GC column deterioration. Similarly, contamination of reverse phase LC columns presents as a minor consideration. For water soluble pesticides, however, steam distillation may present almost as a process for contaminant removal. The steam generated in the distillation process condenses to liquid and is recycled through a boiler in a closed system. Water is continuously washing through the oil collected, extracting much of any water soluble, co-distilled contaminants. In all, however, contamination of distilled oils with volatile, moderately polar pesticide residues is a common occurrence.

Table 1 lists some of the chemicals that are found in some of the major essential oils in Australian production.
Parsley (distilled) -pinene -pinene sabinene myrcene -phellandrene limonene -phellandrene p-cymene -terpinolene p-menthatriene ,p-dimethylstyrene carotol tetramethoxy allyl benzene elemicin myristcin apiole Fennel (distilled) -pinene myrcene -phellandrene limonene -phellandrene 1,8-cineole cis--ocimene fenchone estragole trans-anethole Peppermint (distilled) -pinene -pinene sabinene myrcene limonene 1,8-cineole cis--ocimene -terpinene 3-octanol sabinene hydrate menthone menthofuran isomenthone -bourbonene linalool menthyl acetate neomenthol caryophyllene terpinen-4-ol menthol pulegone isomenthol germacrene D piperitone Boronia (solvent extract) -pinene camphene -pinene sabinene -3-carene limonene -phellandrene terpinolene ethyl octanoate 2,6-dimethyl- 3,7 octadiene-2,6- diol dihydro -ionone dodecanol -ionone sesquicineole dodecyl acetate methyl jasmonate methyl epi jasmonate heptadec-8-ene

Table 1. Chemical composition of essential oils

Practical considerations for laboratory procedures in pesticide analyses

A simple guide to beginners to the field of pesticides analysis is listed. It is not intended to be a comprehensive guide to all sources of information and suggestions for specific procedures are to be instigated in addition to standard protocols common to good laboratory practices.

Collect as much information concerning the chemical nature of the pesticides and reagents to be used in all procedures. This information provides a starting point from which to assess the methodologies most suited to the chemical type and creates an awareness of significant safety issues including toxicity and potentially hazardous materials. Sources of information include: Merck index; pesticide handbooks; material safety data sheets; - available in coordinated databases marketed in comprehensive CD forms - provided by chemical companies such as Sigma Aldrich - available through several web sites, including http://ace.orst.edu/info/extoxnet/ standard literature data base search engines

Internationally standardised analytical methods usually specify detection limits to at least 1 mgkg-1 and can extend to the gkg-1 level. In the preparation of samples and standards, the potential for contamination is very high. Procedures within an analytical laboratory must adhere to strict guidelines to prevent contamination and achieve reproducibility. Listed below are a few recommended precautions: All glassware should be washed with appropriate cleansers such as alkaline Extran with neutralising washes of Galley then repeated washing with distilled water. Heating glassware at 440 is also an option though vessels may become brittle. Solvents should be commercially sourced and designated pesticide-grade or redistilled in decontaminated glass stills. At the commencement of each day of analysis, all surfaces should be wiped clean before chemicals and standard solutions from storage areas are introduced into the working area. Disposable bench liners are useful for reducing contamination risk. Be aware of the surface which have come into contact with pesticides and pesticide solutions. When

syringes are used to dispense standard solutions, place on a dedicated tissue between applications and discard the tissue at the end of each procedure. Prepare all samples to be screened for pesticide residues prior to any procedures which involve pesticide standard solutions such as those used to spike samples to establish standard curves and estimate recoveries. If practicable, complete the sample preparation and seal the vials from which the final aliquot is to be sub-sampled prior to the introduction of standard solutions to the working area. Dedicate syringes to preparations of standard curves and the spiking of samples with pesticide solutions. Have a separate set for the work-up of samples to be screened. Fortification of solvents and samples should proceed from the more dilute to the highest concentration, to minimise risk of contamination. Operators should observe all safety guidelines to comply with material safety data sheets (MSDs) protocols.

Categorisation of Pesticides
In the development of any pesticide, a particular mode of action, such as cholinesterase inhibition, inhibition of cell division or excessive stimulation of weed growth, is usually conferred by the inclusion of functional groups into the structure of the chemical. Distinct classes of pesticides are formed based on the chemical nature of these functional moieties. The chemical type of a pesticide also has implications in terms of their potential to contaminate essential oils. The functional groups unique to each class of pesticides, then, may confer the properties which affect:

translocation through plants and soils, important in terms of incorporation into plant material and potential for root uptake by current and sequential crops;

stability and residual time, which relates to their potential to still be present at harvest; extractability, related to the solubilities and volatility during essential oil production.

In addition, the chemical properties of pesticides have implications in the design of analytical methodology specific to their separation and detection.

Chlorinated chemicals
The organochlorine pesticides are an extensive groups of chemicals which include chlorotriazoles, which function as systemic fungicides, and chlorotriazines, which are broadspectrum residual herbicides, used for pre and post emergence weed control. Within the essential

oil industry these chemicals are at the highest risk of presenting as contaminants in products due to their systemic nature and the similarity of their chemical properties, such as polarity and volatility, to those of sesquiterpenes and oxygenated sesquiterpenes common to many essential oils. The aromatic qualities of the sesquiterpenes and oxygenated sesquiterpenes have ensured that industrial extraction protocols are designed to maximise their yield, with the concomitant effective extraction of many organochlorines. Many organochlorines are very stable, which in addition with their translocation and absorption properties, are prone to bioaccumulation. None the less, many organochlorines used within the essential oil industry have high efficacy with little to no residue at harvest time. In terms of analytical methodology the organochlorines are often amenable to GC and LC. However, as many have polarities similar to essential oil components, they are often the most difficult to pre-concentrate or separate from the matrix. The halogenation does, however, provide for the specificity germane to detection by Electron Capture Detectors (ECD) and distinctive isotope patterns in mass spectral analysis (MS).

Clean-up - The effectiveness of standard clean-up methodology is limited for the reasons already outlined. With sufficient dedicated resources, however, modern solid phase extraction (SPE) products may allow for the development of an effective pre-concentration step for any particular organochlorine residue in essential oils. The similarity in properties of many of these types of pesticides to the components within essential oils, makes the development of a cost-effective multi-residue screen using standard SPE techniques difficult. Liquid / liquid partition can remove many of the waxes and non-polar components of concretes, and to a lesser extent, distilled oils. The inclusion of such as preliminary clean-up can significantly reduce the loading in to GC injectors. For LC systems, particularly those employing columns with non-polar phases such as C18, the life of the guard column and columns can be significantly extended and the need for washing of the columns with extended runs can be reduced. However the simple liquid / liquid partition step described on page 91 is only a clean-up step with no corresponding preconcentration of target analyte. Preliminary development of an SPE methodology is described on page 92, which despite some poor recoveries, provides for a pre-concentration of target analytes and allows for the introduction of larger sample volumes into instrumentation.

Analysis - The majority of organochlorines are sufficiently volatile and thermally stable to be amenable to gas chromatography. The interaction with the liquid phase of the capillary columns is often specific to the analyte, such that many of the triazines elute with excellent

chromatographic characteristics, presenting sharp, well-defined peaks. The triazoles, on the other hand, may present with poor peak shape and significant tailing, especially as the column ages trial and error of each, will quickly distinguish which analytes have poor chromatographic properties and some experimentation with different liquid phases should be investigated. In general, however, the standard phenyl substituted silicones are effective in the separation of many organochlorines. As with gas chromatography, organochlorines are, in general, amenable to reverse phase high pressure liquid chromatography (RP HLPC) with elution profiles and chromatographic properties specific to each analyte determined by trial and error.

Experiments undertaken for equipment assessment GC ECD Detection limits established 0.1 to 5 mgkg-1 (retention time insufficient for unequivocal peak identification) GC HRMS Detection limits established 0.01 to 1 mgkg-1 Detection limits established 0.01 to 1 mgkg-1 (ionisation and response specific for each pesticide) page 56 page 45

(highly specific and quantitative, specialised equipment required) LC MSMS pages 72, 74

Properties typical of a commonly used organochlorine as exemplified by simazine Simazine is a selective triazine herbicide used to control broad-leaved weeds and annual grasses. It acts to inhibit photosynthesis (Kidd et al., 1991, Weed Sci. Soc. Am., 1994). It is moderately persistent with an average field half-life of 60 days (Wauchope et al. 1992). In high pH soils, residual activity may remain for a year after application (2 to 4 kgha-1). Its low water solubility, however, makes it less mobile, limiting its leaching potential (Weed Sci. Soc. Am., 1994). Plants absorb simazine mainly through the roots, with little or no foliar penetration. From the roots, it is translocated upward to the stems, leaves, and growing shoots of the plant (Kidd et al. 1991, Weed Sci. Soc. Am., 1994).

10

Physical Properties: Chemical Name: 6-chloro-N2,N4-diethyl-1,3,5-triazine-2,4-diamine

N

C2 H 5 NH
N

Cl
N

NHC 2 H 5 simazine 6-chloro-N,N-diethyl-1,3,5-triazine-2,4-diamine

Molecular Weight: 201.70 Solubility: water - 5 mgL-1 @ 20C, soluble in methanol, chloroform, and diethyl ether Melting Point: 225-227C (Kidd et al. 1991) Vapour Pressure: 0.000810 mPa @ 20C Partition Coefficient: 1.9600 Adsorption Coefficient: 130 (Wauchope et al. 1992)

Organophosphates
The organophosphate pesticides used predominantly in the essential oil industry are low molecular weight chemicals (< 350) often with low ratios of carbon to oxygen and sometimes chlorine atoms, which confers a degree of polarity as evidenced by their high solubility in water and polar organic solvents such as acetone. This group of pesticides which act as potent cholinesterase inhibitors, generally have lower persistence and bioaccumulation compared with organochlorines, but are regarded as highly toxic. Examples dealt with specifically in this study are monocrotophos, acephate and chlorpyrifos. All are soluble in water and the organic solvents used in essential oil production. The polarities of many of the organophosphates, as for the organochlorines, are similar to those of the oxygenated monoterpenes and sesquiterpenes. The presence of a phosphorus atom is the singular feature which delineates them from the greater body of pesticides. Specifically then, delineating organophosphates into a distinct class is useful only in the context of their chemical stability ie. likelihood of residual time, extractability in normal operations of essential oil production and lability in analytical processes. As a functional moiety able to be exploited for analytical methodology, the phosphorus atom only confers the specificity compatible with flame photometric detection (FPD) in the phosphorus mode or a nitrogen, phosphorous detector (NPD). In most other respects the suitability of GC, LC and the related methods of detection, has the same potential and disadvantages as any other of the pesticide classes. Toxicity, ionisation

11

potential and polarity specific for each organophosphate must be assessed, with considerations given to their increased propensity to degradation. Analytical methodology should be adopted on the basis of, range of application, expense and robustness.

Clean-up - The effectiveness of standard clean-up methodology is limited as the polarities of organophosphates are similar to those of essential oil components. Although modern SPE products may allow for the development of an effective pre-concentration step for any particular organophosphates, the co-elution of these pesticides with essential oil components makes the development of a cost-effective clean-up technique for multiple pesticides in a single screen difficult. In addition, the total number of organophosphates used widely in the essential oil industry is insufficient to warrant a separate, dedicated screen for the class. However a simple liquid /liquid partition step is described for a range of pesticides in solvent extracted concretes, including some organophosphates, on page 91. The method described results in a dilution of the pesticides and oil components into a larger volume of solvent but reduces the loading of nonpolar components into GC injectors and onto LC columns in subsequent analysis. Page 92 details a preliminary work-up for a clean-up and pre-concentration step using SPE.

Analysis - The majority of organophosphates are sufficiently volatile to be amenable to gas chromatography. However, when applied to the most widely used methyl silicone, non-polar liquid phases used in capillary GC, many of these analytes have poor chromatographic properties, with peak tailing especially pronounced as the column ages. Trial and error will quickly distinguish which analytes have poor chromatographic properties and some experimentation with different liquid phases should be investigated. Organophosphates have been found to be amenable to RP HPLC with elution profiles and chromatographic properties specific to each analyte determined by trial and error. Some organophosphates, such as monocrotophos, are degraded in low molecular weight alcohols and this susceptibility should be taken into account in the design of analytical methodology.

12

Experiments undertaken for equipment assessment GC ECD (organophosphates which are also halogenated) Detection limits established 0.1 to 5 mgkg-1 (retention time insufficient for unequivocal peak identification) GC NPD GC HRMS Preliminary assessment Detection limits established 0.01 to 1 mgkg
-1

page 45

page 47 page 56

(highly specific and quantitative, specialised equipment required) LC MSMS Detection limits established 0.01 to 1 mgkg-1 page 70, 74

Properties typical of commonly used organophosphates as exemplified by acephate and monocrotophos. Acephate is an organophosphate foliar spray insecticide used for control of a wide range of biting and sucking insects. The residual systemic activity is around 10-15 days at the recommended application rate (Thomson, 1992). Physical Properties:

O CH 3 CNH

O P

OCH3

SCH 3 acephate acetylphosphoroamidothioic acid O,S-dimethyl ester

Appearance: Colourless to white solid (Montgomery, 1993), Molecular Weight: 183.17 Solubility: water 650, acetone 151, ethanol < 50, ethyl acetate 35, benzene 16, hexane 0.1 (all in g/100 ml at 20C) (Worthing, 1987) Melting Point: 93 C (Kidd et al., 1991) Sufficiently volatile for GC Monocrotophos is a systemic insecticide and acaricide which has a low environmental persistence. It has a half-life of 1.3 to 3.4 days on plant foliage (Chambers et al., 1992). Physical Properties:
O (CH3O)2 P O CH3 H CONHCH3 monocrotophos Dimethyl 1-methyl-3-(methylamino)-3-oxo-1-propenyl phosphate

Monocrotophos is a reddish brown crystalline solid with a mild odour. M. W.: 223.2

13

Water Solubility: Soluble in water, acetone and alcohol (Meister, 1992) Melting Point: 54-55 C Partition Coefficient: -0.22

Urea Chemicals
Phenyl-substituted ureas are used extensively in agriculture as selective herbicides, mainly for pre-and post- emergence and they act by inhibiting photosynthesis. Commonly used substituted ureas are linuron and diuron, which have low residual action and persistence. Both are soluble in the organic solvents used in the essential oil industry and sufficiently labile to codistil with oils in steam distillation.

Clean-up - The solubility of the substituted ureas in water is a parameter which may be exploited in clean-up methodologies. The polarities and chemical distinction of the ureas are also features which may be exploited using SPE and other chromatographic products. The total number of this class of pesticide used in the essential oil industry is limited, however, such that the development of a specific extraction protocol would not be cost effective. However, the inclusion of the urea based chemicals in a protocol which may effectively pre-concentrate other water soluble pesticides, such as the herbicides with acidic moieties including dicamba and fluazifop acid, may be cost effective. Several experiments have been conducted for the acidic moiety pesticides (page 77) and may have direct application to the substituted ureas.

Analysis - The application of GC to phenylurea pesticides is difficult because these compounds are thermally unstable and rapidly degrade to isocyanates and amines (Buchert et al. 1975, Deleu, R. et al. 1979, Mattern, G. C. et al. 1989). Thermal reactions in the detector and on the columns result in a lack of reproducibility and incomplete thermal degradation preclude the monitoring of the degradation products for a quantitative screen. However, the breakdown products have been monitored by GC ECD and high resolution mass spectrometry (HRMS) and residues have been detected in oils produced from field trials using this technology. Linuron and diuron degrade to the same thermal breakdown product and so are indistinguishable using this analysis method. Derivatisation to compounds which are more thermally stable would provide for a more reliable screen. The application of RP HPLC is also problematic. Under the LC conditions trialed using acetonitrile/phosphate buffer, both urea herbicides were degraded. Detection by ion trap MS/MS was also limited. Linuron was ionised by atmospheric pressure chemical ionisation (APCI), but

14

the response was poor. Trials are continuing to optimise the mobile phase, specifically the pH, as urea derivatives are sensitive to slight variations in this parameter.

Experiments undertaken for equipment assessment GC ECD GC HRMS Preliminary experimentation Detection limits established 1 mgkg
-1

page 45 page 56

(not specific as ion produced is identical for linuron and diuron) LC MSMS Detection limits not established page 67

(poor chromatographic properties & poor ionisation in +ive mode APCI)

Properties typical of commonly used, urea based herbicide as exemplified by linuron Linuron is a substituted urea, pre- and post-emergence herbicide used to control annual and perennial broadleaf and grassy weeds. It works by inhibiting photosynthesis in target weed plants. Linuron is slightly to moderately soluble in water, and is not readily broken down in water (U.S. Nat. Lib. Med., 1995). It is more readily absorbed by roots from soil application, than by leaves from foliar application (Weed Sci. Soc. Am. 1994). The rate at which it is absorbed, translocated, and subsequently broken down (or metabolised) differs with various plant species (Weed Sci. Soc. Am. 1994).

Physical Properties:

Cl

NHCONCH 3 OCH 3 Cl

linuron 3-(3',4'-dichlorophenyl)-1-methoxy-1-methylurea)

Appearance: Linuron is an odourless, white crystalline solid (Kidd et al, 1991). Chemical Name: 3-(3,4-dichlorophenyl)-1-methoxy-1-methylurea Molecular Weight: 249.11

15

Water Solubility: 81 mgL-1 @ 25C, slightly soluble. Soluble in aliphatic hydrocarbons and acetone and moderately soluble in ethanol (Kidd et al, 1991) Melting Point: 93-94C (Kidd et al, 1991) Vapour Pressure: 2 mPa @ 24C Partition Coefficient: 3.0043 (Kidd et al, 1991) Adsorption Coefficient: 400 (Wauchope et al. 1992)

Carbamate Derivatives
The carbamates are N-substituted esters of carbamic acid and act as cholinesterase inhibitors that confer insecticidal activity. Their effects are generally less intense than the organophosphates and they have low persistence in the environment. Solubilities of the carbamates can vary quite dramatically. Although the likelihood of the co-extraction of different carbamates will vary with varying representatives of this chemical class, the potential to co-extract with concretes and absolutes is high. This is particularly true for the carbamate pesticides commonly encountered in the Australian industry, carbendazim and carbaryl.

Clean-up - The solubilities of carbamates are quite varied, such that it is difficult to develop one clean-up procedure which is equally effective for all types. The carbamates used in the essential oil industry include carbendazim and carbaryl which are of intermediate polarity such that it is again difficult to remove co-extracting essential oil components in analytical methodologies.

Analysis - The majority of N-substituted carbamates are thermally unstable and therefore not amenable to GC. The breakdown product of carbaryl, naphthalene, has been monitored, but low levels of this latter chemical may be found to be endogenous to essential oils. Derivatisation to thermally stable products presents as the only option if GC is the preferred analytical methodology. LC is more suited to the analysis of this chemical type.

Experiments undertaken for equipment assessment LC MSMS reverse phasae HPLC and with ionisation in +ive APCI page 69

16

Properties typical of commonly used carbamate as exemplified by carbaryl.

