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CHEM. (VOL. 69, NO. 3, 1986)

443

DRUG RESIDUES I N A N I M A L TISSUES

Liquid Chromatographic Determination of Monensin in Chicken Tissues with Fluorometric Detection and Confirmation by Gas Chromatography-Mass Spectrometry
K E I G O TAKATSUKI, S H I G E R U S U Z U K I , and ISAMU USHIZAWA Miyagi Prefectura1 Institute of Public Health a n d Environment, 4-7-2, Saiwai-cho, Sendai, Miyagi 983, J a p a n
An accurate, sensitive method is described for the determination of monensin residue in chicken tissues by liquid chromatography (LC), in which monensin is derivatized witb a fluorescent labeling reagent, 9-anthryldiazomethane (ADAM), to enable fluorometric detection. Samples are extracted with methanol-water (8 2), the extract is partitioned between CHCL and water, and the CHCI, layer is cleaned up by silica gel column chromatography. Free monensin, obtained by treatment with phosphate buffer solution (pH 3) at OC, is derivatized with ADAM and passed through a disposable silica cartridge. Monensin-ADAM is identified and quantitated by normal phase LC using fluorometric detection. The detection timit is 1 ppb in chicken tissues. 1.8% at 1 ppm, 56.7 I7.1% at 100 ppb, and Recoveries were 77.6 I 46.5 3.7% at 10 ppb fortification levels in chicken. Gas chromatography-mass spectrometry is capable of confirming monensin methyl ester tris trimethylsilyl ether in samples containing residues >5 ppm.

METHOD
Apparatus (a) Hornogenizer.-Biotron type BT 10 20 350D (Biotrona Co. Ltd, Switzerland). (b) Wrist-action shaker.-Model-8-1-W (Yayoi Co. Ltd, Tokyo, Japan). (c) Chrornatographic co1umn.-Glass, 15 m m id with coarse fritted disk and Teflon stopcock. (d) Rotary evaporator.-Model N-1 (Tokyo Rikakikai Co. Ltd, Tokyo, Japan). (e) Tapered reaction vial.-Amber Reacti-vial, 0.3 mL volume (Pierce Chemical Co., Rockford, IL). (f) Liquid chrornatograph.-Model 6000A or 510 solvent delivery system, U6K injector (Waters Associates, Miiford, MA). (g) Radial cornpression systern.-Z module (Waters Associates). (h) LC co1urnn.-Nova-Pak C,, fitted in Z module and pPorasil(3.9 mm id x 30 cm) (Waters Associates). (i) UV detector.-Model UVIDEC 100 11UV spectrophotometer (Japan Spectroscopic Co. Ltd, Tokyo, Japan). U) Fluorescente detector.-Model FP-210 spectrofluorometer (Japan Spectroscopic Co. Ltd). (k) Recorder.-Model YEW 3066 (Yokogawa Electric Works Ltd, Tokyo, Japan). O ) 1ntegrator.-Model C-R3A Chromatopac (ShimadzuCo., Kyoto, Japan). (m) Gas chrornatograph-rnass spectrorneter.-Model JGC20K gas chromatograph, coupled to Model JMS-D300 mass spectrometer via single stage jet separator, JMA-3100 mass data analysis system (JEOL Ltd, Tokyo, Japan). The system was operated using hard disk-based computer software as supplied. Ionization voltage 70 eV, ionization current 300 pA, ion multiplier 140. (n) GC co1urnn.-Glass column of 2 mm id x 50 cm, packed with 2% OV-101 on 80-100 mesh Chromosorb WHP. Column temperature 300C. Reagents (a) Chernica1s.-Ethyl ether, ethanol, methanol, CH,CN, CH,C12, anhydrous Na2S04:pesticide grade (Wako Chemicals Co. Ltd, Osaka, Japan). (b) 9-Anthra1dehyde.-Technical grade, 90% purity, remainder is anthracene (Aldrich Chemical Co., Milwaukee, WI). (c) Hydrazine hydrate.-Reagent grade (Wako Chemicals Co. Ltd). (d) 9-Anthraldehyde hydrazone.-Prepared from 9anthraldehyde and hydrazine hydrate according to method of Nakaya et al. (12). (e) Active Mn02.-Prepared from KMn0, and MnSO, according to method of Attenbun-ow et al. (15). (f) Saturated ethanol solution of K0H.-Add ca 1 g KOH to 10 mL ethanol and shake. Use supemate. (g) Filter paper.-Whatman No. 1.

Monensin is a monocarboxylic polyether antibiotic having antimicrobial and anticoccidial activity (1-3). It has been used for the treatment of coccidia in chickens since 1971 (4); and more recently it was found to effectively increase feed efficiency and weight gain in beef cattle (5). Therefore, an analytical method which can detect residual low levels (ppb) of monensin in biological tissues is needed for screening drug residues in animal tissues. The colorimetric method for determining monensin uses a color reaction with vanillin under acidic conditions (6). This method is only applicable to feeds, premixes, and fermenta:ion broth, and lacks adequate sensitivity for determining :races of monensin in animal tissues. Today, antibiotics are determined almost exclusively by nicrobiological methods. These methods are rather lengthy, x e not sufficiently sensitive, and lack specificity because of :nterfering compounds. A thin layer bioautographic method can detect 10 ppb monensin in animal feeds (7) and has been pplied to detect residual levels in chicken tissues (8-11). :dowever, its semiquantitative nature, inconsistent results at : O ppb levels (7), and the relatively tedious analytical procedure limit this method for routine analyses of drug residues n animal tissues. For these reasons, a new liquid chromatographic (LC) -iethod was developed which uses 9-anthryldiazomethane iDAM) (12-14) as a fluorescent labeling reagent for the - arboxyl group. The preparation of ADAM (14) was modified. '.fonensin is extracted from the sample, cleaned up, and lsrivatized with ADAM. The fluorescent ADAM derivative f monensin is identified and quantitated by using LC with 2 aorometric detection. This method allows the determination i monensin in biological tissues with a high degree of specicity and accuracy at ppb levels. The determination limit is ppb in tissue samples. Furthermore, a confirmation method with gas chromatography-mass spectrometry (GC-MS) is presented, which may -s used to confirm monensin in tissue samples containing -:sidues >5 ppm. In this method, monensin methyl ester tris -Amethylsilyl ether is prepared and the mass spectrum is 5tained by GC-MS.
Received May 24, 1985. Accepted September 6 , 1985.