SAMPLE PREPARATION TECHNIQUES

FOR BIOANALYSIS
Wahyu Utami, M.Sc., PhD, Apt
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Bioanalysis
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The choice of sample preparation method is

dependent on the
• Sample
• Sample size
• Type of sample (e.g. blood vs. urine vs. lachrymal fluid),
• Analyte
• Type of analytes (LMW vs. HMW compounds),
• Concentrations of the analytes in the sample
• Stability of the analytes (whether analytes are protein bound)
• Method of Analysis
• The type of analysis (untargeted vs. targeted analysis)
• Sensitivity and specificity of the analytical method.
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• The measurement of defined groups

Targeted of chemically characterized and
biochemically annotated metabolites.
Analysis
(Targeted Metabolomics) • The main purpose is the quantitation
of a restricted number of analytes or a
specific compound.

• Comprehensive analysis of all the

Untargeted measurable analytes in a sample
including chemical unknowns.
Analysis
(Untargeted Metabolomics) • The main purpose is identification and
quantitation of various compounds of
interest.
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Sample collection or sampling

• The first step in all biomedical and clinical analyses.

• Sampling comprises collection, handling, delivery,

processing, and storage of the biological sample prior to
sample preparation.

• The commonest biomedical and clinical samples are body

fluids (blood, urine, saliva, bile, cerebrospinal fluid, and
amniotic fluid), faeces, and tissue biopsies.

• The correct timing and the duration of sample collection

are important for correcting the physiological variables.
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Sampling

Pre-analytical parameters can seriously affect bioanalysis.

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Sample collection requires knowledge of:

• the expected concentrations of the analytes in the sample (to
correct sample to be taken);

• the stability of the analytes, particularly to temperature, light,

pH, and biochemical changes due to enzyme actions (the
sample can then be collected on ice, kept frozen, protected
from light by covering with tin or aluminium foils, adjusted to a
suitable pH or an enzyme inhibitor added, as appropriate;

• the physiological variations of the analytes due to physical

activity, stress or biological (diurnal and circadian) rhythms.
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Why is Sample Preparation Required?

• Sample is • Sample
Concentration

Compatibility

Cleanliness
• Target
analyte(s) not matrix
not compatible components
concentrated with or will interfere
enough for would be with the
quantitative harmful to analysis
detection. your
chromatogra
phic system
Goals of Sample Preparation
• Minimize Risk
• Minimise interference from biological matrices, such as protein,
peptides, lipids, inorganic salts, or surfactants from the formulations
of compound.
• Eliminate sample to sample variability (more reproducible
quantitation, more robust assays)
• Decrease assay variability
• Increased sensitivity
• Sample concentration
• Removal of interferences
• Cleaner Samples
• Increased instrument uptime
• Improve method robustness
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Possible Effects of Biological Sample

Components
Issue Result
Poor peak shapes, co-elution, no Difficulty in identifying, quantifying
resolution components
Mechanical issues (particulates, LC/GC column lifetime issues
blockages)
Increased instrument downtime Reduced productivity, increase in
sample run time / cost
Interferences Ion suppression in LC-mass
spectrometry
Peak integration issues
Overall lower sensitivity Inability to meet detection limits
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SAMPLE PREPARATION
METHOD
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Sample Preparation Method

• Direct injection

Less selective
• Dilution
• Filtration
• Basic particulate removal from ALL kinds of samples
• Useful when additional step of lipid content removal is needed
• Protein Precipitation (PPT)
• Liquid-Liquid Extraction (LLE)
• Straightforward sample preparation technique
• Useful for in-house or commercial extraction
• Solid supported liquid extraction (SLE)

More selective
• Increased productivity using liquid/liquid extraction principle and the
concept of automation
• Ideal for aqueous sample or samples
• Solid-Phase Extraction (SPE)
• Ultra-clean sample preparation for analysis when high selectivity and
sensitivity are required
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Direct sample injection

• Liquid samples such as urine, bile or serum.
• The samples are injected directly (into column LC) or after a
quick centrifugations or filtrations.
• Requires the sample to contain a high enough concentrations
of the analyte for detections.
• Precipitates need to be treated using acid, e.g.HCl
• Major problem: the presence of a large amount of proteins
which may adsorb or precipitate on the column causing
blockage.
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Dilution (Dilute & Shoot)