O CH3
N

carbaryl 1-naphthyl-N-methylcarbamate

Carbaryl is a wide-spectrum carbamate insecticide and an acaricide. Degradation of carbaryl in crops occurs by hydrolysis within the plants. It has a short residual life of less than 2 weeks. Physical Properties: Carbaryl is stable to heat, light, and acids. It is not stable under alkaline conditions. Chemical Name: 1-naphthyl-N-methylcarbamate CAS Number: 63-25-2 Molecular Weight: 199.25 Water Solubility: 40 mgL-1 @ 30C. Soluble in dimethylformamide, acetone, cyclohexanone. (U.S. Environ. Prot. Agency, 1988) Melting Point: 142C Vapour Pressure: <5.3 mPa @ 25C Partition Coefficient: Not Available Adsorption Coefficient: 300 (U.S. Environ. Prot. Agency,1988)

Dithiocarbamates
Dithiocarbamates are broad spectrum pesticides used widely in the essential oil industry to control fungal diseases such as rust. They are non-systemic, contact fungicides which remain on the surface of the plants until degraded or washed off with rain or abrasion. Dithiocarbamates are heat labile and degrade to a number of products including ethylenethiourea (ETU), which is soluble in water and readily absorbed and metabolised in plants. ETU is suspected to have goitrogenic, carcinogenic, mutagenic and tetragenic properties (Graham, 1973, 1972). The dithiocarbamate fungicide most commonly used in the essential oil industry is Dithane, whose active ingredient is mancozeb. As a polymeric salt of ethylenebisdithiocarbamic acid, containing 20% manganese and 2.5% zinc, it is insoluble in most organic solvents and water, is non-volatile and labile. It would seem unlikely that residues would contaminate

17

essential oil, whether solvent extracted or distilled. This, of course, does not preclude the possibility of ETU contamination and neither does it remove the need to confirm the absence of residues of the parent dithiocarbamate, considering the high and frequent applications implemented in commercial crops.

Clean-up - The lack of solubility of mancozeb and related dithiocarbamates in water and organic solvents, in addition to precluding their co-extraction in the production of essential oil, also presents as a chemical property which may be exploited in potential clean-up techniques. The standard method of analysis is the headspace analysis of carbon disulfide produced by digestion of residues in acidified stannous chloride, with analysis by GC and detection by FPD (Cullen, 1964; Keppel, 1969). It could be assumed that this methodology would remove the bulk of the components of essential oils. However, sulfur chemicals are endogenous to many essential oils, such as 4-methoxy-2-methyl-2-mercaptobutane in blackcurrant oil (Rigaud et al., 1986). Many of these components are associated with quality oils and extraction protocols are adapted to maximise yields. Levels of sulfur compounds in blackcurrants are as high as 50 mgkg-1 in some clonal material (Garland et al., 2002). Headspace analysis of the acidified stannous chloride digest of peppermint oil, known to be free of dithiocarbamate pesticides, found background levels of carbon disulfide of the order of 5 mgkg-1 (page 86). Research, instead, has focussed on the extraction of residues of thiocarbamates with EDTA, which acts as a chelating agent to solubilise the metal complexes into partly neutralised sodium hydroxide solutions (pages 87, 89). In the analysis of essential oils this presents as an excellent clean-up protocol, assuming the interface of the oil and aqueous extraction solution is sufficient to effect reproducible recoveries. Ethylenethiourea, as for many of the organochlorines and organophosphates, is soluble in the solvents used to produce essential oils and many of the problems associated with these chemicals also apply to the clean-up of ETU from essential oils.

Analysis - The most widely used method of carbon disulfide production using acidified stannous chloride is not sufficiently specific for the detection of residues of thiocarbamates and background levels of endogenous sulfur compounds preclude this method for qualitative and quantitative analysis. Detection of ethylene diamine, a side product of this reaction was not successful (page 86). Research has been conducted whereby EDTA / NaOH solutions are used to extract residues of the metal complexes of the fungicides which form sodium salts of the N, Nethylenebis(dithiocarbamate). Two methods for the quantification of these salts include

18

derivatisation using methyl iodide and analysed by LC MSMS, which had limited success (page 87), and the application of Inductively Coupled Plasma Optical Emission Spectrophotometer (ICP-OES). ICP OES was successfully applied to measure the manganese from mancozeb, which had been chelated in partly neutralised NaOH / EDTA solution (page 89).

Experiments undertaken for equipment assessment GC FPD Carbon disulfide produced by acidified stannous chloride (not specific) LC MSMS Preliminary investigation of EDTA extraction and methyl iodide derivatisation of N, N-ethylenebis(dithiocarbamate) ICP OES page 107 page 46

Elevated levels of manganese used as a marker for mancozeb contamination Page 111

Properties typical of commonly used dithiocarbamate as exemplified by mancozeb Mancozeb is used to protect many fruit, vegetable, nut and field crops against a wide spectrum of fungal diseases and is used in the essential oil industry to control rust. Mancozeb is of low soil persistence, with a reported field half-life of 1 to 7 days (Wauchope, et al., 1992). Mancozeb rapidly and spontaneously degrades to ETU in the presence of water and oxygen (U.S. Environ. Prot. Agency, 1988). ETU may persist for longer, on the order of 5 to 10 weeks (Wauchope, et al., 1992).

Physical Properties:

22+

-Mn- SCNCH 2 CH2 NCS H H x x:y 10:1

Zn

Chemical Name: manganese ethylenebis(dithiocarbamate) (polymeric)

Molecular Weight: 266.31 Water Solubility: 6 mgL-1 -Practically insoluble in most organic solvents (Kidd et al. 1991) Melting Point: Decomposes without melting @ 192 C Vapour Pressure: Negligible @ 20 C Adsorption Coefficient: >2000

19

Pesticides with Acidic Moieties

This class of broad leaf weed killers include a large range of carboxylic acid herbicides. Some of the pesticides are applied in chemical formulations as esters, which decompose to the acidic form under alkaline or acidic conditions. Translocation of the acidic pesticides then takes place in the roots of treated plants. The breakdown of most alkanoic acids is rapid. The acid forms are highly water soluble and less likely to be extracted during the production of essential oils than the parent esters, which are soluble in organic solvents.

Clean-up - Relevant aspects of the analysis of the ester forms of many of these chlorinated herbicides are similar to those already discussed. The polarity and solubility characteristics closely mimic those of the oxygenated monoterpenes and sesquiterpenes which constitute many essential oils, making separation from such matrices difficult. The excellent retention characteristics under GC and LC, combined with the specificity afforded by ECD of the chlorinated moieties of this chemical class, and by high resolution MS and ion trap MS, are often sufficient to allow for the analysis of oils without residue pre-concentration. The acidic forms of this class of pesticide, however, are water soluble, which confers a physical parameter on which clean-up protocols may be designed. Aqueous extractions of essential oils for the pre-concentration and removal of interfering matrix components present as the most promising approach (page 75). In an alkaline solution, the carboxylic acid moiety of the herbicides is de-protonated, such that the polarity of resultant ion moderates any non-polar characteristics of the remaining organic molecular framework of the residues. This facilitates extraction across the interface between an organic oil phase and an aqueous solution. Ion exchange chromatography also presents as a promising method of pre-concentration of aqueous extracts of essential oils (page 100).

Analysis - The parent esters of pesticides with acidic moieties follow the same considerations as previously discussed for the organochlorines. They are directly amenable to GC and elute in the same time frame as many of the oxygenated sesquiterpenes. Acidic moieties are not amenable to GC. However, many derivatisation steps, such as methylation or trimethylsilylation can convert acidic pesticides to esters compatible with GC. Phenoxy based herbicides are detected in essential oils using HRMS without clean-up (page 58) and preliminary experiments with GC ECD of the ester derivatives will allow for detection to 1 mgkg-1 (page 45 and page 99). However, the exploitation of the water solubility of acidic moieties to clean-up oil residues from oil matrices will not be compatible with many of these derivatisation processes, unless the

20

residues are re-extracted from the aqueous phase. Anion exchange discs were used to trap deprotonated acid based pesticides. The discs were dried then the acids derivatised and eluted with methyl iodide (page 99). Alternatively, a re-protonation of the acidic herbicide residues, by acidifying the alkaline extracting solution, would facilitate the re-extraction of the herbicides back into an organic solvent such as dichloromethane. Limited success in terms of recovery has been achieved in preliminary experiments investigating such an extraction protocol. The most promising line of research, however, is the extraction of acidic herbicides into an aqueous solution with analysis by LC interfaced with APCI in the negative mode and detection using ion trap MSMS (page 77)

Experiments undertaken for equipment assessment GC ECD Detection limits established for parent esters - 5 mgkg-1 (not sufficiently specific) Preliminary method development using ion exchange discs GC HRMS Detection limits established for parent esters - 0.1 - 1 mgkg (acidic moieties require derivatisation) ; LC MSMS Excellent chromatography good ionisation in -ive APCI
-1

page 45

page 99

page 58 page 78

Properties typical of commonly used acidic herbicide as exemplified by dicamba Dicamba is a benzoic acid herbicide. It can be applied to the leaves or to the soil to control annual and perennial broadleaf weeds. It is moderately persistent in soil having a typical half-life of 1 to 4 weeks (Wauchope et al., 1992). The rate of biodegradation increases with temperature and increasing soil moisture, and tends to be faster when soil is slightly acidic. Dicamba is rapidly taken up by the leaves and roots of plants, and it is readily translocated to other plant parts. It some plant species, dicamba accumulates in the tips of mature leaves (Weed Sci. Soc. Am., 1994)

21

Physical properties

COOH Cl OCH3

Cl
dicamba (3.6-dichloro-2-methoxybenzoic acid)

Chemical Name: 3,6-dichloro-2-methoxybenzoic acid Molecular Weight: 221.04 Water Solubility: 6500 mg/L @ 25C. Soluble in acetone, dichloromethane, ethanol, toluene and Xylene. (Kidd et al., 1991) Melting Point: 114-116C Vapour Pressure: 4.5 mPa @ 25C Partition Coefficient: -0.5376 Adsorption Coefficient: 2 (salt)

Quaternary Nitrogen Herbicides

The pesticides of this class used most frequently in Australia are paraquat and diquat. They are used for broadleaf weed control. They are quick acting, non-selective, contact poisons which are also translocated through the plant. Paraquat and diquat degrade slowly when exposed to sunlight but are otherwise quite resistant to microbial degradation and can persist indefinitely when bound to soil particles. The dichloride salts are stable to heat in acidic or neutral solutions but are hydrolysed by alkaline solutions. Paraquat and diquat are very soluble in water, have very low solubility in the lower alcohols and are insoluble in hydrocarbons. The likelihood of residues in solvent extracted essential oils is therefore very low. The processes associated with the production of steam distilled oils, where the oil is collected from a stream of condensed water and volatiles, would act to continuously extract water soluble chemicals. As for all potential contaminants, however, it is necessary to conclusively certify that no detectable pesticide residues are present in oils.

Clean-up - As with most of the pesticides which are unlikely to contaminate essential oils, the chemical properties which preclude their extraction in oil production are also properties which may be used to extract them from essential oils should contamination occur. Very few essential

22

oil components extract into water, whereas, providing there is a sufficient area of phase interface, paraquat and diquat should easily move into an aqueous solution from an organic phase.

Analysis - The quaternary ammonium herbicides are not amenable to GC. LC is limited usually requiring the inclusion of ion-pair reagents or the analytes must be derivatised. This presents limitations as to compatible detectors, as ion pair reagents preclude the use of some ionisation sources in LC MS interfaces. A highly sensitive and specific methodology using direct injection of water extracted samples into an electrospray ionisation (ESI) module configerated with an MS ion trap has been developed.

Experiments undertaken for equipment assessment LC MSMS Not compatible to LC owing to high absorption on surfaces (detection limit to 0.01 mgkg-1 ) ; page 85

Properties typical of commonly used quaternary nitrogen pesticide as exemplified by paraquat Paraquat is a quaternary nitrogen herbicide widely used for broadleaf weed control. It is a quick-acting, non-selective compound, that destroys green plant tissue on contact (and by translocation within the plant). It is a highly toxic compound and is highly persistent in the soil environment, with a reported field half-life of greater than 1000 days (Wauchope et al., 1992). The reported half-life for paraquat in one study ranged from 16 months (aerobic laboratory conditions) to 13 years (field study) (Rao et al., 1980). Ultraviolet light, sunlight, and soil microorganisms can degrade paraquat to products which are less toxic than the parent compound.

Physical Properties:
2+

H3C

N
paraquat

CH3

2Cl

Chemical Name: 1,1'-dimethyl-4,4'-bipyridinium dichloride (Kidd et al., 1991) Molecular Weight: 257.20 Water Solubility: 700 gL-1 @ 20C. The dichloride salt is sparingly soluble in lower alcohols (Kidd at al, 1991) Melting Point: Decomposes @ 300C Vapour Pressure: Negligible @ room temperature (paraquat dichloride)

23

Partition Coefficient: 4.4683 Adsorption Coefficient: 1,000,000 (estimated) (Wauchope et al., 1992)

24

Analytical Equipment
Gas Chromatography
The separation of volatile oil components using temperature and pressure gradients through capillary columns in gas chromatography has become one of the widely applied technologies in analytical chemistry. The relatively low cost, amenability to automation and robustness has ensured the adoption of this technology by most analytical laboratories. The volatile fraction of solvent extracted concretes or absolutes can be 20 to 80% of the total extract whilst steam distilled oils are almost 100% volatile. This would imply that many oils could be loaded directly onto a GC column without clean-up, providing the detector has sufficient sensitivity and specificity. The loading limit on capillary columns depends on the stationary phase and its thickness. The loading capacity for a standard dimethyl polysiloxane (methyl silicone), non-polar phase GC column is in the vicinity 20 g of oil in a 1 L split injection (0.6 g in a 30:1 split). Assuming a pesticide residue contamination level of 1 mgkg-1, as little as 0.02 ng of the target analyte will be present in that same 1 L injection. Most non-specific detectors such as flame ionisation detection (FID) cannot detect much below 0.1 ng of any one component. In samples which have not been subject to clean-up procedures, then, GC must be coupled to sensitive and specific detectors.

25

Plate 1. Gas Chromatograph

Detectors assessed in this manual include: flame ionisation - high detection limits & non-specific flame photometric detection - high specificity to sulfur and phosphorus electron capture detection - halogenated chemicals NPD detector chemicals containing nitrogen and phosphorus mass selective detection - general screening limited to detection ~1 to 5 mgkg-1 without clean-up high resolution mass spectrometry - highly specific, with confirmational ions usually available for each analyte, low detection limits with minimum clean-up

26

The limitations of gas chromatography in the analysis of essential oils which have not undergone clean-up and pre-concentration relates to the need to load large amounts of oil components into the systems to detect low levels of pesticide residues. For solvent extracted concretes or absolutes (such as boronia and blackcurrant) large amounts of non-volatile components, including waxes and tannins, may not volatilise in the GC injection chamber or may not elute from the column. Many non-volatile components are condensed onto the silanised glass wool within the injector liner of the GC and this can be removed after a number of injections, replaced or cleaned and re-silanised before re-use. However, the loading of excessive amounts of essential oil components can lead to column damage, resulting in poor resolution, peak tailing and low recovery of analytes. There are many types of GC injectors that afford a measure of control to reduce the loading of components onto the column, such as septum equipped, programmable injectors (SPI). Pre-columns can also prolong the viability of GC columns and periodical baking of the column through a slow temperature gradient, with a long hold at the maximum, can rejuvenate poor performing columns which have been subject to excessive loading. A section of the column can also be removed to improve performance. However, if matrix components co-elute with the target analyte and saturate the detector signal, the parameters which can be optimised are limited to temperature gradients and pressure. Alternatively separation may be achieved using GC columns with different immobile phases or the target analyte may be amenable to derivatisation. Retention characteristics of target analytes under standardised GC conditions are often insufficient for an unequivocal identification as these can vary with column conditions. Standard curves, confirming retention times, should be run immediately before and after a sample is tested for pesticide contamination. The sample may also be re-chromatographed, after being fortified with the target compound. This will show an enhanced peak for retention time confirmation. Retention times can be expressed as values relative to retention time of a standard reference compound. Selection of a chemical with similar structural and physical properties to the target analyte will ensure that slight changes in experimental conditions will change the retention characteristics of the reference compound to the same degree as those effected on the target analyte.

27

Despite reproducible and standardised methods the retention characteristics are often insufficient for unequivocal peak assignment. Methods for confirming peak identity include;

application of clean-up techniques to remove interfering peaks; reference to mass spectral data, comparing relative retention times on capillary columns having different liquid phases;

adjustment of experimental conditions to improve peak resolution; derivatisation and application of different detectors specific to different functional groups on the target compound, such as halogenation.

The following sections detail the experiments undertaken in the assessment of a range of GC compatible detectors, including ECD, NPD, FPD, benchtop MSMS and HR MS.

Assessment of GC ECD in the Analysis of Pesticide Residues in Essential Oils.

Electron Capture Detectors (ECD) are specific to halogenated chemicals. Both essential oils and solvent extracts are predominantly hydrocarbons and oxygenated hydrocarbons. The bulk of the matrix of an essential oil, contaminated with halogenated pesticide residues, should not register by ECD. As discussed previously, however, any one component of an essential oil can constitute in excess of 10% of the oil, compared to pesticide contamination levels which are often around 0.0001%. Obviously, co-elution of a major oil component would mask the specificity of the ECD through saturation of the detector with a component 105 times more concentrated than the halogenated pesticide. The retention characteristics of the target analyte relative to the major components of essential oils are therefore of critical relevance. When considering the suitability of GC ECD to the analysis of a new target analyte, the determination of the retention characteristics relative to essential oil components can provide an indication as to whether the analyte response will be masked by co-eluting extractives. Gas chromatographic retention indices (Kovts' indices) relate the retention time of a particular analyte to the retention time of a series of CnH(2n+2) hydrocarbons. It is useful to pre-determine the retention indices of the components of the relevant essential oils. The introduction of a new pesticide into a GC screen can then be easily assessed by first determining the retention indices for the new analyte and relating them to the established indices for the essential oil components. This will allow an assessment as to whether the pesticide residue will elute in a time window separated from other major essential oil components.

28

To assess the application of GC ECD to the detection of pesticide residues in the matrix of essential oils, Kovt's indices were established for the major components of essential oils and for the halogenated pesticides commonly used in the industry. In the experiments detailed in the following pages, it was shown that the majority of the components of distilled oils have Kovt's indices below 1500, whilst the majority of pesticides have Kovt's indices above this figure. This indicated that co-elutions of essential oil peaks with the target analyte would be minimal. Despite the high degree of specificity of the GC ECD, and the adequate retention and response properties of some of the halogenated pesticides tested, it was evident that the retention times alone were insufficient to unequivocally identify peaks. GC ECD can only be used as a screen to determine that no gross contamination of essential oils has occurred. The high number of components of boronia extract with retention indices greater than 1500 effected the masking of pesticide residue peaks. None the less, method validations were conducted for eight halogenated pesticides in fennel, parsley and peppermint distilled oils and boronia extracts.

Experiments Undertaken in the Process of Method Development Example 1. Retention Indices of Components of Essential Oils
Aim : To establish the Kovt's indices of essential oils on a HP 5MS column under the GC conditions to be used in standard GC ECD pesticide analysis using GC FID Experimental : GC FID has the capacity to detect a 1L split injection of a 1 mgmL-1 solution. A 1 mgmL-1 solution of a range of CnH(2n+2) hydrocarbons was prepared and analysed in the same column, and under identical conditions of pressure and temperature, as those to be used in the proposed GC ECD analysis.

Analytical parameters Instrumentation

Injection: Column: Carrier gas: Head Press.: Oven Temp: Injection Temp: Detector:

Hewlett Plackard 5890 gas chromatograph Hewlett Plackard Flame Ionisation Detector Processing Software - HP Chem 1 L, split automatic injections 30 m HP 5MS, 0.22 mm id, 0.25 m film thickness Instrument grade nitrogen 10 psi 1 min. at 60C, then programmed at 20C/min to 290C for 10 min. 260C FID 280C

29

Results: Figure 1, 2, 3 and 4 are the GC FID chromatograms for injection of 20 mgkg-1 solutions of parsley, fennel and peppermint oils and boronia oils.

Figure 1 - GC FID of distilled parsley oil

Figure 2. - GC FID of distilled fennel oil

30

Figure 3 - GC FID of distilled peppermint oil. Figure 4 - GC FID of solvent extracted boronia oil

31

Table 2 records the retention times of mixed 1 mgmL-1 solutions of hydrocarbon standards ranging from C8H18 to C36H74. The Kovt's indices are calculated using the formula t'R(A) - t'R(N) I ab = 100N + 100n -------------------t'R(N+n) - t'R(N)

Where I is the retention index on phase a at temperature b and t'R(N) and t'R(N + n) are the adjusted retention times of n - paraffin hydrocarbons of carbon numbers N and (N+ n) that are respectively smaller and larger than the adjusted retention times of the unknown, t'R(A) (ref 1.)
Standard Peak nC15H32 nC16H34 nC17H36 nC18H38 nC20H42 nC21H44 nC22H46 nC24H50 nC28H58 nC34H70 Ret. Time (mins) 14.804 16.126 17.381 18.582 20.794 21.834 22.817 24.682 28.697 35.435 Kovt's indices 1500 1600 1700 1800 2000 2100 2200 2400 2800 3400

Table 2. Retention times of CnHn+2 hydrocarbons The retention times of the major components and the calculated Kovts' indices are listed in Table 3.

32

Parsley oil -pinene -pinene myrcene -phellandrene -terpinolene menthatriene tetramethoxyallyl benzene myristicin apiole Peppermint oil 1,8-cineole menthone menthol pulegone isomenthol germacrene D piperitone

ret. time 6.40 7.13 7.50 8.03 8.94 9.47 15.57 16.53 21.17 ret. time 7.96 10.21 10.71 11.49 12.22 14.26 15.11

Kovts indices 674 743 778 827 913 962 1535 1626 2061 Kovts indices 821 1032 1079 1152 1221 1412 1492

Fennel oil -pinene -phellandrene limonene -phellandrene fenchone estragole trans-anethole

ret. time 6.33 7.45 7.86 7.86 9.07 10.71 12.52

Kovts indices 667 773 812 812 925 1079 1249

Boronia extract -pinene -pinene terpinolene -ionone dodecyl acetate methyl epijasmonate heptadec-8-ene waxes & high M.W. chem.

ret. time 6.30 7.02 7.83 15.07 15.40 16.41 17.37 23 - 34

Kovts indices 665 733 808 1488 1520 1614 1704 2233-3266

Table 3. Kovts indices for major components of essential oils and extracts. Figures 5 to 8 record the GC ECD traces of injection of 20 mgkg-1 solutions of essential oils.

Figure 5. GC ECD of distilled parsley oil

33

Figure 6. GC ECD of distilled peppermint oil

Figure 7. GC ECD of distilled fennel oil

34

Figure 8. GC ECD of solvent extracted boronia oil

Conclusion: Evident in Figures 5 to 7 is that the components of the distilled essential oils which effect an ECD response, have Kovt's Indices less than 1500. The solvent extracted boronia extract shown in Figure 8, however, has interfering components eluting through to 35 minutes.

Experiments Undertaken in the Process of Method Development Example 2. Retention Indices of Pesticides in Essential Oils
Aim: To establish the Kovt's indices for pesticides on a HP 5MS capillary column Experimental: An acceptable limit of detection for a basic screen would approach 1 mgkg-1. The loading capacity of standard non-polar capillary columns such as HP1 or HP5MS, is in the vicinity of a 1 L split injection of a 20 mgmL-1 solution of an essential oil. A sample containing 0.02 g of an active ingredient of a pesticide, in 20 mg of oil constitutes a 1 mgkg-1 solution. Without clean-up techniques, GC ECD would need to be able to detect 0.02 ng of analyte in a 1 L injection. For each halogenated pesticide a concentration of ~2 g of analyte in 1 mL of solution is equivalent to 100 mgkg-1 in a 20 mg sample of oil. This concentration should be sufficiently high for easy detection so as to determine the retention time of each analyte and provide some indication as to the likely response under GC ECD conditions.

35

Table 4 lists the pesticides commonly used in the essential oil industry which contain at least one halogen in their molecular structure.

Halogenated, GC amenable pesticides tebuconazole propiconazole linuron diuron simazine oxyflurofen chloroprofam bromacil terbacil procymidone difenoconazole ethofumesate dimethenamid chlorpyrifos norflurazon haloxyfop esters fluazifop esters glyphosate - derivatised mecoprop - derivatised MCPA - derivatised Dicamba - derivatised trichlopyr - derivatised clopyralid - derivatised fluroxypyr - derivatised

Table 4. Halogenated pesticides used within the essential oil industry Acetone solutions (2 gmL-1) of each halogenated, GC amenable pesticide were injected in a gas chromatograph with detection by ECD under the following conditions. Analytical parameters Instrumentation Hewlett Packard 5890 gas chromatograph Hewlett Packard Electron Capture Detector Processing Software - HP Chem 1 L, split automatic injections 30 m HP 5MS, 0.22 mm id, 0.25 m film thickness Instrument grade nitrogen 10 psi 1 min. at 60C, then programmed at 20C/min to 290C for 10 min. 260C ECD 260C

Injection: Column: Carrier Gas: Head Press.: Oven Temp: Injection Temp: Detector:

The effects of the matrix on the detection of halogenated pesticides were then assessed. Distilled oils (20 - 30 mg) were weighed into GC vials. Boronia extracts were warmed and mixed thoroughly to ensure an even distribution of all oil components. Sub-samples (20 - 30 mg) were weighed into 2 mL GC vials. Vials were spiked with 10 L of an acetone solution containing a 0.2 mgmL-1 mix of each pesticide standard.

Results: ECD was found to be successful in the detection of terbacil, bromacil, haloxyfop ester, propiconazole, tebuconazole and difenaconazole. Figure 9 shows the chromatograms obtained by GC ECD.

36

Figure 9. GC ECD traces of 2 gmL-1 solutions of halogenated pesticides

pesticide linuron ethofumesate simazine terbacil fluazifop ester fluroxypyr ester procymidone propiconazole tebuconazole difenaconazole ret. time 20.652 20.596 18.099 19.147 23.388 24.951 22.146 24.563 25.00 32.620 Kovt's indices 2013 2007 1773 1871 2270 2416 2153 2380 2421 3136

Table 5. Kovts indices calculated for the pesticides shown in Figure 9. Figures 10 to 13 shows the GC ECD chromatograms for spiked parsley, peppermint, and fennel oils and boronia extract.

37

Figure 10. GC ECD chromatogram of parsley oil fortified with 100 mgkg-1 mixed pesticide standard

Figure 11. GC ECD chromatogram of peppermint oil fortified with 100 mgkg-1 mixed pesticide standard

38

Figure 12. GC ECD chromatogram of fennel oil fortified with 100 mgkg-1 mixed pesticide standard

Figure 13. GC ECD chromatogram of boronia extract fortified with 100 mgkg-1 mixed pesticide standard

39

Discussion: The application of GC ECD to the detection of halogenated pesticides in essential oils, without clean-up techniques, is limited. Detection limits are unlikely to be less than 0.1 mgkg-1. The large number of interfering peaks from co-eluting extract components makes the unequivocal identification of unknown peaks impossible. Method validation has been undertaken for the peaks which showed good response and chromatographic properties.

Experiments Undertaken in the Process of Method Development Example 3. Method Validation for Detection of Pesticides by GC ECD in Parsley, Peppermint and Fennel Oils and Boronia Extracts.
Aim: From the results presented in Figures 10 to13, a preliminary method validation experiment was designed to include simazine, terbacil, bromacil, haloxyfop and fluazifop esters, propiconazole, tebuconazole and difenaconazole.

Experimental: Four 20 mg aliquots of each oil were spiked with standard solution mixes to produce a range of concentrations of 0.01 to 10 mgkg-1. Samples were analysed using the same parameters as listed on page 40. Repeat injections at each concentration of fortified oil were analysed to determine repeatability. Results: The detection limits, linearity as expressed by the r2 value of a linear regression, and the repeatability as expressed by the r.s.d.s are listed in Table 6.

40

Pesticide

ret. time

std. curve coefficients x

2

curve fit 2 r

detection limits solvent gmL

-1

fennel

pep.mint

parsley

bor.

r.s.d. %

simazine terbacil bromacil haloxyfop ester fluazifop ester propiconazole tebuconazole difenaconazole

18.02 18.92 20.55 22.05 23.31 26.00 21.92 32.70 9.19E-09 5.12E 1 -2.92E-09
-1

1 2.29E-09 9.56E 0
-1

1 0.5 1 5 0.5 1 0.5 0.5 0.5 5 0.5 1 1 1 50 5 1 1 5 1 5 5 1 22.3 2.2 13.7 12.2 4.3 7.7

5.50E-05 8.39E-06 6.34E-05 1.85E-05 2.26E-06 5.15E-05

1.000 1.000 0.995 0.999 0.997 0.998

0.02 0.01 0.02 1 0.02 0.01 0.02

Table 6. Detection limits and repeatability for the analysis of halogenated pesticides in essential oils and extracts by GC ECD

Discussion: Analysis of pesticide residues in essential oils and extracts by application of GC ECD are not specific enough to allow for an unequivocal identification of any contaminant. As a screen, ECD will be effective to establish that no gross contamination is present in oils or extracts in the absence of a co-eluting peak. In the situation where a peak is recorded with the correct elution characteristics, and which is enhanced when the sample is fortified with the target analyte, a second means of contaminant identification would be required, such as high resolution mass spectrometry. ECD is a screen for contamination and will not provide a positive identification for any pesticide contaminant.

Assessment of GC FPD
GC FPD was not found to be particularly suitable for the analysis of pesticide residues in essential oils and extracts without considerable clean-up procedures. Tests were undertaken with acephate, methedimos and monocrotophos. Poor chromatography, due to thermal degradation and poor interaction with the liquid phase of the GC columns, precludes this detection method in specific screens for the analysis of residues in essential oils. Digestion of dithiocarbamate pesticides with acidified stannous chloride produces carbon disulfide. The gaseous product can undergo head-space sampling with analysis by GC FPD in the sulfur mode. This well established technique for the detection of dithiocarbamate residues was applied to the analysis of essential oils, though with several adaptations to the sampling method for the gaseous end product. The detection of mancozeb in peppermint oil using acidified stannous chloride digestion, with partitioning of carbon disulfide into n-octane was trialed (adapted from methodology published by Woodrow 1995). Recoveries were as low as 10%, with

41

detection limits of around 10 mgkg-1. Many essential oils have endogenous sulfur components and analysis of carbon disulfide by acid digestion of samples, not previously treated with dithiocarbamate chemicals, gave background levels as high as 5 mgkg-1. A by-product of acidified stannous chloride digestion of dithiocarbamates is ethylenediamine. The quantification of the amount of ethylenediamine produced was trialed. Derivatisation of ethylenediamine with BSTFA to convert the product to a GC amenable analyte was successful. However, detection of ethylenediamine produced from the digestion of mancozeb standard was unsuccessful. Residual stannous chloride or hydrochloric acid may have promoted the depletion of the derivatising agent. Detection of the carbon disulfide using headspace analysis with detection by FPD has been trialed. Mancozeb was digested in the matrix of peppermint leaves and oils and compared to solvent only digestions. Detection of mancozeb at concentration levels equivalent to 20 mgkg1

gave a r.s.d. of 10.6%. However, peppermint oil samples that had not been treated with

mancozeb gave positive results for the production of carbon disulphide using the digestion methods described. The presence of endogenous sulfur chemicals precludes this methodology for the analysis of mancozeb in most essential oils. Nonetheless, the methodology for the analysis of peppermint leaves is included in the appendix 1. The response for carbon disulfide by GC FPD was non-linear and less sensitive than that recorded for phosphorus compounds using the 524 m filter.

Assessment of GC NPD in the Analysis of Pesticide Residues in Essential Oils.

The NPD is based on the ionisation of nitrogen and phosphorus in a thermionic source within a jet flow of hydrogen and air. The NPD collector contains a small alumina cylinder coated with a rubidium salt (active element) which is heated electrically. The ionised organic molecules are collected and the resulting current is measured. The specificity afforded by the efficient ionisation of organic molecules containing nitrogen or phosphorus may allow for the NPD to selectively respond to nitrogen and phosphorous containing pesticides, preferentially detecting them from amongst the C H and O containing components of essential oils. Much of the points noted under the assessment of GC ECD apply for GC NPD with the probability of excessive amounts of the components quenching the signal. None the less experiments were undertaken to determine the potential of GC NPD to detect pesticides in essential oils.

42

Experiments Undertaken in the Process of Method Development Example 4. The Detection of Pesticides by GC NPD in Peppermint Oil.
Aim: To undertaken an assessment of the response of GC NPD to pesticides in the matrix of peppermint oil.

Experimental:

The pesticides included in this experiment were monocrotophos, simazine,

propazine, cyanazine, propiconazole, tebuconazole and difenaconazole. The retention times of the proposed analytes were established by injecting 1 mgkg-1 solutions of each pesticide into a GC with detection by FID. The column and GC parameters used to establish the retention times were replicated when transferring analysis to the GC NPD. An injection (1 L) of a 20 gmL-1 mixed solution of monocrotophos, simazine, propazine, cyanazine, propiconazole, tebuconazole and difenaconazole was analysed by GC NPD using the conditions listed. Retention times were used to identify peaks. Solutions of 200, 20 and 2 ngmL-1 in acetone were prepared for direct comparison to equivalent solutions but also containing 20 LmL-1 of peppermint oil. This translated to 20, 10 and 1 mgkg-1 pesticides concentrations in peppermint oil. Analytical parameters: Instrumentation: Varian 3300 gas chromatograph. Varian NPD. Processing Software Varian Starr Workstation. 1 L, splitless manual injections 30 m HP5MS, 0.22 mm id, 0.25 m film thickness Instrument grade helium 10 psi 60C (1 min.) -10C/min-280C (15 min.) 260C NPD 300C 3.1 mV -4.0 mV

Injection: Column: Carrier gas: Head Press: Oven Temp: Injection Temp: Detector: Filament voltage Voltage offset

Results: Figure 14 show the chromatogram of a 10 gmL-1 acetone solution of monocrotophos, simazine, propazine, cyanazine, propiconazole, tebuconazole and difenaconazole. Figures 15 and 16 show the GC NPD traces of pesticide fortification levels of 1 mgkg-1 in solvent only and in peppermint oil respectively.

43

Figure 14. Pesticides (10 gmL-1) in acetone analysed by GC NPD.

Figure 15. Pesticides (1 mgkg-1) in solvent analysed by GC NPD.

44

Figure 16. Pesticides (1 mgkg-1) in peppermint oil analysed by GC NPD The areas obtained for each fortification levels of pesticides in acetone are compared to the response of equivalent concentrations of each pesticide in peppermint oil in Table 7. The relative response of pesticides in oil compared to pesticides in solvent expressed as a percentage is also listed. The oil has limited effect on the retention time but suppresses the response of the NPD by over 80 % for all the pesticides. The earlier eluting isomer of propiconazole is suppressed by 99%. However, despite the quenching of the response, the selectivity of the NPD is encouraging and the analyte peaks stand well above the background signal.

simazine propazine cyanazine propiconazole propiconazole tebuconazole

ret. time mins 18.445 18.645 21.344 25.335 25.506 25.883

solvent only 8505 12091 3444 13760 2238 2335

ret. time mins 18.429 18.630 21.327 25.319 25.487 25.864

peppermint oil 1186 2139 729 190 344 436

% response pepp.oil to solvent only 14 18 21 1 15 19

Table 7. Relative response of nitrogen and phosphorous containing pesticides fortified at a level equivalent to 1 mgkg-1 relative to oil in solvent and peppermint oil.

45

Assessment of Benchtop GC MSMS

Despite the high expectations based on the sensitivity and selectivity of the application of benchtop gas chromatography daughter / daughter mass spectrometry, severe limitations relating to the configuration of the system, precluded its application in the analysis of pesticide residues in essential oils without considerable clean-up techniques. The equipment accessed by this project was a Varian Starr 3400 CX GC coupled to a Varian Saturn 4D MS/MS. Unlike other mass spectrometry systems, the effluent from the gas chromatograph elutes into ion trap before ionisation. In some mass spectrometers, only ions enter directly into the ion trap. The introduction of a 1 L split injection may contain as much as 20 g of essential oil components. With a split ratio of 1 : 50 the injector will deliver 0.4 g onto the column. The entire eluant then enters the trap such that the isolation of specific target parent ions is difficult. The Saturn MS/MS, under the control of the 'Autogain', collects a specific number of ions. Ionised components of the background matrix are often in sufficient abundance to fill the ion trap prior to the ionisation of the target analyte. In addition essential oil components would quickly accumulate within the ion trap. The mass spectrometer is required to be shut down for several days for the trap to be cleaned. Although several of the pesticides relevant to the essential oil industry were trialed in solvent solutions, with encouraging results, it was not considered practical to continue method development within the matrix of essential oils without comprehensive clean-up methodologies.

46

Plate 2. Benchtop GC MSMS

Assessment of GCHRMS in the Analysis of Pesticide Residues in Essential Oils.

HRMS has the capacity to measure the m/z ratio to such an accuracy as to be able to determine the molecular mass of fragments to 4 and 5 decimal places. This provides a high degree of specificity and when used in the selected ion monitoring mode (SIM) can detect target analytes even when they are in a much lower abundance amongst co-eluting matrix components. Very few analytical laboratories have access to this sophisticated and expensive equipment. In addition, a high level of expertise is required to maintain and operate such an instrument. However, with the difficulties inherent in the analysis of pesticide residues within essential oils GC HRMS affords a capacity for specific and sensitive detection for all GC amenable analytes irrespective of the presence or absence of halogens, thiols, amines or phosphates.

47

Plate 3. Gas Chromatograph, High Resolution Mass Spectrometer

The Application of GC HRMS in the Analysis of GC Amenable Pesticides.

The MS and retention characteristics of the active ingredient of each pesticide are first assessed. Each analyte is run by GC MS in full scan mode. The retention time is recorded and the ions most suitable for monitoring are selected on the basis of their abundance and diagnostic potential. Ions with masses similar to those originating from co-eluting components of essential oils can be selected, as the degree of mass discrimination afforded by high resolution allows distinction of the ions separated by as little as 10 ppm in mass or 0.05 atomic mass units (amu) at m/z 500. If the relative retention and basic MS of analytes are known, then the first full scan run can include a range of pesticides for the determination of retention time and ions suitable for monitoring. Figure 17 shows a sample run of acephate, simazine, dichlorpyrifos, dimethanamid, sethoxydim, oryzalin, bromacil, norflurazon and diuron by GC TIC. As a case example, Figure 18 shows the MS of sethoxydim, which elutes at 10:53 minutes on the GC temperature ramp used. The ions selected for monitoring were 191 and 219, corresponding to the fragmentation

48

ions C11H13NO2 and C13H17NO2. The masses for the ions to be monitored by high resolution SIM were calculated to be 191.0946 and 219.1259.