• Simple sample dilution
• Advantages
• Fast and easy
• High throughput
• Limitations
• Interferences are not removed
• Concentration is reduced
• Instrument and column contamination
• Matrix interferences – ion suppression or poor peak shapes
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Filtration
• Basic sample prep method for ALL kinds of samples
• Can be the 1st choice of sample prep or 2nd secondary step
• Mechanical filtration for visible interference removal
• – Syringe filters
• – Syringeless filters
• – Agilent Captiva (cartridge and 96-well plate formats)
• – Agilent Captiva ND (cartridge and 96-well plate formats)
• Mechanical filtration + extraction by sorbent for lipid removal
• – Agilent Captiva ND Lipids (cartridge and 96-well plate
formats)
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Protein Precipitation (PPT)

• The proteins can be precipitated by addition of acid or a
water-miscible solvent like acetonitrile.
• PCA and trichloroacetic acid (TCA) have been widely
used to precipitate cellular macromolecules and to extract
cellular content.
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Liquid-Liquid Extraction

• Inorganic salts • Labour-intensive

Advantages

Disadvantages
easily removed • Large volumes of
• Short method organics
development time • Difficult to
• Low cost automate
• Flexible for a • Variable results
variety of sample • Expensive, clean
types glassware
• Easy to perform • Emulsion formation
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Solid Supported Liquid Extraction (SLE)

• Extraction mechanism: same as traditional
liquid/liquid extraction (LLE)
• Simple, time-saving process
• Apply aqueous sample to the solid bed
• Extract with water-immiscible solvent (MTBE
<methyl tert-butyl ether>, dichloromethane, ethyl
acetate)
• Analyse extract or evaporate and reconstitute as
needed
• Convert LLE methods to SLE to save time and
money, and increase throughput.
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Solid-Supported LLE - Benefits

• No emulsions → easier extractions
• No special glassware → lower cost per sample
• Less time, minimal method development → faster
implementation
• Reduced technique dependence → better ruggedness
• Increased reproducibility → better results
• Automatable → enables batch processing
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Solid Phase Extraction (SPE)

• Types of SPE
• Reversed phase SPE
• Cation exchange SPE
• Anion exchange SPE
• Mixed mode SPE
• Specialty SPE
• Capabilities
• Very selective
• Highly clean samples
• Wide range of applicability
• Automation friendly
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A Typical SPE Sequence

• Step 1:
Condition the cartridge
• Step 2:
Apply sample (e.g. food extract, water, plasma)
• Some compounds “retain”
• Step 3:
• Wash of the cartridge, interference removal
• Wash of the cartridge, additional interference removal
• Step 4:
Apply a different liquid to “elute”

 The extract is cleaner, in a different liquid, and typically more

concentrated.
 Some sorbent technologies let you reduce the number of steps for
easier, faster extractions
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Why Choose SPE?

• Flexible - match a broad spectrum of sample and target
compound types to different sorbents and forms.
• Wide array of formats and sorbents for lower detection limits
and longer instrument uptime from cleaner extracts.
• Increase sample throughput with automation-friendly formats.
• Easy adoption of methods due to high number of publications
and applications
• Get the right answer the first time with highest accuracy and
confidence.
• Best balance of sample cleanliness, accuracy of results, and
cost-per-sample.
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How do Sample Prep Options Compare?

• Solid Phase Extraction
• Often very clean, allows for trace analysis, built in
concentration Investment
• Potential for the most selectivity (and hence
cleanliness)

• Liquid/Solid Extraction Complexity

• Relatively clean and inexpensive

• Filtration

Cleanliness
• Dilute and shoot (guard columns or retention
gaps)
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Sample preparation techniques according to their respective selectivity &

enrichment capabilities.
The sphere volumes are proportional to the cost and complexity of the approach.
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Different Methods for Different

Purposes: Decision Making Process
PPT PLR Plate LLE SPE
Highest Selectivity v
Direct Inject v v v
Simple v v v
High throughput v v v v
PL Removal v v
Highest Sensitivity v
Clean Extracts v v
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Typical Process
1. The simplest method which meets the assay needs is
usually chosen
• PPT or LLE are common starting points
2. For challenging assays (low detection limits, closely
related endogenous constituents, inhalation products,
peptides, etc) SPE may be first choice
3. Exact technique chosen will depend on outcome of
study and how much risk can be tolerated