Figure 17. TIC of pesticides by GC HRMS

Figure 18. MS of sethoxydim

49

Having determined the retention times and suitable mass fragments for monitoring, the mass spectroscopist establishes an instrument run whereby the HRMS is programmed to switch between ions to be monitored within time windows spanning the retention time relevant to each particular analyte included in the screen. In HRMS the response of ions monitored will change with instrument tuning which must be optimised each day of use. An internal standard is necessary. In early method development octadecane was selected and the ion monitored was 254.2973. However, the solubility of this hydrocarbon presented some limitations. When samples were prepared in polar solvents, as a result of extraction protocols etc., octadecane was observed to separate, preventing quantitative determination. Endosulfan was selected as an internal standard as the chemical structure and properties are similar to that of other pesticides. It chromatographs well on capillary GC and it is not commonly used in the essential oil industry. Should this analyte be present in oils, the high level at which the standard is spiked should overwhelm any background contamination and not significantly affect quantification. The ion selected for monitoring of the internals standard was 194.9534. Figure 18 shows a typical GC HRMS run established to monitor diuron, dimethenamid, simazine, acephate, norflurazon, bromacil, chlorpyrifos and sethoxydim. Method development has been undertaken for 19 pesticides that can be directly analysed by GC HRMS. Table 8 lists the analytes, the ions monitored and general comments with regard to their chromatographic behaviour. The pesticides are listed in order of elution under the conditions trialed. The actual retention times are omitted as this parameter is subject to variations depending on column type, temperature and pressure gradients etc.

50

Figure 18. GC HRMS chromatogram of 9 pesticides

51

Active ingredient linuron diuron acephate monocrotophos terbacil chloropropham int. std. prometryn simazine ethofumesate int. std. dimethenamid pendimethalin bromacil chlorpyrifos oxyflurofen fluroxypyr propiconazole tebuconazole norflurazon sethoxydim

Trade name Linuron Krovar Orthene Nuvacron Sinbar Allicide octadecane Gesaguard Gesatop Tramat endosulfan Frontier Stomp Krovar Lorsban Goal Fusilade Tilt Folicur Solicam Sertin

Ion mass 188.9555 188.9555 136.0164 127.0160 161.0117 213.0557 254.2973 241.1361 201.0781 286.0875 194.9534 230.0406 252.0984 204.9613 196.9202 252.0398 282.0742 259.0291 250.0743 303.0386 219.1259

Confirmation ion 186.9584 186.9584 97.0527

Chrom. properties degrad. prod. degrad. prod. poor poor good good good

Det. limit peppermint mgkg 1 1 1 0.1 0.1 0.05 0.05 0.1 0.02 0.05 0.05 0.05 0.05 0.01 0.05 0.02 0.1 0.05 1
-1

Det. limit boronia mgkg 0.1 1 1 1 0.1 0.05 0.05 0.1 0.02 0.05 0.05 0.05 0.05 0.02 0.05 0.02 0.5 0.05 1
-1

226.1126 186.0546

good med. good good

232.0377 281.1376 198.9172 300.0035 393.1344 261.0262 252.0714 191.0946

good good good good good good diastereomers some tailing good some tailing

Table 8. Analysis of 19 pesticides by GC HRMS - ions monitored and chromatographic characteristics

Experiments Undertaken in the Process of Method Development Example 5. Method Validation of the Analysis of Tebuconazole, Propiconazole, Simazine, Terbacil, Bromacil and Oxyflurofen in Boronia Extract by GCHRMS
Aim: To validate the methods developed for the detection of 6 pesticides in the matrix of boronia extract using GC HRMS.

Experimental: Standards were purchased from Sigma Aldrich and approximately 25 mg of each were weighed into 25 mL volumetric flasks and made up to volume with acetone. Solutions of 10gmL-1 and 1 gmL-1 were prepared by diluting 250 L and 25 L of the 1 mgmL-1 solutions, respectively, into 25 mL of acetone. Standard curves were prepared by weighing 22 x ~20 mg of boronia concrete into GC vials. Vials were fortified with the standard solutions as listed in Table

52

9, to allow for 6 replicate samples at 3 concentrations to establish repeatability. Endosulfan (5 g) was added to each vial as an internal standard. Hexane (200 L) was added to each, the lids were placed firmly on top, without sealing, and the mixtures gently swirled to dissolve the concrete. The bases of the vials were gently warmed by briefly placing them on the base of an oven where required.
Concentrations (mgmL ) 20 10 1 0.1 0.05 0.02 0
-

no. 1 6 6 1 6 1 1

10gmL 40 20

-1

1gmL

-1

0.1gmL

-1

endosulfan 5 5 5 5 5 5 5

20 20 10 4 0

Table 9. Spiking protocol for the establishment of standard curves and repeatability experiments Methanol (0.8 mL) was added to each vial, which were then sealed and shaken vigorously to ensure complete mixing. After a settling period of ~ 5 minutes the lids were removed and 4 drops of distilled water were added to each vial. The lower layer was filtered through cotton wool into 200 L glass GC vial inserts for analysis. Recoveries were determined by establishing a standard curve in boronia solutions which had been subject to the same partition clean-up step described above, but spiked after that process and using the same fortification levels as listed in Table 9. In addition a standard curve was prepared by fortifying methanol / water solutions with standard pesticide solutions to concentrations equivalent to 0.05, 1, and 10 mgkg-1 in oils. Endosulfan (5 g) was added and the vials sealed for analysis.

Analytical parameters: Samples were analysed on a HP 5890 Gas Chromatograph directly coupled to a Kratos Concept ISQ Mass Spectrometer. The GC was equipped with a BPX5 fused silica capillary column (25 m, 0.22 mm i.d., 0.25 m film thickness). Splitless injections of 1L of sample were analysed using a carrier gas flow program of 30 psimin-1 from 25 to 40 psi., held for 0.1 min, then at 30 psi. min.-1 to 25 psi, then at 1 psi. min.-1 to 35 psi. The GC injection temperature was 260C and the oven temperature programmed from 60C to 290C at 20C min.-1. Ion monitored for terbacil was 161.0117 between 5:00 and 7:50 mins. Ion 194.9534 was monitored for internal standard, endosulfan, between 7:50 and 8:12 minutes. For oxyflurofen, ion 252.0398, monitored between 8:12 to 9:00 minutes, tebuconazole, ion 250.0743, propiconazole,

53

ion 259.0210, bromacil, ion 204.9613 and simazine, ion 201.0871. A dwell time of 300ms/ion and 50 ppm voltage sweep were employed for all ions. Resolution of 10,000 (10% valley definition) and the ion m/z 242.9856 from perfluorokerosene was used as the lock mass for all analytes and the internal standard. Electron ionisation was undertaken at a source temperature of 210C and an electron energy of 70 eV, with an accelerating voltage of 5.3 kV.

Results: Table 10 records the detection limits and the relevant statistical data for the detection of the 6 analytes by GC HRMS.
analyte tebuconazole propiconazole oxyflurofen bromacil terbacil simazine det. limit (mgkg-1) 0.5 0.05 0.1 0.05 0.05 0.1 x coefficient 0.043 0.054 0.146 0.046 0.050 0.445 r2 0.998 0.995 0.999 1.000 0.995 0.899 r.s.d. (at 1 mgkg-1) 17 9 13 12 14 9 recovery (at 1 mgkg-1) 95 85 72 82 107 90

Table 10. Detection limits, recoveries and r.s.d.s for pesticides analysed by GC HRMS. Discussion: The chromatographic properties presented the primary limitation to low detection levels. The poor peak shape of tebuconazole, for example, resulted in the interference from background noise at low levels such that the detection limit was 10 fold higher than that obtained for propiconazole. R.S.D.s increased as the concentration at which they were obtained decreased.

The Application of GC HRMS in the Analysis of Pesticide Residues with Acidic Moieties.
The commercially produced formulations available for growers usually contain the ester form of the carboxylic acid specific to this group of pesticides. When the esters come in contact with the soil the ester is cleaved to form the acid. The parent ester is often present in more than one form and may include methyl, ethyl or ethoxy ethyl derivatives. The ester form of the active ingredient can often be included in the general screen for GC amenable pesticides. The fluazifop and fluroxypyr esters have been incorporated into GC HRMS and ECD methodologies. The acidic forms of this group of pesticides, however, require derivatisation to render them amenable to analysis by GC. Of the many methylating reagents available, ether solutions of diazomethane are convenient and effective. The reaction of the herbicide trichlopyr and diazomethane is shown.

54

Cl

OCH2 COOH CH2 N2

Cl

OCH2 COOCH3

Cl
trichlopyr

Cl

ether/10mins

Cl
trichlopyr methyl ester

Cl

[(3,4,6-trichloro-2-pyridinyl)oxy]acetic acid

The preparation of the diazomethane solution is presented in appendix 3. Following the inclusion of a derivatisation step method development is the same as that undertaken for the other GC amenable pesticides. A TIC is obtained, either be GC with a mass selective detector (MSD) or on the GC HRMS. The retention times and the ions to be monitored are accurately calculated and a suitable screen is programmed within which the mass spectrometer switches between the ions to be monitored in each time window. If the parent ester of the carboxylic acid is a simple methyl ester, the derivatisation of the acid with diazomethane will produce an analyte identical to that which originally present in the commercial formulation. Often cleavage from the ester to the acid form in the field situation is incomplete and derivatised acid residues are indistinguishable from residues of the ester. In this circumstance the sample should be analysed twice, with and without derivatisation. The amount detected in the underivatised sample can then be subtracted from the amount detected in the methylated sample. This will give the amount of acid contaminating any one sample. Figure 19 shows the chromatogram for the analysis of haloxyfop, trichlopyr, MCPA, dicamba and clopyralid, with endosulfan included as an internal standard.

55

Figure 19. GC HR MS of herbicides with acidic moiety

Table 11 shows the analytes, ions monitored and detection limits for haloxyfop, trichlopyr, mcpa, dicamba and clopyralid.

56

Active ingredient haloxyfop trichlopyr dicamba mcpa clopyralid

Trade name Verdict Garlon Dicamba MCPA Lontrel

Ion 316.0352 209.9280 214.0397 202.9666 173.9513

Chrom. good good good good good

Det. limit mgkg 0.5 0.1 0.1 0.1 0.1

-1

Table 11. GC HRMS of derivatised residues of pesticides with acidic moieties

Liquid Chromatography
Liquid chromatography presents as a less deleterious separation process for thermally labile chemicals, has application for non-volatile components and has more flexibility in terms of adjustable parameters. These include flow rate, polarity of both mobile and stationary phases and gradient profile of the liquid phase. High pressure liquid chromatography (HPLC) systems can be configerated with multiple pumps, providing numeous options of solvent combinations within one chromatographic run. HPLC systems can be interfaced with a range of detection systems, however, this manual only includes the assessment of detection using ion trap MS.

Assessment of HPLC in the Analysis of Pesticide Residues in Essential Oils

In ion trap MS, the effluent from the LC system is nebulised and can be ionised by a range of methods as described below. The charged constituents of the aerosol are then transferred to the mass separation system by the action of a high vacuum. The stream of ionised molecules passes through a system by which many of the non-ionised particles are eliminated, into an ion trap where the electromagnetic field oscillates to reject ions without the correct m/z ratio. The concentration of the target analyte may then be measured. However, co-eluting ions may confuse the background and so daughter ions can be formed from the target analyte by increasing the energy of the selected ions in the ion trap, such that the collision of the ions effect a controlled breakdown of the parent ion to form daughter ions. The second level of fragmentation, known as MSMS, is normally very specific to the target ion and the level of interfering ions is greatly reduced. The ionisation methods interfacing liquid chromatography to ion mass detectors assessed are: Thermospray (TSP)- Proton transfer reactions may be stimulated by using a filament or discharge electrode, termed filament assisted TSP or discharge assisted TSP respectively. Electrospray (ESP)- The LC effluent is sprayed in the vicinity of a small ion sampling orifice. Ionisation is effected by applying a high voltage to the needle through which the spray exits.

57

Atmospheric pressure chemical ionisation (APCI).- Ionisation occurs by molecular collisions. The primary ions may be solvent molecules ionised by a corona discharge or desolvated buffer ions.

Plate 4. Waters 2690 HLC and Ion Trap Mass Spectrometer

Retention of Essential Oil Components on HPLC Columns

Clean-up methods were not to be considered within the initial framework of the analytical methodology. Without clean-up techniques the threshold of detection is limited by the amount of oil which can be safely loaded onto the HPLC system without the loss of column functionality. The potential of oil components to be retained within the column under a given mobile phase protocol may lead to a shorter working life of the column and the infusion of oil components into the MS system, itself, has the potential to permanently contaminate the ion trap. The retention characteristics of essential oils on a standard reverse phase (RP) HPLC C18 column were therefore assessed. A Total Ion Current (TIC) by MS of the effluent from a LC column will only detect chemicals which have been ionised. Ultraviolet (UV) detection was applied to determine the total elution profile of essential oils and extracts introduced into the HPLC and to ensure that

58

components do not remain on the column. In addition, the flow from the HLPC was diverted to a collection vessel and the sample analysed by GC MS.

Experiments Undertaken in the Process of Method Development Example 6. Behaviour of the Components of Parsley, Peppermint and Fennel Distilled Oils on RP HPLC Columns.
Aim: To assess the retention characteristics of essential oils on RP HPLC columns using acetonitrile / ammonium acetate buffer mobile phase

Experimental: Four 200 L (~200 mg) aliquots of each of peppermint, fennel and parsley oil were dispensed with an Eppendorf into LC vials and 800 L of analG acetone was added. Injections of 20 L were introduced into the HPLC system detailed below. The flow from the column was diverted to a Waters photo diode array detector (PDA).

Analytical parameters: LC - A Waters 2690 separation module (Alliance) system equipped with a NOVAPAK C18, 3.9 x 150 mm column was used to establish a mobile phase with gradient of 50:50 acetonitrile / 0.1M ammonium acetate buffer ramped to 90:10 over 15 minutes.

Results: Figures 20 to 22 show the LC UV traces of the essential oils analysed under the conditions listed.

59

3.50

3.00

2.50

2.00 AU 1.50

1.00

0.50

0.00 2.00 4.00 6.00 8.00 10.00 12.00 Minutes 14.00 16.00 18.00 20.00 22.00

Figure 20. HPLC UV trace of distilled fennel oil (270 nm)

1.40

1.20

1.00

0.80 AU 0.60

0.40

0.20

0.00

-0.20 2.00 4.00 6.00 8.00 Minutes 10.00 12.00 14.00

Figure 21. HPLC UV trace of distilled parsley oil (220 nm)

60

3.50

3.00

2.50

2.00 AU 1.50

1.00

0.50

0.00 2.00 4.00 6.00 8.00 Minutes 10.00 12.00 14.00

Figure 22. HPLC UV trace of distilled peppermint oil (270 nm)

The fractions collected from the HPLC were subject to GC MS analysis to confirm the elution of the majority of essential oil components from the LC system.

Discussion: The main components of distilled essential oils do not remain on the RP

column under the parameters tested.

HLPC MSMS Method Development
In HPLC, the flexibility afforded by the interchangeable nature of the stationary phase of the column, the solvents, the solvent gradients and the flow rates allows for adaptations to accommodate specific analytes. Optimisation of these parameters for each analyte would be a difficult undertaking. In the assessment of the HPLC equipment a reverse phase NOVAPAK C18, 3.9 x 150 mm column was selected with acetonitrile / ammonium acetate buffer as the mobile phase. This system may not necessarily be optimal for each analyte, or for the separation of pesticide residues from essential oil components, but this commonly used system served to give workable results from which more specific methods may be developed.

61

The first steps in method development are the determination of the mass spectrum of the analyte and the establishment of tune file optimising the collision energy and isolation width specific for each parent and daughter ion for each ionisation source. To undertake the optimisation a 10 gmL-1 solution is infused directly into the MS using a worm driven syringe at a rate of 0.8 mLmin-1. Table 12 records the parameters established for a selection of pesticides analysed by ESI and APCI in the positive and negative modes.
ESI linuron oxyflurofen propiconazole prometryn tebuconazole sethoxydim simazine endosulfan bromacil terbacil haloxyfop ester fluazifop ester fluroxypyr ester NR - no response parent ion NR NR 342 242 308 328 202 NR NR 215 (-ive) 434 384 369 159 316 328 255 15 25 19 27 6 6 5 6 159 200 NR 282 124 17 18 5 4 21 20 6 5 daughter ion collision energy isolation width (amu) no response no response notes

Table 12. Mass ions and collision energies detected by fragmentation of pesticide parent molecules by ESI

Preliminary results listed in Table 12 indicate that ESI has limited application. No response was recorded for some of the most commonly used pesticides in the essential oil industry such as linuron, oxyflurofen and bromacil. For analytes such as tebuconazole the molecular mass ion was detected but no useful daughter ions were produced. Hence MS/MS could not be used to increase the specificity and lower background noise. Bromacil and terbacil were successfully ionised in the negative mode using ESI but combined with the poor sensitivity or lack of ionisation for the great majority of analytes trialed, ESI appears to have limited application for a wide ranging screen. All the chemicals were trialed using APCI in the negative and positive mode. Table 13 shows the general parameters established.

62

APCI Linuron Oxyflurofen Propiconazole Prometryn Tebuconazole Sethoxydim Simazine Endosulfan Bromacil Terbacil Haloxyfop ester Fluazifop ester Fluroxypyr ester NR - no response

parent ions 251 363 342,343.344 242,243,244 308,310 328,329,330 202,204 NR 261,259(-ive) 215,217,218 NR NR NR

daughter ions 182 316 159,161,163 200,201 282,283 124 203,205 161,163

collision energy 19 21 21

isolation width (amu) 6 5 6

notes poor response poor response

19 19

5 5

Table 13. Mass ions and collision energies detected by fragmentation of pesticide parent molecules by APCI

Results indicated that APCI had limited application for the detection of linuron, oxyflurofen and the esters of the acid moiety pesticides. Again, poor results were obtained for tebuconazole, with no daughter ions generated. However, excellent results were obtained for prometryn, sethoxydim, simazine, bromacil and terbacil. Also, compared to ESI, APCI was slightly more sensitive for the commonly used pesticides, tebuconazole and propiconazole and showed a response, though poor, to linuron and oxyflurofen. In addition, APCI is the preferred ionisation method for several reasons;

APCI uses less nitrogen gas compared to ESI, making overnight runs less costly; APCI does not have the high back pressure associated with ionisation by ESI such that APCI can be run in conjunction with UV-VIS without risk of damaging the UV cell, which is pressure sensitive. Chromatography Once the ions to be monitored by mass spectrometry were established the retention characteristics of the analytes were assessed using the acetonitrile / ammonium acetate buffer elution profile on the RP column. The chromatographic characteristics of the triazole pesticides, propiconazole and tebuconazole were poor. In addition, bromacil, simazine and terbacil eluted in a very narrow time frame under the mobile phase conditions tested. Terbacil had a higher

63

response in the negative mode by APCI. The MS trap has the capability to switch from the positive mode to the negative several times within a single run. However, the similar elution characteristics of simazine, which is ionised in the positive mode, precluded the monitoring of these three pesticides in the same screen as there was insufficient separation to allow for switching between the ionisation modes. Overall, however, the mobile phase has general application to most of the pesticides trialed. Fine tuning of the phase gradient, adjustments of the pH to provide better resolution of the haloxyfop esters and triazine pesticides and trials of alternative mobile phases are all aspects which warrant further experimentation. The preliminary method developments described form the foundation on which comprehensive method development can continue. The process of establishing the ionisation properties and optimising tuning files, collision energies and isolation widths was undertaken for a range of chemicals to be included in a general screen. An example of method development is shown below.

Experiments Undertaken in the Process of Method Development Example 7. Method Development for the Analysis of 14 Pesticides by HPLC MSMS Using APCI in the Positive Mode.
Aim: To undertake method development for acephate, carbaryl, cyanazine, dicamba, dimethoate, difenoconazole, ethofumesate, glyphosate, pirimicarb, pendimethalin, procymidone, mcpa, monocrotophos and propazine.

Experimental: As previously described, standards of all the target analytes were prepared to concentrations of 10 gmL-1 in acetone and each were introduced into the MS via the continuous flow worm drive with the mobile phase of 50/50 0.1M ammonium acetate buffer / acetonitrile flowing from the HPLC at 0.8 mLmin-1. The ionisation potential, [M + H]+ ion, daughter ion, collision energy (CE) and isolation width were determined for each compound. Target compounds which had good ionisation potential in positive mode APCI were then analysed by HPLC MS using the mobile phase previously established.

64

Results: Table 14 lists the optimised parameters for the analytes to be included in the screen.

analyte acephate carbaryl cyanazine dicamba dimethoate difenaconazole ethofumesate glyphosate pirimicarb pendimethalin procymidone MCPA mecoprop monocrotophos propazine

M.W. 183 201 240 220 229 405 286 169 238 281 283 200

parent ions 184 202,203 241,243 219, 221 230 406,408 287 no ion. 239,240 282,283 282,284(-) 199,201(-) 213,214 224,225

daughter ions 143 143,145 214,216 175,177 199 337,339,341 259,241,207 182,195 212,213 161,163 141,143 141,143 193,167,98 188,190

band width 3 4 5 8 4 6 4 3 4 6 5 4 5 5

C.E. 13 20 18 17 20 22 20 22 20 23 18 18 22 18

notes buffer adduct=loss signal good ionisation but lost in LC pot. Int. std. daughter ion weak, try -ive good ionisation but lost in LC good signal - double peak 3 daughters similar inten.=poor possible ESI or -ive APCI strong signal strong signal but lost in LC weak signal-alternative LC poor signal +ive & -ive strong signal & LC peak

229

230,232

Table 14. Parameters established for the analysis of pesticides by APCI in the positive mode. Discussion: The results listed above formed the basis for the inclusion of monocrotophos, pirimicarb, propazine and difenaconazole into the standard screen already established. Acephate, carbaryl, dimethoate, ethofumesate and pendimethalin all require further work for enhanced ionisation and/or improved elution profiles. Negative ionisation mode for APCI gives improved characteristics for dicamba, procymidone, MCPA and mecoprop but this ionisation mode was compromised by the acetate buffer. Analytes that ionised in the negative APCI mode were incorporated into a separate screen which included bromacil and the pesticides with an acidic moiety. The thirteen pesticides included in this general screen were monocrotophos, simazine, cyanazine, pirimicarb, propazine, sethoxydim, prometryn, tebuconazole, propiconazole, difenoconazole and the esters of fluroxypyr, fluazifop and haloxyfop. Bromacil and terbacil were not included as both require negative ionisation and elute within the same retention time window as simazine, which requires positive ionisation. The method validation was conducted for three oils, peppermint, parsley and fennel.

65

Experiments Undertaken in the Process of Method Development Example 8. Method Validation for the Analysis of 10 Pesticides in Distilled Oils
Aim: To validate the method developed for the analysis of 10 pesticides in peppermint, fennel and parsley oil.

Experimental: Four 200 L (~200 mg) aliquots of each of peppermint, fennel and parsley oil were dispensed with an Eppendorf pipette into GC vials and 800 L of acetone was added. Stock solutions (1 mgmL-1) of the 10 pesticides were prepared in volumetric flasks in acetone, which were then diluted to 100, 10 and 1 g/mL standard solutions. Oils were fortified with solutions of the mix of the 13 pesticides to the equivalent of 0.1, 0.5, 1.0 and 10 mgkg-1. All fortified oils were spiked with 20 L of 100 gmL-1 solutions of cyanazine as an internal standard. The oils were analysed by LC using the following conditions.

Analytical Parameters: A Waters 2690 separation module (Alliance) system equipped with a NOVAPAK C18, 3.9 x 150 mm column was used to establish a mobile phase with gradient of 50:50 acetonitrile / 0.1M ammonium acetate buffer ramped to 90:10 over 15 minutes. A Finnigan MAT LCQ was used to monitor the ions listed in Table 15 within the relevant time windows.
analyte M.W. of major isotope 223 201 240. 238 229 327 241 307 341 406 centre of parent m/z window [M+H] 224 202 242 239 231 328 242 n/a 344 408 isolation collision product tentative identification [M+H-CH3NH2] [M+H-HCN] [M+H-C3H6] [M+H-C3H6] [M+H]
+ + + + + + +

retention time (mins) 1.50 2.56 2.45 3.11 4.12

+

monocrotophos simazine cyanazine pirimicarb propazine sethoxydim prometryn tebuconazole propiconazole difenaconazole

width (amu) energy (CE) ions monitored % 5 22 193 4 5 3 5 5 5 n/a 6 6 18 18 22 18 17 20 n/a 21 22 124 214,216 182 188,190 282 200 308,310 159,161 337,339

[M+H-C2H5ClN] [M+H-C3H7N]

[M+H-C2H6O]
+

3.90 5.15 5.50 6.58,6.66 7.62,7.81

[C7H4Cl2+H]

[M+H-C2H3N3]

Table 15. Parameters and conditions for the monitoring of ions monitored by MS.

66

Results: Figure 23 shows the HPLC MS trace for the analysis of pesticides listed in Table 15.

RT: 0.01 - 5.17 RT: 1.49 AA: 4137884 monocrotophos NL: 7.47E5 m/z= 192.5-193.5 F: + c SRM ms2 224.00 [ 165.50 - 194.50] RT: 2.56 MA: 2522163 simazine NL: 4.14E5 m/z= 123.5-124.5 F: + c SRM ms2 202.00 [ 122.50 - 125.50] NL: 3.52E5 m/z= 212.0-218.0 F: + c SRM ms2 242.00 [ 212.50 - 217.50] RT: 3.13 AA: 9862826 NL: 1.42E6 m/z= 181.5-182.5 F: + c SRM ms2 239.00 [ 181.00 - 196.00] NL: 2.90E6 m/z= 187.5-188.5 F: + c SRM ms2 231.00 [ 186.50 - 189.50]

100 50 0 100 50 0 100 50 0 100 50 0 100 50 0 0.5 1.0

RT: 2.45 MA: 1986999

pirimicarb

RT: 4.11 MA: 23349203

propazine

1.5

2.0

2.5 Time (min)

3.0

3.5

4.0

4.5

5.0

RT: 1.74 - 10.27 RT: 4.09 MA: 10313193 sethoxydim RT: 5.18 MA: 30994994 NL: 1.13E6 m/z= 281.5-282.5 F: + c SRM ms2 328.00 [ 280.50 - 283.50] NL: 4.19E6 prometryn m/z= 199.0-200.8 F: + c SRM ms2 242.00 [ 198.50 - 201.50] NL: 8.64E5 m/z= 307.5-308.5 F: + c SIM ms [ 306.50 309.50] NL: 2.03E5 m/z= 158.5-159.5 F: + c SRM ms2 344.00 [ 157.50 - 160.50] NL: 1.96E6 m/z= 336.5-337.5 F: + c SRM ms2 408.00 [ 335.00 - 339.00]

100 50 0 100 50 0 100 50 0 100 50 0 100 50 0 2 3

RT: 5.54 MA: 8354032

folicur

RT: 6.68 MA: 2375398

tilt

RT: 7.62 MA: 32135613

difenoconozole

6 Time (min)

10

Figure 23. HLC MS trace for the analysis of 10 pesticides.

67

Tables 16, 17 and 18 present the detection limit achieved, the coefficients for the standard curves, the 'r' square value and the r.s.d.s calculated for the responses recorded at the 1 mgkg-1 detection level in fennel, parsley and peppermint respectively. r.s.d. = (standard deviation / mean) x 100.
Fennel analyte monocrotophos simazine pirimicarb propazine sethoxydim prometryn tebuconazole propiconazole difenoconazole det. limit gkg 20 10 10 5 25 10 100 100 10
-1

x coefficient 0.932 0.585 2.12 4.858 2.493 6.869 1.926 0.509 7.248

r2 1.000 0.999 1.000 1.000 1.000 1.000 0.999 0.999 1.000

r.s.d. (at 1 mgkg ) 3 6 7 4 4 6 7 5 3

-1

Table 16. Method validation statistics for the analysis of pesticides in fennel distilled oil.
Parsley analyte monocrotophos simazine pirimicarb propazine sethoxydim prometryn tebuconazole propiconazole difenoconazole det. limit gkg 50 20 20 0.5 50 0.5 200 100 10
-1

x coefficient 0.904 0.547 1.995 4.45 2.54 5.827 1.865 0.553 7.134

r2 1.000 1.000 1.000 1.000 1.000 1.000 0.998 0.997 1.000

r.s.d. (at 1 mgkg ) 9 4 8 6 9 6 13 19 6

-1

Table 17. Method validation statistics for the analysis of pesticides in parsley distilled oil.
Peppermint analyte monocrotophos simazine pirimicarb propazine sethoxydim prometryn tebuconazole propiconazole difenoconazole det. limit gkg 20 20 5 10 25 10 200 50 10
-1

x coefficient 1.029 0.645 2.335 5.374 2.69 7.737 2.125 0.558 7.993

r2 0.999 1.000 1.000 1.000 1.000 1.000 0.998 0.996 1.000

r.s.d. (at 1 mgkg ) 7 11 4 3 5 7 16 11 4

-1

Table 18. Method validation statistics for the analysis of pesticides in peppermint distilled oil.

68

Table 19 below records the r.s.d.s for repeat injections of fennel, parsley and peppermint distilled oils fortified with the thirteen pesticides. Predictably, the values increase as the detection limit is approached.
10 mgkg Fennel monocrotophos simazine pirimicarb propazine sethoxydim prometryn tebuconazole propiconazole difenoconazole Parsley monocrotophos simazine pirimicarb propazine sethoxydim prometryn tebuconazole propiconazole difenoconazole Peppermint monocrotophos simazine pirimicarb propazine sethoxydim prometryn tebuconazole propiconazole difenoconazole 4 4 4 2 6 4 6 5 3 7 11 4 3 5 6 16 11 4 9 20 5 34 10 5 15 26 5 7 14 38 14 8 22 8 6 4 4 4 6 2 1 4 4 9 4 8 6 9 6 13 19 6 9 8 2 3 6 5 19 21 5 11 12 15 21 14 20 7 5 8 7 7 6 7 4 7 6 3 6 7 4 4 6 7 5 3 11 15 9 4 13 5 12 13 8 15 48 28 4 14 17 19
-1

1 mgkg

-1

0.5 mgkg

-1

0.1mgkg

-1

Table 19. R.S.D.s of repeat injections of fortified fennel, parsley and peppermint distilled oils. Discussion: HPLC MSMS provides an effective screen for pesticides in essential oils for ten analytes with detection limits to 0.01 mgkg-1, excluding propiconazole and tebuconazole, which can only be detected to levels of 0.5 mgkg-1. The type of oil analysed had minimal effect on the response function as expressed by slope of the standard curve. The parsley oil selected for background matrix for the method validation has background levels of prometryn such that the standard curve did not pass through zero. These levels are in the order of 0.3 mgkg-1. Overall method development experiments have found contamination of most parsley oils with this

69

pesticide. Location of an oil, not contaminated with prometryn, will allow for a repeat of this validation experiment.

Experiments Undertaken in the Process of Method Development Example 9. Method Validation for the Analysis of 10 Pesticides in Boronia concrete
Aim: A separate screen for detection in boronia extract of all the analytes listed in example 8 was developed. A simple partition step was included in the extraction process to remove non-polar components of the waxy extract. Experimental: Stock solutions of 1 mgmL-1 of each of the 10 pesticides were prepared in acetone using volumetric flasks. These were used to prepare mixed solutions of 100, 10 and 1 gmL-1 by volumetric serial dilution. Standard acetone solutions of 10 gmL-1 and 100 gmL-1 were used to fortify 0.5 g of boronia concrete (weighed into 9 mL, 13 x 100 mm disposable test tubes) to produce a calibration curve of 0.25, 1, 10 and 50 mgkg-1 relative to oil weight. Cyanazine (25 L of a 100 gmL-1 solution) was added as an internal standard. The boronia concretes were dissolved in 1 mL of hexane. The total volume of acetone in each tube was brought to 250 L. The samples were vortexed and 1 mL of methanol/water (5:1) was added. The mixtures were again vortexed for 3 x 20 second bursts then centrifuged for 5 minutes at 3000 rpm to effect a partition. The aqueous layer was sub-sampled using a disposable pipette and filtered through cotton wool into a 200 L GC vial insert. The samples were analysed by LC MS/MS using the listed conditions. For determinations of recoveries, 1mL of methanol/water (5:1) were spiked at concentrations equivalent to those prepared for the standard curve. 250 L of acetone was added to ensure the polarity was similar to the samples in boronia matrix. Samples for recovery determinations were analysed under the same conditions as the standard solutions.

Analytical parameters: LC - A Waters 2690 separation module (Alliance) system equipped with a NOVAPAK C18, 3.9 x 150 mm column preceded by a Alltech Econosphere C18, 5 m guard cartridge, was used to establish a mobile phase with gradient of 50:50 acetonitrile / 0.1M ammonium acetate buffer ramped to 90:10 over 15 minutes. A Finnigan MAT LCQ APCI source had a vaporiser temperature 450C; sheath gas (nitrogen) at a pressure of 60 psi. with auxillary gas at 20 psi.. Capillary temperature was 150C with a voltage of 16 V. The corona current was 5 A. The parent m/z, collision energies and product ions monitored are listed in Table 15.

70

Results & Discussion: The clean-up step used in the analyses of boronia required an assessment of the suitability of cyanazine as an internal standard. The low recovery of 41.2 0.7% of cyanazine is offset by the excellent reproducibility. Table 20 lists the recoveries for the target analytes across 4 concentration levels.
mg/kg analyte monocrotophos simazine pirimicarb propazine sethoxydim prometryn tebuconazole propiconazole difenaconazole n 4 4 4 4 4 4 4 4 4 50 mean SE 123 4 38.4 0.5 101 1 38.6 0.5 35 1 72.6 0.8 105 2 24.4 0.4 72 1 10 mean SE 155 7 34 1 107 2 43.3 0.8 21.6 0.3 66 1 105 3 17.4 0.5 74 0.5 1 mean SE 178 11 34 2 115 2 47.5 0.5 20.7 0.9 74 2 119 3 15 1 75 2 0.25 mean SE 214 11 25 3 109 4 49 1 17 2 81 2 88 7 nil 66 2

Table 20. Percentage recoveries for target analytes in clean-up step for pesticides in boronia The high recoveries recorded for monocrotophos in boronia preclude the application of this screen in quantitative experiments. The signal for ion m/z 193 is enhanced in the matrix of boronia, probably as a result of co-eluting components in the boronia extract. As the level of monocrotophos approaches the ppb level, the relative contribution of the co-eluting contaminant increases. The recoveries for propiconazole, on the other hand are very low. However, the repeatability of that low recovery, with an r.s.d. of 7% at 1 mgkg-1 still allows for the application of this analytical method for the detection of gross contamination of propiconazole in boronia concrete. Recoveries for simazine, propazine and sethoxydim are also less than 50%, however, the consistency in the percentage of analyte recovered across the range of fortification levels allows for the application of this analytical method to the screening of these three analytes in boronia extract. Table 21 presents the detection limits achieved, the coefficients for the standard curves, the 'r2' values and the relative standard deviations for the detection of all nine pesticides investigated in boronia concrete.

71

Boronia concrete det. limit analyte monocrotophos simazine pirimicarb propazine sethoxydim prometryn tebuconazole propiconazole difenaconazole mg/kg 0.15 0.1 0.02 0.01 0.1 0.01 0.2 0.2 0.02 coefficient 0.140 0.038 0.298 0.393 0.115 0.717 0.287 0.030 1.108 x lin. reg. fit (r ) 0.993 1.000 0.998 1.000 0.994 1.000 0.990 0.997 1.000
2

%rsd 50 mg/kg 2 3 4 3 3 3 3 6 6 10 mg/kg 2 3 3 3 4 2 2 10 6 1 mg/kg 8 11 6 4 7 3 9 18 1 0.25 mg/kg 10 22 5 6 17 2 16 n/a 8

Table 21. Method validation for pesticides boronia oils by LC MSMS using ionisation with APCI in the positive mode.

The application of HPLC MSMS to the Analysis of Pesticides with Acidic Moieties.
The process for method development for the detection of this class of pesticide followed the same protocols described in the preceding section. Tuning files were established optimising the fragmentation and ionisation of haloxyfop, fluazifop, fluroxypyr acids and their parent esters. Parameters were established by direct infusion of 1 gmL-1 acetone solutions into the MS. Fluazifop possessed good ionisation characteristics using ESI. The elution profile for the esters and the acids under the acetonitrile / ammonium acetate buffer RP chromatography showed acceptable resolution but the responses for haloxyfop and fluroxypyr esters were poor with haloxyfop registering a response thirty times lower than that of fluazifop. This variation in response was partly due to the haloxyfop ester being present in the standards as both the ethyl and methyl esters, effectively splitting the relevant peak. Table 22 records the ions monitored and the relevant retention times.

72

analyte fluazifop-butyl fluazifop acid haloxyfop ethyl haloxyfop acid fluroxypyr methyl heptyl fluroxypyr acid

MW 383 327 433,435 361,363 366,368,370 255,256,258

MS 384 (+) 326 (-) 434/436 (+) 360/362 (-) 361 367,369,371 (+) 253,255,257 (-)

MSMS 328 254 316,318 288,290 288,290 255,257 233,235

ret. time 11.1 10

isol. window & C.E. 5 amu 19% 3 amu 17% 6 amu 25% 4 amu 26%

12.7

6 amu 18% 6 amu 17%

Table 22. MS parameters for the detection of the parent ester and acid forms of fluazifop, haloxyfop and fluroxypyr.

Poor chromatography and response necessitated an improved mobile phase and the effect of pH on elution characteristics was considered the most critical parameter. The acidic nature of the active ingredient of this class of pesticide presented a moiety which may be exploited in an extraction protocol. The inclusion of an aqueous extraction, however, precluded the simultaneous analysis of the parent esters with the carboxylic acid forms. The development of a screen for the pesticides with an acidic moiety proceeded with the inclusion of an extraction step whilst the esters were to be included in the general screen using APCI in the positive mode.

Extraction Protocol for Pesticides with Acidic Moieties

Under basic conditions, the acid moiety of this class of pesticide is de-protonated facilitating the extraction of the analytes from the essential oil into an aqueous layer. Preliminary experiments whereby distilled oil samples were extracted with 0.01M NaOH, which was acidified and back extracted with dichloromethane (DCM) had limited success. LC UV analysis of the DCM confirmed that very little essential oil residue was co-extracted through the process but recoveries were poor. The pesticides within this class are weak acids. The 0.01M NaOH was replaced with 0.01M NaHCO3, which was adjusted to a pH of 10 with NaOH. In addition, the acidification and subsequent back extraction with DCM was removed as it was considered that only a limited amount of essential oil components should be soluble in an aqueous extract which was analysed directly. To prevent excessive dilution of the pesticide extracts the ratio of essential oil to NaHCO3 solution was lowered to 4 : 1. Recoveries and reproducabilities were improved with these modifications.

73

The inclusion of an internal standard negates the errors introduced by inaccurate subsampling and dilutions. The agents 2,4-D and 2,4,5-T were proposed as suitable internal standards and both were introduced in the MS using direct infusion to determine the ionisation and fragmentation parameters. Table 23 records the parameters established for 2,4-D, 2,4,5-T, and for the acidic pesticides dicamba, MCPA and mecoprop.
analyte M.W. of major isotope 220 254 220 200 254 214 361 centre of parent m/z window [M+H] n/a 254 221 200 255 214 361 isolation collision product tentative identification [M-H]
+ + + + + + +

retention time (mins) 2.16 2.50 4.13 4.51 5.86 6.02 8.89

dicamba fluroxypyr 2,4-D MCPA 2,4,5-T mecoprop haloxyfop

width (amu) energy (CE) ions monitored % n/a n/a 219,221 6 6 5 8 4 4 17 20 18 20 18 26 233,235 161,163 141,143 195,197,199 141,143 288,290

[M-H-H2O]

[M-H-C2H2O2] [M-H-C2H2O2] [M-H-C2H2O2] [M-H-C3H4O2] [M-H-C2H4O2]

Table 23. MS parameters for the monitoring of 2,4-D, 2,4,5-T dicamba, MCPA and mecoprop.

The parameters established in the preliminary investigations were used in the validations for the detection of pesticides with acidic moieties were conducted. The densities of parsley oil were quite variable such that multi-layers were present when non-polar/aqueous partitions were established during the extraction process. Validations were conducted for peppermint and fennel distilled oils only.

Experiments Undertaken in the Process of Method Development Example 8. Method Validation for the Analysis of Acidic Pesticides by HPLC MSMS.
Aim: To conduct method validation for the extraction and analysis for residues of pesticides with acidic moieties in fennel and peppermint distilled oils.

Experimental: Commercially produced oils were combined to produce a blended oil with uniform characteristics. Aliquots (20 x 5 mL) of each of peppermint and fennel oils were dispensed into 50 mL test tubes. Extracting solvent (1 mL of 0.01M NaHCO3 adjusted to pH 10 with NaOH) was added and the mixtures were vortexed for 4 x 15 second bursts. The emulsions were

74

transferred to 13 x 100 mm borosiliate glass tubes and centrifuged at 2500 rpm for 15 minutes. The aqueous layers were transferred to a 10 x 75 mm borosilicate glass tubes using pasteur pipettes. Hexane (1 mL) was added to each and the mixtures were vortexed for 2 x 15 second bursts to remove residual oil contaminants. The mixtures were centrifuged at 2000 rpm for 5 minutes and 250 L of the aqueous layers were quantitatively transferred to a GC vial inserts. Internal standard (10 l of a 50 gmL-1 solution of 2,4,5-T) was added to each and the samples analysed using the conditions listed.

Analytical Parameters: A Waters 2690 separation module (Alliance) system equipped with a NOVAPAK C18, 3.9 x 150 mm column was used to establish a mobile phase with gradient of 50:50 acetonitrile / 0.1M ammonium acetate buffer (adjusted to 3.8 with acetic acid) ramped to 90:10 over 15 minutes. A Finnigan MAT LCQ was used to monitor the ions listed in Table 22 and 23 within the relevant time windows.

Results: Figure 25 shows the trace for the analysis of acidic pesticides by HPLC MS.
RT: 0.01 - 11.81
100
2.16 dicamba 2.53
2.50 fluroxypyr
2.91
NL: 1.56E6 m/z= 218.3-219.3+220.3-221.3 F: - c SIM ms [ 218.00 - 284.00] NL: 1.73E6 m/z= 232.5-233.5+234.5-235.5 F: - c SRM ms2 254.00 [ 232.00 - 236.00] NL: 2.02E6

100

100

4.13 2,4D
4.44
4.51 MCPA

m/z= 160.7-161.7+162.7-163.7 F: - c SRM ms2 221.00 [ 160.00 - 164.00] NL: 2.57E6 m/z= 140.7-141.7+142.7-143.7 F: - c SRM ms2 200.00 [ 140.00 - 144.00]

100

100

5.86

NL: 5.76E5

2,4,5-T

m/z= 194.7-195.7+196.7-197.7+198.7-199.7 F: - c SRM ms2 255.00 [ 193.00 - 201.00] NL: 1.85E6

100

6.02 mecoprop

m/z= 140.7-141.7+142.7-143.7 F: - c SRM ms2 214.00 [ 140.00 - 144.00]

0 100

8.89

haloxyfop

NL: 3.15E6 m/z= 287.7-288.7+288.7-289.7 F: - c SRM ms2 361.00 [ 287.00 - 291.00]

6
Time (min)

10

11

Figure 25. HPLC MS trace of analyse of acidic pesticides.

Table 24 records the detection limits, the slopes of the linear regression describing the standard curves established, 'r2' value pertaining to the fitted curves and the reproducibilities as

75

described by the r.s.d.s for the analysis of the acidic forms of dicamba, fluroxypyr, mcpa, mecoprop and haloxyfop acid. Table 25 records the r.s.d.s for each pesticide at each fortification level in fennel and peppermint oil.
analyte Fennel dicamba fluroxypyr MCPA mecoprop haloxyfop Peppermint dicamba fluroxypyr MCPA mecoprop haloxyfop 0.1 0.01 0.01 0.01 0.01 3.86E-06 3.28E-06 2.78E-06 6.89E-06 1.63E-06 0.999 0.996 0.998 0.999 0.999 13 15 18 20 14 det. limit mgkg 0.1 0.1 0.01 0.01 0.01
-1

x coefficient 4.25E-06 3.20E-06 2.28E-06 3.99E-06 3.24E-06

%rsd (at 1 mgkg )

-1

0.999 0.995 0.992 0.995 0.997

2 7 4 6 6

Table 24. Detection limits correlation co-efficients and standard curve statistics for the detection of pesticides with acidic moieties in fennel and peppermint distilled oil.
10 mgkg Fennel dicamba fluroxypyr MCPA mecoprop haloxyfop Peppermint dicamba fluroxypyr MCPA mecoprop haloxyfop 5 2 5 4 8 13 15 18 20 14 42 10 6 18 13 9 31 7 51 9 8 9 11 12 2 7 4 6 6 16 8 6 8 5 22 42 50
-1

1 mgkg

-1

0.5 mgkg

-1

0.1mgkg

-1

Table 25. R.S.D.s for the detection of pesticides with acidic moieties in fennel and peppermint distilled oil. Discussion: The aqueous extraction and detection by HPLC MSMS in the negative mode of pesticides with acidic moieties has excellent detection limits, is reproducible and gives a linear relationship between analyte concentration and peak area.

76

The Application of Direct MSMS to the Analysis of Quaternary Ammonium Salts

The quaternary ammonium salts form a distinctive class of pesticides. The method development for the two main products used in the Australian essential industry, paraquat and diquat, initially follow the same processes as the preceding methodologies, however, the problems encountered are very specific to this chemical type. Acetone / water solutions (50:50) of paraquat and diquat (1 mgmL-1) were diluted to 10 gmL-1 and introduced into the MS using a continuous flow worm driven syringe at a flow rate of 0.8 mLmin-1. Initial trials of ESI showed excellent ionisation response. Parameters established are listed in Table 26.
analyte diquat molecular formula C12H12N2
2+

M.W. 184

parent ion (+ive) 183

+

daughter ion 157

isolation width 4

collision energy 17.1%

paraquat

C12H14N3

2+

186

186

171

1.5

17.1%

Table 26. MS parameters for the detection of diquat and paraquat.

The application of liquid chromatography to the separation of paraquat and diquat presented difficulties. The effects of low solubility in organic solvents and the predisposition of both analytes to absorb on to most surfaces was manifest by their complete loss when analysed by the standard HPLC protocols. An alternative approach to the analysis of quaternary ammonium salts was required. Paraquat and diquat are both very soluble in water and virtually insoluble in hydrocarbons. A water extraction of essential oils would be expected to effectively remove paraquat and diquat from an oil extract with very little of the oil components co-extracting. The power of mass spectrometry allows for the monitoring of ions specific to the target analyte despite the collection of large amounts of background ions. An aqueous extract of paraquat and diquat from essential oils may contain so few co-extracted oil components that direct injection of samples into the injection loop of the mass spectrometer may be conceivable. The associated loss of analyte within the LC system would be avoided. A post-column injection would still require the continuous flow of mobile phase from the LC system through the injection loop of the mass spectrometer, however the approach would also have the added advantage of a quick analysis turn around time. This would offset the loss of automation that direct injection into the loop of the mass spectrometer would represent.

77

The adsorption problems also had consequences for the reproducibility of direct manual injections into the mass spectrometer. Repeated 20L loop injection of a 0.5 mgmL-1 water solution of paraquat and diquat showed a rapid decrease in response over 5 minutes, with decreases extremely pronounced for diquat as illustrated in Figure 26.

RT: 0.01- 47.76 1 00 90 80 Relative Abundance 70 60 50 40 30 20 1 0 0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 20 22 24 26 Time (min) RT: 2.35 A A : 583405 RT: 5.66 A A : 7951 52 RT: 9.42 A A: 839050 RT: 24.56 A A : 740456

diquat
RT: 21 .48 A A : 61 2331 RT: 33.60 A A : 67351 6 RT: 30.89 A A : 557462 RT: 38.78 A A : 71 3276 RT: 41 .49 A A: 622433 RT: 44.98 AA : 61 71 1 8

RT: 1 .85 1 RT: 1 8 8.1 RT: 1 4.74 A A : 71 2836 A A : 734053 A A: 668402

NL: 1 .29E5 TIC F: + p SRM ms2 1 83.00 [ 1 56.00 - 1 59.00]

RT: 27.04 A A : 61 8054

RT: 36.40 A A : 585527

RT: 42.73 AA: 1 33825

28

30

32

34

36

38

40

42

44

46

paraquat
NL: 1 .22E5 m/z= 1 56.0-1 73.0 F: + p SRM ms2 1 86.00 [ 1 70.00 - 1 73.00]

RT: 0.01- 47.75 1 00 90 80 Relative Abundance 70 60 1 0.68 50 40 30 20 1 0 0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 20 22 24 26 Time (min) 28 30 32 34 36 38 40 42 44 46 2.38 2.61 4.72 4.90 8.1 6 8.48 1 0.96 1 .05 1 1 3.30 1 3.48 1 3.75 1 5.91 20.04 20.27 21 .60 23.25 25.96 23.48 23.62 26.23 35.00 26.55 27.56 29.72 30.22 32.61 35.36 40.46 37.89 40.69 43.1 6 1 3.07 1 6.69 1 9.86 23.07 26.1 0 34.77 32.1 5 1 6.92 1 7.29 1 7.79 37.57 40.27 42.80 46.33 42.89 46.1 9 29.35 7.47 4.1 7

Figure 26. Repeat injections of paraquat and diquat solutions.

In addition to the loss of analyte by absorption other problems encountered were shorting across the ionisation source which was possibly related to the acetonitrile / ammonium acetate buffer. The replacement of acetonitrile with methanol alleviated this problem. The adsorption of quaternary ammonium salts onto surfaces has been encountered by other research groups. Kaniansky et al., (1994) reported the introduction of serious analytical errors in the analysis of paraquat and diquat at low concentrations by adsorption losses of the analytes in sample storage containers. These were eliminated by spiking samples with diethylenetriamine (DETA). DETA was found to preferentially accumulate on the surfaces of sample containers, competitively binding on the adsorption sites. Table 27 presents the areas for repeat injections of the fennel oil extracted in glassware pre-conditioned with 5% of a 0.1M DETA solution compared to extractions in glassware not conditioned.

78

paraquat parent ion daughter range mgkg

-1

diquat 183 (peak area) - DETA 5860 12206 4145 6302 9840 156-159 + DETA (peak area) - DETA

186 170-173 + DETA 23234 15028 17824 32109 35127 32640 20810

0.01

mean std. Dev. R.S.D. + deta / -deta 0.1

25253 7986 32 3.3 186155 206904 273962 236205 183563 231997 173454

7671 3273 43 149364 112825 106703 107986 97298 88943 110520 20859 19 124055 22701 18 2.4 374669 505016 474457 420107 385911 763011 490703 438389 407946 525012 162305 31 52322 10907 21 144892 126952 146982 99221 102230 54473 44854 43708 48236 70337

mean std. Dev. R.S.D. + deta / -deta 0.5

213177 36064 17 1.9

mean std. Dev. R.S.D. + deta / -deta linear regression r2 0.999 0.998

432032 56317 13 0.8 0.986

0.984

Table 27. Effect of the inclusion of DETA treated glassware in the area response for the analysis of paraquat and diquat.

The inclusion of a DETA solution pre-treatment of all glassware has effected: a 3.3 fold increase in paraquat response at 0.01 mg/kg-1 fortification level; a 2.4 fold increase in diquat response at the 0.1 mgkg-1 fortification level;

79

an increase in the repeatability of the analysis for paraquat as expressed by the relative standard deviation (r.s.d.), lowered from 43 to 32%; an increase in the linearity of the standard curve at low concentrations as expressed by the improved 'r2' values for both paraquat and diquat. In addition to this experiment, 100 mgkg-1 samples of paraquat and diquat in fennel oil stored

for two weeks in un-treated glassware and glassware pre-treated with DETA solutions were compared to freshly prepared standard extracts. Areas for the peaks of the respective treatments are presented in Table 28.
0.1 mgkg
-1

paraquat 186 170-173 + DETA 2 weeks < 12 hours 8866 213177 (peak area) - DETA bdl 110520

diquat 183 156-159 + DETA 16903 124055 (peak area) - DETA bdl 52322

parent ion daughter range

Table 28. Effect on the area response of the inclusion of glassware pre-conditioned with DETA on the stability of paraquat and diquat in storage.

Results in Table 28 confirm that despite the improved response and reproducibility afforded by the use of glassware pre-treated with DETA, depletion of both paraquat and diquat in solutions necessitates the preparation of standards and samples on the day of analysis. Reproducibility in the analyses of paraquat and diquat was further improved by including the following measures:

pre-conditioning of all glassware to be used in the extraction procedures; extraction of all oil samples with DETA solutions; the inclusion of DETA in all solutions, including standard stock solutions; changing of mobile phase to 50 : 50 100 M DETA / methanol; minimising of contact with all metallic surfaces in the flow path, including the replacement of the metal injection loop with teflon.

80

Experiments Undertaken in the Process of Method Development Example 10. Method Validation for the Detection of Paraquat and Diquat.
Aim: To determine the repeatability of the detection of paraquat and diquat using HPLC MSMS Experimental: Fennel oil (16 x 5 mL) were weighed into 10 mL glass tubes, previously washed in a 5% solution of 0.1M DETA and dried. Freshly prepared 10 gmL-1 and 50 gmL-1 solutions of paraquat and diquat were used to spike each of 5 mL of fennel oils to produce fortification levels of 0.1, 0.5 and 1.0 mgkg-1. The fortifications were repeated four times at each concentration to determine repeatability of the method. The oils were extracted with 2 mL of 0.1M DETA solution and vortexed for 3 x 20 second bursts. The tubes were centrifuged at 2500 rpm for 15 minutes and the aqueous layers were transferred into 10 x 74 mm 'Kimbal' tubes. The solutions were washed with 0.75 mLs of DCM.

Analytical parameters: A Waters 2690 separation module (Alliance) system equipped with a NOVAPAK C18, 3.9 x 150 mm column was used to establish an isocratic mobile phase of 50 : 50 DETA : methanol mobile phase. A Finnigan MAT LCQ was used to monitor the parent ion 186 for diquat with the daughter ion in range 170 to 173 and the parent ion 186 for paraquat with the daughter ion range of 156 to 159. Manual injections to fill a 25 L teflon loop were interposed into the LC mobile phase post-column and introduced directly into the MSMS. Each injection was repeated six times for each of the four repeats of each fortification level and the areas averaged. Results: Table 29 records the results obtained.
diquat (mgkg ) 0.0 0.1 0.5 1.0 paraquat (mgkg ) 0.0 0.1 0.5 1.0
-1 -1

g 0 0.5 2.5 5 g 0 0.5 2.5 5

Response (mean area) 0 9528314 14776066 18640057 Response (mean area) 0 4494099 14490788 17807392

r.s.d. 14 6 8 r.s.d. 14 12 10

Table 29. Repeatability of the detection of paraquat and diquat using HPLC MSMS

81

Discussion: The results in Table 29 show a non-linear relationship between analyte concentration and peak area for the analysis of paraquat and diquat by HPLC MSMS. A curve fit describing the shape of the curve with a second order polynomial gave a 'r2' value of 0.971 and 1.000 for paraquat and diquat respectively. At low concentrations adsorption problems seem more pronounced for paraquat, such that the response for this analyte is half that seen for diquat at the 0.1 mgkg-1 level. The problems encountered for the detection of paraquat and diquat at levels of 1 mgkg-1 have, to the greater extent, been overcome. Problems affecting linearity of the response at low levels are evident but the method has been shown to be robust and efficient.

The Application of LC MSMS to the Analysis of Mancozeb

This methodology is still in developmental stages, however, the details of progress to date may be useful as an indication as to the applicability of LC MS/MS to the analysis of dithiocarbamates. The widely accepted method for the analysis of dithiocarbamate pesticides, that is headspace GC FPD analysis of carbon disulfide produced by the acidified stannous chloride digestion of dithiocarbamates (page 46), is not suitable for the analysis of essential oils. Sulfur chemicals are endogenous to many essential oil crops and will produce carbon disulfide when subjected to acidic digestion. Gustafsson and Thompson (1981) developed a HPLC UV method for the determination of dithiocarbamates using a EDTA / NaOH extraction followed by methylation with methyl iodide. This method was adapted with the intention of using detection by HPLC MS/MS.

S
EDTA

S SNa SNa
TBAS CHCl3 -hexane (3:1) aq.(pH 6.5-8.5)

CH2 CH2

NH C NH C

CH2 CH2

NH C NH C

SCH 3 SCH 3

mancozeb

NaOH

When mancozeb is extracted in an NaOH / EDTA solution the salt produced is disodium N, N'ethylenebis(dithiocarbamate), which is transferred across the aqueous organic interface into a methyl iodide hexane / chloroform solution with the phase transfer reagent, tetrabutylammonium hydrogen sulfate (TBAS), to produce dimethyl ethylenebis(dithiocarbamate) (DMED). DMED was first synthesised using the method described by Gustafsson and Thompson (1981). The product was then dissolved in acetone and introduced into the MS via the worm drive syringe effecting direct infusion into the MS at a flow of 0.8 mLmin-1. The parent m/z ion

82

was 240.8 which when subject to a collision energy of 17% produced the daughter ion of m/z 192.8. The analyte was then introduced into the HPLC to be chromatographed on a NOVAPAC C18 column with a 50 : 50 methanol : 0.1M ammonium acetate buffer mobile phase with a gradient of 90 : 10 over 15 minutes. The analyte eluted at 5.2 minutes. Mancozeb was dissolved in an EDTA / NaOH solution and TBAS was used to transfer the solvated product into the chloroform / hexane layer containing methyl iodide. However, when introduced into the HPLC MS, TBAS contaminated the entire system, such that any signal from the target analyte was overwhelmed. This was overcome by the washing the organic layer with water. Analyses by LC MS/MS gave positive results for the mancozeb derivatives extracted from fortified NaOH / EDTA solutions down to the 0.25 mgkg-1 level. However, when essential oil was spiked with mancozeb and extracted with NaOH / EDTA solutions and transferred into the organic layer for derivatisation with MeI, no DMED was detected. Further experimentation found that pre-washing the oil with NaOH / EDTA improved the yield of DMED showing that the components of the oil, which are interfering in the analytical procedure, are soluble in NaOH / EDTA and once removed allow for a successful extraction and derivatisation of mancozeb. It was also concluded from further tests that increasing the concentration of the phase transfer reagent, TBAS, was detrimental but that increasing the concentration of the derivatising agent, methyl iodide, resulted in a marked increase in DMED response. This would indicate that the essential oil components co-extracted with the mancozeb may be competitively binding with methyl iodide The separation of the ethylenebis(dithiocarbamate) salt from the aqueous phase was attempted using specialised empore discs to allow for direct methylation without the need for phase transfer reagent. Adapting the method of Wells et al. (2000), Emporite Anion-SR exchange discs were to be used to selectively bind the salt produced by NaOH / EDTA solvation of mancozeb. The discs were then dried at 60C and the bound salt methylated with methyl iodide in acetonitrile. In preliminary experiments the target analyte was not detected in any of the extracted samples. It was found that the precursors and/or the target product were thermally labile and that without heat, derivatisation could be accomplished. However, this methodology was not taken to conclusion, despite showing some promise as a highly specific analytical technique. Instead attention was diverted to the monitoring of manganese levels in oils to be used as an indication of mancozeb contamination as detailed in the following pages.

83

Assessment of the application of ICP OES to the screening of essential oils

ICP OES is used for elemental analysis. Samples, usually dissolved in aqueous solutions, are introduced into a plasma formed by ionised gas in an oscillating magnetic field that reaches extremely high temperatures. Atoms entering the plasma emit light (photons) with each element producing a characteristic wavelength. This light is recorded by one or more optical spectrometers and when calibrated against standards, provides a quantitative analysis. Obviously most pesticides, which normally are present as contaminants at very low concentrations, would not produce enough of any one element to register above background noise in a crude extract. However, metals such as manganese, are trace elements in most vegetative materials and are only present at very low levels. Mancozeb contains manganese and zinc in a ration of 10 : 1. Elevated levels of these elements in samples treated with the pesticide may be indicative of mancozeb contamination. Initial trials were instigated to determine base levels of endogenous manganese / zinc ratios and trial the effectiveness of standard digestion procedures. Digestions are traditionally performed using nitric acid:perchloric acid (5:1). Samples are heated to 100C for 2 hrs then heated to 240C and refluxed for 4 hrs. Sample volumes are then reduced to 1-2 mL on a heating block under a stream of air, cooled and made up to 25 mL in volumetric flasks using distilled water then filtered. This methodology was applied to the digestion of essential oils. Fennel reacted moderately with the acid but formed amorphous lumps that were difficult to digest and spontaneously combusted toward the end of the digestion. Parsley reacted violently with the acid and combusted instantly resulting in the loss of the sample. Peppermint, on the other hand, reacted only moderately. It was concluded that in addition to perchloric acid digests being dangerous and unlikely to meet OH&S standards, essential oils do not react consistently under the conditions trialed. Digestions using H2SO4:H2O2 were trialed in samples of parsley, peppermint, fennel and boronia oils. Sulphuric acid charred the oils such that a black solid mass formed in the bottom of the tube. No ICP analysis was possible. Due to the difficulty in digesting essential oils and the limited quantity of oil available for analysis it was concluded part of the method proposed by Gustafsson and Thompson (1987) could be adopted. A solution of 0.25M EDTA / 0.45M NaOH was used to complex mancozeb residues allowing free manganese ions to pass into the aqueous phase. This method was found to be successful, however, solutions had to be diluted 1 : 20 as the carbon content of co-extracted oil components was too high. Despite this, increased mancozeb fortification levels coinciding with higher levels of manganese detected. The manganese : zinc ratio recorded was not that expected

84

from the breakdown of mancozeb. Higher than expected levels of zinc may have been due to contamination of glassware/reagents etc. High carbon content was overcome by using a partly neutralised solution of 5 mM EDTA / 9 mM NaOH. Experiments were conducted to test the parameters of the method developed in parsley, peppermint and fennel distilled oils and in boronia concrete.

Processes undertaken in the process of method development Example 11. Method undertaken for the monitoring of manganese by ICP OES to screen for mancozeb in fortified essential oils.
Aim: To establish detection limits and linearity of response for the analyses of mancozeb in essential oils using ICP OES

Experimental: Samples (5 mL) of peppermint, fennel and parsley were spiked with mancozeb (suspended in acetone) at levels of 0.1 0.25, 1.0 and 10.0 mgkg-1. These were extracted with 2 x 5 mL of a 1 in 50 dilution of 0.25M EDTA / 0.45M NaOH and adjusted to pH 9.6. Boronia extract (0.5 g) was dissolved in 5 mL of hexane prior to the sub-sampling of 5 mL such that only 2.5 grams of boronia matrix was extracted. Fortifications of boronia extracts were conducted as for that undertaken for distilled oils. Solutions were vortexed for 3 x 20 seconds and centrifuged for 10 minutes at 2500 rpm. The EDTA extracts were combined and washed with 3 mL of hexane to remove any oil contamination. Analytical parameters: Samples were submitted to the Central Science Laboratory, University of Tasmania, for metals analysis. Mn and Zn were monitored using an 'IRIS" Inductively Coupled Plasma Optical Emmission Spectrometer (ICP-OES, Thermo Jarrell Ash (Franklin, Ma, USA)). Instrument tuning, optimisation and calibration were performed daily. Standard mixtures were prepared in 5% (v/v) HNO3, with quantitation by means of external calibration. Each calibration protocol typically consisted of a blank and 3-5 standards covering the concentration range 0 to 200 mgL-1. Commercial reference preparations (AccuStandard Inc, ICPMO143-5 and ICPMO165-5, New Haven, CT, USA) were analysed regularly as quality control samples (accurate to <10% for all elements considered). Sample uptake time to the ICP-OES was ~60 seconds, while the rinse time between samples was also ~60 seconds.

Results: Table 30 records the results obtained.

85

Sample Name Parsley blank -1 Pars_100gkg -1 pars_250 gkg -1 pars_1000 gkg -1 pars_10000gkg Peppermint blank -1 pep_100 gkg -1 pep_250 gkg -1 pep_1000 gkg -1 pep_10000 gkg Fennel blank -1 fen_100 gkg -1 fen_250 gkg -1 fen_1000 gkg -1 fen_10000 gkg Boronia blank -1 Bor_100 gkg -1 Bor_250 gkg -1 Bor_1000 gkg -1 Bor_10000 gkg

Manganese 0 0.01 0.02 0.07 0.79 0 0.01 0.02 0.07 0.85 0 0.01 0.02 0.06 0.79 0 0.00 0.01 0.04 0.53

zinc 0.01 0.00 0.00 0.03 0 0.00 -0.01 0.00 0.05 0 -0.04 -0.03 -0.03 -0.01 0 0.00 0.00 0.00 0.01

Table 30. Manganese levels in dilute 5mM EDTA / 9mM NaOH extracts of essential oils and boronia extracts fortified with mancozeb. The results indicate that the detection limit for mancozeb in distilled oils approaches 0.1 mgkg-1 whilst a level of 0.25 mgkg-1 is achievable in the matrix of boronia extracts. The boronia results recorded a higher manganese blank. This may be due to endogenous manganese or possible mancozeb contamination. Contamination is more likely in solvent extracted concretes than in distilled oils. This experiment provides the basis for a sensitive, economical screen. The extraction and analyses using adaptations of this basic methodology from the surface of peppermint leaves is described in appendix 2.

86

Preliminary clean-up methodology

Liquid / liquid extraction of pesticides from solvent extracted oils
Direct analysis of solvent extracted concretes such as boronia and blackcurrant is limited as the complex mixtures usually include low polarity hydrocarbons and waxes which contaminate and reduce sensitivity in most analytical machines. A simple partition clean-up step can be effective in removing non-polar components. This allows for the introduction of the samples onto LC systems with reduced risk of blocking the guard columns or non-polar phase LC columns. Many of the non-polar compounds removed are non-volatile so the inclusion of a liquid/liquid extraction can also reduce the loading into the injector of GC based analytical methodologies.

Experiments Undertaken in the Process of Method Development Example 12 The application of liquid / liquid partition in the preliminary clean-up of pesticides in solvent extracted oils.
Aim: To determine the effectiveness and recoveries of pesticides extracted from boronia concrete using liquid / liquid partition. Experimental: Standard acetone solutions of 10 gmL-1 and 100 gmL-1 of the pesticides were used to fortify 0.5 g of boronia concrete (weighed into 9 mL, 13 x 100 mm disposable test tubes) to produce fortification levels of 0.25, 1, 10 and 50 mgkg-1, relative to oil weight. Cyanazine (0.025 mL of a 100 gmL-1 solution) was added to each as an internal standard. The total volume of acetone in each tube was brought to 250 L. The boronia concretes were dissolved in 1 mL of hexane. The samples were vortexed and 1 mL of methanol : water (5:1) was added. The mixtures were again vortexed for 3 x 20 second bursts then centrifuged for 5 minutes at 3000 rpm to effect a partition. The aqueous layer was sub-sampled using a disposable pipette and filtered through cotton wool into a 200 L GC vial insert. The samples were analysed by LC MS/MS using the listed conditions. To allow for an estimation of recoveries fortified oil extracts were to be compared to aliquots of 1mL of methanol:water (5:1) which had been fortified at concentrations equivalent to those prepared for the standard curve. Acetone (250 L) was added to each to ensure the polarity was similar to the samples in boronia matrix. Samples, prepared to determine recoveries, were analysed under the same conditions as the standard solutions.

87

Results & Discussion: Table 31 lists the recoveries at each fortification level using

the liquid / liquid partition method described.

mg/kg analyte monocrotophos simazine pirimicarb propazine sethoxydim prometryn tebuconazole propiconazole difenaconazole n 4 4 4 4 4 4 4 4 4 50 mean SE 123 4 38.4 0.5 101 1 38.6 0.5 35 1 72.6 0.8 105 2 24.4 0.4 72 1 10 mean SE 155 7 34 1 107 2 43.3 0.8 21.6 0.3 66 1 105 3 17.4 0.5 74 0.5 1 mean SE 178 11 34 2 115 2 47.5 0.5 20.7 0.9 74 2 119 3 15 1 75 2 0.25 mean SE 214 11 25 3 109 4 49 1 17 2 81 2 88 7 nil 66 2

Table 31. % Recoveries of pesticides using liquid / liquid partition. The high recoveries recorded for monocrotophos in boronia preclude the application of this screen in quantitative experiments. The signal for ion m/z 193 is enhanced in the matrix of boronia, probably as a result of co-eluting components in the boronia extract. As the concentration level of monocrotophos approaches the ppb level, the relative contribution of the co-eluting contaminant increases. The recoveries for propiconazole, on the other hand are very low. However, the repeatability of that low recovery, with an RSD of 7% at 1 mgkg-1 still allows for the application of this analytical method for the detection of gross contamination of propiconazole in boronia concrete. Recoveries for simazine, propazine and sethoxydim are also less than 50%, however, the consistency in the percentage of analyte recovered across the range of fortification levels allows for the application of this analytical method to the screening of these three analytes in boronia extract.

Preliminary Development of Solid Phase Extraction of Pesticides from Essential Oils.

Solid phase extraction (SPE) has limited application for pesticides in essential oils, as the elution characteristics of the components are often very similar to the analytes we wish to separate from the oils. The large array of parameters which can be varied in SPE methodology make the number of possible chromatographic conditions unmanageable. However, thin layer chromatography (TLC) provides a quick effective method for selection and optimisation of solvent regimes. Within this section TLC was used to establish a basic method for the separation of a range of pesticides. Successful solvent / media combinations were then scaled up for application in SPE.

88

Experiments Undertaken in the Process of Method Development Example 13 The application of Thin Layer Chromatography for Development of a Solvent Combination Suitable for the Separation of Pesticides from Essential Oil Components.
Aim: The behaviour of oil components and pesticides were first trialed using TLC. Commercially available, activated silica TLC plates were used to test the separation of parsley oil, peppermint oil and boronia extracts trialed under a range of solvent combinations.

Experimental: Thin layer chromatography aluminium sheets layered with silica gel 60 F254 supplied by Merck were cut into 15 x 4 cm sections. Pencil lines were inscribed on the silica above the level of the chromatography chamber sumps and dilute aliquots of fennel oil, parsley oil, peppermint oil and boronia extracts were spotted onto the base line with glass capillaries. A range of solvent combinations was trialed. The retention characteristics and U.V absorbances of a range of pesticides were also established. Solvent combinations trialed included hexane / acetone, hexane / ether and chloroform / methanol in varying ratios. After resolution of each plate the solvent front was marked and the Rf calculated for the oil components and each pesticide where

Rf = elution distance of target analyte elution distance of solvent front

Results & Discussion: Table 32 records the Rf of pesticides for some example solvent combinations.

89

hexane/acetone
pesticide difenaconazole linuron bromacil terbacil simazine procymidone ethofumesate chloropropham fluroxypyr ester sethoxydim mecoprop pendimethalin pirimicarb 50/50 0.8 0.7 0.8 0.8 1.0 1.0 1.0 0.9 0.9 not tested not tested not tested not tested

chloroform/
methanol 50/50 0.7 0.9 0.5 0.5 0.6 1.0 0.8 0.9 0.6 not tested not tested not tested not tested

hexane/ether
50/50 0.0 0.2 0.1 0.0 0.2 0.4 0.0 0.2 0.1 not tested not tested not tested not tested

hexane/ether
90/10 0.0 0.0 0.0 0.0 0.0 0.2 0.0 0.2 0.1 0.1 0.2 0.4 0.0

Table 32. Chromotagraphic characteristics of selected pesticides resolved on TLC under differing solvent regimes. For all oils tested hexane / diethyl ether (90 / 10) effected separation of oil components. The movement of oil components and the relative immobility of pesticides presented the hexane / ether solvent combination as the most favourable.

Experiments Undertaken in the Process of Method Development Example 14. SPE Method Validation
Aim: In the experiment described previously it had been established that the target pesticides remained on silica columns when hexane / ether (90 / 10) is used as a mobile phase. The analytes are then eluted from the column with acetone. A validation of the application of this silica based SPE to the clean-up of essential oils was undertaken.

Experimental: 1 mL of peppermint oil and boronia concrete were spiked with 0.01, 0.1, 1 and 10 g of monocrotophos, simazine, cyanazine, pirimicarb, propazine, propiconazole, tebuconazole and difenaconazole to give fortification levels of 0.01, 0.1, 1 and 10 mgkg-1. 5 L of a 1 mgmL-1 solution of cyanazine was added to all as an internal standard. Prepacked "Bond Elut" TM MEGA BE-SI 6 mL columns, containing 1 g of silica, were purchased from Varian. Columns were pre-conditioned with 2 mL of hexane / ether (9:1). Fortified oil (100 L) was applied to the pre-conditioned columns and 4 x 1 mL of the hexane / ether was used to remove oil components from the column. Acetone (4 mL) was used to elute the retained pesticides. The acetone fraction was collected into 9 mL Kimbal disposable test tubes and dried under nitrogen on a heating block. Acetonitrile (200 L)

90

was added and the solution transferred to 200 L insert in a 1 mL LC vial. The extractions were repeated 4 times for each concentration. To assess the effect of oil on recoveries 100 L of solvent spiked with the equivalent level of pesticides as that prepared in oil were also passed through silica columns. This experiment was also conducted in quadruplicates. The responses recorded for fortified solvents and peppermint oils applied to SPE were compared to samples fortified with equivalent concentrations of pesticides but which had not passed through columns. This was undertaken to obtain a measure of signal suppression due to oil components alone and overall recovery of pesticides from the acetone fraction. Also included in this experiment was an assessment of the amount of volatiles eluted in each fraction relative to the total amount in oil not subject to SPE. To obtain a measure of total volatiles in the oils used in the SPE validation experiment 25 L of peppermint oil (22 mg) was dissolved in 1 mL of acetone and spiked with 1.236 mg of the internal standard, octadecane, and analysed directly. A further 100 L of the peppermint oil was applied to the SPE column as described. The fractions collected of ~4 mL of hexane / ether (9:1) and ~4 mL of acetone were each spiked with 4.927 mg of octadecane and subject to analyses by GC flame ionisation detection (FID). The volatiles, defined as all peaks eluting before the octadecane under the conditions described, were expressed as pecentage recoveries relative to the amount of oil analysed by assuming a 1 : 1 response ratio to the known quantity of internal standard added. This process was repeated with boronia oil to obtain a measure of the volatile material removed by SPE from the polar fraction containing the target pesticides.

Analytical parameter: Samples were analysed by LC MS/MS using the conditions detailed on page 70. The percent volatiles recovered in each SPE fraction were determined by GC FID under the following conditions. GC column: carrier gas injection volume: injector temp: detector temp: initial temp: head pressure : Hewlett Packard 6890 Hewlett Packard 5MS 30m, i.d 0.32m instrument grade nitrogen 1L (split) 250C 280C (FID) 50C (3 min), 10Cmin-1 to 270C (7 mins) 10psi.

Results & Discussion: Table 33 records the %volatiles recovered in each SPE fraction relative to the amount determined in the neat oil.

91

sample parameter wt of oil (mg) wt of volatiles calculated (mg) %volatiles % menthol sample parameter wt of oil (mg) wt of volatiles calculated (mg) %volatiles %post C18 peaks by GC FID %yield total GC-able peaks

peppermint oil neat 21.8 15.5 71.3 49.3 boronia concrete neat 12.2 0.8 6.7 15.6 22.3 hexane/ether fraction 97.4 3.3 3.3 12.3 15.6 acetone fraction 97.4 2.7 2.8 3.6 6.4 hexane/ether fraction 87.1 22.1 25.4 0.0 acetone fraction 87.1 30.1 34.6 84.9

Table 33. Recoveries of volatile components form peppermint and boronia oil in SPE fractions.

A total of 71.3% of the volatiles in peppermint oil are detectable by GC FID under the conditions listed. 84% of those volatiles come through in the 2 fractions collected with 58% eluting with the pesticides in the acetone fraction. This result, which at first appears discouraging, reveals on closer analyses that 85% of the volatiles in the acetone fraction is menthol. Excluding menthol, only 4.5 mg of the original 87.4 mg or 4.6% of volatiles coelute with the target pesticide. In some circumstances menthol can be selectively vented to waste. In boronia extract only 22.3% of the oil is detectable by GC FID. Of that percentage only 19% remained in the acetone fraction along with the pesticides. The results presented for boronia, however, are of limited relevance as boronia concrete, which is a solvent based extraction using low polarity solvents such as hexane, has a high proportion of waxes and low polarity components which are not volatile within the GC environment. Peppermint oil on the other hand is produced by steam distillation and has a higher proportion of volatile components.

92

Table 34 lists the recoveries for the internal standard, cyanazine

n Response in solvent without SPE Response in peppermint oil without SPE Response in solvent with SPE Response in peppermint oil with SPE Response in boronia oil with SPE 4 4 16 16 16 area 4.23E+08 1.66E+08 1.03E+08 3.80E+07 3.17E+07 %C.V. 2 2 2 22 18 %response 100 39 24 9 7

Table 34. Recoveries of cyanazine from SPE with hexane / ether mobile phase

The results listed in Table 34 indicate that cyanazine is not suitable as in internal standard for the analyses of pesticides using silica columns with the mobile phase tested. When cyanazine is analysed within the matrix of peppermint oil, 61% of the response is lost. This is further exacerbated when only 24% of the analyte is recovered from the silica column. The combined effect results in only 9% overall response to cyanazine. The %C.V. of 22% is too high to allow for the application of a correction factor to relate the final result for cyanazine on silica to the result obtained for the analyte not applied to silica. The retention times of sethoxydim within the LC MS/MS were not consistent and did not elute within the time window established for the target ion, m/z 282. Results are not reported for this analyte. Note should be made, however, that in peppermint oil, and not subjected to SPE, the response for sethoxydim was found to be linear with increasing analyte concentration and that 15% of that signal is suppressed when compared to the same fortification level in solvent only at 10 mgkg-1. Prometryn also did not elute from the silica column but the response of this analyte when analysed in peppermint oil without SPE was 78% (S.E. 4% n=4) relative to that recorded when analysed in solvent only. Table 35 records the signal suppression caused by the presence of peppermint oil which is calculated by the formula

% response

area of signal in peppermint oil x 100 area of signal in solvent only

mgkg 10 1 0.1

-1

monocrotophos 69.0 76.6 70.2 0.0

simazine 68.6 67.9 56.3 0.0

pirimicarb 53.4 78.1 96.1 377.9

propazine 62.4 66.3 64.1 0.0

tebuconazole 56.7 80.9 0.0 0.0

propiconazole 69.7 83.2 242.2 0.0

difenaconazole 66.4 77.9 90.2 0.0

0.01

Table 35. Percentage response of pesticides in peppermint oil relative to solvent only injections

93

Table 36 lists the responses of the target analytes applied to SPE in 100 L of solvent, relative to the same concentrations of analytes not subject to SPE. The recoveries are reported as percentages. Tables 37 and 38 list the recoveries of pesticides from SPE in peppermnt oil and boronia extract respectively.
mgkg 10 1 0.1 0.01
-1

monocrotophos 26.90.7 372 292 416

simazine 46.30.9 562 34.00.7 321

pirimicarb 32.30.9 523 352 487

propazine 432 533 461 645

tebuconazole 452 58.00.7 611 0.0

propiconazole 472 611 472 453

difenaconazole 442 57.50.5 471 512

Table 36. Percentage recoveries of pesticides in solvent only when applied to SPE.
mgkg 10 1 0.1 0.01
-1

monocrotophos 746 605 626

simazine 59.30.7 52.40.4 493 587

pirimicarb 641 701 822 15412

propazine tebuconazole 57.40.6 65.40.8 601 1313 483 905

propiconazole 68.8 65.9 1783 57189

difenaconazole 642 592 653 677

Table 37. Percentage recoveries of pesticides in peppermint oil by SPE relative to pesticides recovered from SPE without oil.
mgkg 10 1 0.1 0.01
-1

monocrotophos 13711 1275 21011 5147

simazine 493 41.70.6 683 731

pirimicarb 11111 1145 1584 179.70.8

propazine 745 6416 1922 7852.5

tebuconazole 746 762 934

propiconazole 160 9.30.7 15.70.8

difenaconazole 976 952 11911 181.00.6

Table 38. Percentage recoveries of pesticides in boronia oil by SPE relative to pesticides recovered from SPE without oil. The matrix effect of peppermint oil on the analyses of all the pesticides tested was marked. For monocrotophos, simazine and propazine the response is reasonably constant across the concentration ranges tested. However it would appear that for pirimicarb, propiconazole and difenaconazole there may be a co-eluting peak which enhances the signal disproportionately at lower concentrations. Alternatively the responses for propiconazole and difenaconazole may be non-linear as the detection limits are approached, as a peak was not recorded for either analyte at concentrations around 0.01 mgkg-1. The amount of essential oils able to be injected onto an LC MS/MS system is limited as contamination of inlet systems and the ion trap itself is possible. As such only 10 L of the oil solution not subject to SPE was injected into the LC MS/MS. In addition this had been diluted by 4:1. The areas recorded for each peak were multiplied by 2.4, then by 4 to allow for direct comparison with the areas detected by the 10L injection of

94

undiluted spiked solvents. In actuality this would mean that the LC MS/MS is attempting to detect a lower level of pesticide in peppermint oil than in solvent only such that any non-linear behaviour at low levels would be exasperated. This is not the case for pirimicarb, which recorded a strong positive result even in non-fortified oil. This would result in a false positive being recorded for pirimicarb in essential oil screens using LC MS/MS. Excluding the signal suppression observed for pesticides in essential oils the recoveries of pesticides in peppermint oil by SPE using the solvent protocol are low but the standard errors recorded at each concentration level are within an acceptable range indicating that with further work the SPE method developed has potential. Recoveries from fortified boronia concretes detailed in Table 38 are higher than those obtained from peppermint oil further highlighting the effect different matrices can have on SPE methodology. The SPE methodology allows for the processing of larger amounts of essential oil and reduces loading of essential oil components into analytical instrumentation. Standard curves must be established within the matrices of contaminant free essential oil and must undergo the same work-up as that undertaken for the samples to be tested. It would be difficult to identify an internal standard without using isotopes of the analytes to be tested but the inclusion of an internal standard post-SPE would negate the effects of poor instrument precision etc.

The application of ion exchange chromatography to the clean-up of acidic pesticides with detection by GC ECD

In basic solution acidic pesticides are present as negative ions. This presents the potential to bind such pesticides onto ion exchange media whilst interfering matrices, such as components from essential oils, are eluted. The retained pesticides can then be eluted from the ion exchange media in an acidic mobile phase and submitted for analysis. In the following experiments the binding of six herbicides onto ion exchange discs is undertaken with detection by gas chromatography electron capture detection (GC ECD).

95

Experiments Undertaken in the Process of Method Development

Experiment 15

The trapping and derivatisation of acidic pesticides on ion exchange discs.

Aim: The effectiveness of trapping of six commonly used acidic pesticides in basic solutions onto anion exchange discs with subsequent methylation of the fixed acids with methyl iodide was to be tested. Experimental: Standard solutions of 1 mgmL-1 of clopyralid, dicamba, fluroxypyr acid, haloxyfop acid, MCPA and mecoprop were made up in acetone. A 5 mL aliquot of NaHCO3 buffer, adjusted to pH 10 with NaOH, was fortified with the standard solutions at levels equivalent to 10 mgkg-1 and 0.1 mgkg-1. Empore ion exchange discs, provided by Varian, were punched into 13 mm diameter discs and washed under a light vacuum with 5 mL of acetone followed by 5 mL of methanol. Whilst still wet, 5 mL of the fortified buffer solution was passed through the preconditioned discs under gravity. The discs were removed from the holders and dried at 80C for 60 minutes. The discs were then rolled and inserted into a 1 mL GC vial. 1 mL of acetonitrile and 100 L of methyl iodide was added and then heated at 85C for 60 mins. The GC lid was then opened and the solvent removed under a stream of nitrogen. The samples were then reconstituted in 200 L of acetonitrile, transferred to a 100 L GC insert and subject to GC ECD under the following conditions.

Analytical parameters: Instrumentation Hewlett Packard 5890 gas chromatograph Hewlett Packard Electron Capture Detector Processing Software - HP Chem 1 L, splitless automatic injections 30 m HP 5MS, 0.22 mm id, 0.25 m film thickness Instrument grade nitrogen 10 psi 60C (1min.) -20C/min-290C (10 min.) 260C ECD 280C

Injection: Column: Carrier gas: Head Press : Oven Temp: Injection Temp: Detector:

Results & Discussion: Of the six acids included in the experiment only four were successfully methylated using the method outlined. Table 40 lists the areas obtained for the two fortification levels tested. A non-linear relationship between analyte concentration and area detected is evident, an effect inherently associated with ECD.

96

retention time analyte 10mgkg

-1 -1

4.71min dicamba 79026 917

5.18min clopyralid 972898 13180

7.94min fluroxypyr 216267 3131

11.24min haloxyfop 1588199 24770

0.1mgkg

Table 40. ECD response to methylated acidic pesticides derivatised on anion exchange discs.

Experiments Undertaken in the Process of Method Development

Experiment 16

The isolation and derivatisation of acidic pesticides on anion exchange discs.

Aim: The success of the previous experiment in methylating 4 acidic pesticides using anion exchange discs justified the application of the technique to the pesticides in the matrix of essential oils. The stock solutions of dicamba, clopyralid, fluroxypyr and haloxfop were used to fortify peppermint and fennel oil with view to application of the technique described. Experimental: Standard solutions of 1 mgmL-1 of clopyralid, dicamba, fluroxypyr acid and haloxyfop acid were made up in acetone. Aliquots (5 mL) of peppermint and fennel oil were fortified at levels equivalent to 10 and 0.5 mgkg-1. Samples were extracted with 3 mL of NaHCO3 adjusted to pH 10 with NaOH. Empore ion exchange discs, provided by Varian, were punched into 13 mm diameter discs and washed under a light vacuum with 5 mL of acetone followed by 5 mL of methanol. Whilst still wet the 3 mL of the buffer solution extract was passed through the pre-conditioned discs under gravity. The discs were removed from the holders and dried at 80C for 60 minutes. The discs were then rolled and inserted into a 1 mL GC vial. Acetonitrile (1 mL) and 100 L of methyl iodide was added and then the vial was heated to 85C for 60 mins. The GC lid was then opened and the solvent removed under a stream of nitrogen. The samples were then reconstituted in 200 L of acetonitrile, transferred to a 100 L GC insert and subject to GC ECD under the conditions previously listed.

Results & Discussion: Table 41 records the ECD response for the methylated acidic pesticides extracted from peppermint and fennel oil.

97

dicamba sample 10mgkg solvent only 10mgkg peppermint oil 0.5mgkg peppermint oil 10mgkg fennel oil 0.5mgkg fennel oil
-1 -1 -1 -1 -1

clopyralid 5.059min 711900 574182 18148 684607

fluroxypyr 8.908min 438271 786839 14497 588610 58584

haloxyfop 13.121min 269085 85959 5871 217000

4.477min 1576381 1981816 33778 1462200 67826

Table 41. Areas as obtained by GC ECD for acidic pesticides in peppermint and fennel oil methylated on ion exchange discs. The results indicate successful derivatisation of the acids on the ion exchange media. However, a very large contaminant was evident, eluting at around 12.7 mins and elevating the base line through to the next sample run. Figure 27 records the chromatogram as obtained by GC ECD for acidic pesticides fortified at a concentration of 0.5 mgkg-1 in peppermint oil.

Figure 27. Chromatogram of 0.5 mgkg-1 methylated acidic pesticides in peppermint oil by GC ECD

98

The contaminant bleed evident in Figure 27 continued through to subsequent injections, however, much of the contaminant was removed following the elution of the solvent peak from the next injection. This may indicate that the contaminant is moved through the column by acetonitrile. Ideally the source of the contaminant should be isolated and removed from the sample work-up. However, if the source is an indispensable ingredient in the process, an injection of acetonitrile, followed by a 6 minute run to a GC oven temperature of around 100C, may remove the contaminant. Interspersing such a run between samples would ensure the GC was relatively clean between injections. However, the elution of the methylated haloxyfop acid rides the shoulder of the contaminant such that quantification of this analyte using the methodology detailed would be problematic.

99

Literature Cited
Ballee, D. L., Gould, G. E. and Fahey, J. E. Residues of DDT and Analogues in Midwestern Mint Hay and Oil. J. Econ. Entomol. 1970, 63, 1658-1660.

Buchert, A. and Lokke, H., J. Chromatography, 1975, 115 682 -686.

Chambers, J. E. and Patricia Levi. 1992. Organophosphates Chemistry, Fate, and Effects. Academic Press, Inc. New York, NY.

Chemical Information Systems, Inc. Oil and Hazardous Materials/Technical Assistance Data System, Baltimore, MD, 1988.10-16

Cullen, T. E. J. Assoc. Off. Anal. Chem. 1964 . 47, 221

Davies, N. W., Gas Chromatographic Retention Indices of Monoterpenes and Sesquiterpenes on Methyl Silicone and Carbowax 20M Phases, J. Chromatogr., 1990 503 1-24.

Deleu, R. and Copin, A., J. Chromatogr., 1979, 171 263 - 268

Garland, S. M., Menary, R. C. and Claye, C. J., Variation during dormancy and the effect of freezing and postharvest incubation on the chemical composition of blackcurrant buds (Ribes nigrum L.). J. Hort. Sci. & Biotech., 2002, 77, (4) 489-497

Garland, S. M., Menary, R. C. and Davies, N. W. Dissipation of propiconazole and tebuconazole in peppermint crops (Mentha piperita (Labiatae)) and their residues in distilled oils. J. Agric. Food Chem. 1999, 47 294-298

Gould, G. E. Problems in the Control of Mint Insects. J. Econ. Entomol. 1960, 53, 526-527.

Graham, S. L. J. Agric. Food Chem. 1973, 21, 234

100

Graham, S. L., and Hansen, W. H. Bull. Environ. Contam. Toxicol., 1972, 7, 19

Gustafsson, K. H., Thompson, R. A. High-pressure liquid chromatographic determination of fungicidal dithiocarbamates. J. Agric. Food Chem., 1981, 29, 729 - 732

Jennings, W. and Shibamoto,T., Qualitative Analysis of Flavour and Fragrance Volatiles by Gas Capillary Column Gas Chromatography, Academic Press, New York, 1980.

Kaniansky, D., Ivanyi, F. and Onuska, F.I., On-Line Isotachophoretic Sample Pretreatment in Ultratrace Detemination of Paraquat and Diquat in Water By Capillary Zone Electrophoresis, Analytical Chemistry,1994, 66, (11),1817-1824

Keppel, G. E. J. Assoc. Off. Anal. Chem. 1969, 52, 162 - 167

Kidd, H. and James, D. R., Eds. The Agrochemicals Handbook, Third Edition. Royal Society of Chemistry Information Services, Cambridge, UK, 1991

Mattern, G. C., Singer, G. M., Louis, J., Robson, M., and Rosen, J. D., J. Assoc. Off. Anal. Chem., 1989, 72 970-974

Meister, R.T. Farm Chemicals Handbook 1992. Meister Publishing Company. Willoughby, OH. 1992.

Montgomery, J. H. (ed.). Agrochemicals Desk Reference. Environmental Data. Published by Lewis Publishers, Chelsea, MI. 1993.

Rao, P. S. C. and Davidson, J. M. Estimation of pesticide retention and transformation parameters required in nonpoint source pollution models. In Environmental Impact of Nonpoint Source Pollution. Overcash, M. R., Davidson, J. M. Eds. Ann Arbor Science, 1980.

Rigaud, J., Etievant, P., Henry, R. Latrasse, A. Le methoxy-4-methyl-2 butanethiol-2, un constituant majeur de l'arome du bourgeon de cassis (Ribes nigrum L.) Sci. des Aliments, 1986, 6, 213-220

101

Starr, H.; Kiigemagi, U.and Terriere, L. C. Insecticide Residues in Peppermint and their Distillation with Peppermint Oil. J. Agric. Food Chem. 1963, 11, 482-486.

Thomson, W. T. Agricultural Chemicals. Book 1: Insecticides. . Thomson Publications, Fresno, CA. 1992

U.S. Environmental Protection Agency. Guidance for the Registration of Pesticide Products Containing Maneb as the Active Ingredient. Washington, DC, 1988.4-11

U.S. National Library of Medicine. Hazardous Substances Databank. Bethesda, MD, 1995.9-9

Wauchope, R. D., Buttler, T. M., Hornsby A. G., Augustijn-Beckers, P. W. M. and Burt, J. P. SCS/ARS/CES Pesticide properties database for environmental decisionmaking. Rev. Environ. Contam. Toxicol. 1992, 123: 1-157,

Weed Science Society of America. Herbicide Handbook, Seventh Edition. Champaign, IL, 1994

Wells, M. J. M. and Yu, L. Z., Solid-phase extraction of acidic herbicides, J. Chromgr., 2000. 885. (1-2) 237-250

Woodrow, J., Analytical Method for the Dithiocarbamate Fungicides Ziram and Mancozeb in Air. J. Agric. Food Chem., 1995, 43, 1524-1529

Worthing, C. R. (ed.) . The Pesticide Manual: A World Compendium. Eighth edition. Published by The British Crop Protection Council. 1987.

102

Appendix
1. Analysis for Mancozeb in Peppermint Leaves by Detection of Carbon Disulfide Produced by Digestion with Acidified Stannous Chloride. Acidified stannous chloride was prepared by dissolving 1.618 g of SnCl2.2H2O (M.W. = 225.65) in 100 mL of the 5M HCl.. Peppermint leaves were ground under liquid nitrogen in a stainless steel mortar and pestle. 2 g of the ground leaves were weighed into 25 mL headspace vials. 2 mL of distilled water and 2 mL of acidified stannous chloride solution were added. The vials were shaken vigorously and placed in an oven at 90C for 1 hour. The gaseous CS2 trapped in the headspace vial was pressurised and vented to the injection port of a gas chromatograph. The FPD detector had a 393 nm filter specific for the detection of sulphur. The analytical system was calibrated with solutions of known quantities of analytical grade carbon disulphide dissolved in acidified stannous chloride. Each unit of a mancozeb molecule produce 2 molecules of carbon disulfide. The molecular weight of a unit of the mancozeb chain is 266.31 so 1 mole of mancozeb produces 146 g CS2. Standard curves were produced by fortifying leaf samples with known quantities of mancozeb. Note that mancozeb is insoluble in water, but vigorous shaking of mancozeb / water mixtures produces a fine suspension from which sub-samples can be taken with some degree of accuracy. The response for carbon disulfide was non-linear and less sensitive than that recorded for phosphorus compounds using the 524 m filter

103

2. Analysis for Mancozeb in Peppermint Leaves by Detection of Manganese Quantified by ICP OES. Mancozeb is a non-systemic fungicide which remains on the surface of the leaf. Maceration of leaves was therefore not undertaken as the co-extraction of vegetative matrix would not enhance the amount of mancozeb extracted yet would increase the load of carbon compounds, thereby precluding the application of ICP-OES to the monitoring of residual manganese from the mancozeb polymer. Using tweezers to grasp the base of the petiole, 50 leaves were removed from treated peppermint plants and placed in pre-weighed 250 mL 78 x 70 mm polycarbonate vials. The samples were re-weighed. An extra 50 leaves were collected and passed through a Paton Electronic Planimeter to give an estimate of total leaf area sub-sampled. A further

selection of leaves / sample were weighed and then dried at constant temperature and humidity to determine dry weight. As neither maceration nor agitation were to be applied during the

extraction process a desorption isotherm was established to determine how long leaves should be immersed in the extracting solution to effect solvation of mancozeb. De-sorption Isotherm: Following an application of Mancozeb, 5 X 20 samples of 50 leaves were collected to establish a de-sorption profile of mancozeb into 5M EDTA / 9 M NaOH. The leaves were collected into 250 mL, 78 x 70 cm polycarbonate specimen containers sealed with a polypropylene lid. In the laboratory the samples were divided into 5 groups of 4. 150 mL of the 5M EDTA / 9 M NaOH was added ensuring all the leaves were covered. The containers were left for 1 minute, without agitation before the aqueous extract was decanted into plastic 40 mL vials. Another 4 samples were immersed in 150 mL 5M EDTA / 9 M NaOH for periods of 5 minutes whilst the remaining 3 x 4 leaf samples were subject to passive extraction for 10, 15 and 30 minutes. After decanting all samples were submitted for analyses by ICP OES. The desorption isotherm established (Figure 28) was used to determine the optimum time for extraction of mancozeb into the chelating reagent. Unfortunately the isotherm did not reach an asymptote within 30 minutes

104

1.5

mancozeb mg/kg DMB

0.5

0 0 10 20 30 40

minutes

Figure 28. Desorption isotherm of mancozeb into NaOH / EDTA

3. Derivatisation of Residues of Pesticide with Acidic Moieties in Essential Oils Diazomethane

Preparation - Potassium hydroxide, 16 g, was weighed into a 250 mL conical flask. 40 mL of distilled water and 50 mL of diethyl ether was added. The mixture was placed on ice. N-methylN-nitrosourea (3 g) was added slowly and the mixture was stirred for 20 minutes. The mix was transferred to a separating funnel and the aqueous layer drained and washed with a further 25 mL of ether. The ether solutions were combined and stored at -4C until use. Derivatisation - 20 -30 mg of essential oil was weighed into a GC vial. 0.75 mL of the

diazomethane in ether solution was added and the solutions were gently swirled to ensure the oil was completely in solution. The vials were left at room temperature for 10 minutes. 200 L of glacial acetic acid was added to each vial to quench the remaining diazomethane. GC vials were crimp sealed, ready for analysis.

105