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Aims of Sample Preparation
1. Solubilization/homogenization of
solids/emulsions, etc.
2. Disruption of protein binding
3. Addition of internal standard
4. Removal of insoluble residues/interfering
compounds
5. Concentration of analysis
6. Hydrolysis of conjugates
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Guidelines for Sample Preparation
Aim to Avoid
Keep the analysis as simple as possible Unnecessary additives (eg preservatives)
Use high quality reagents, solvents, etc. Use of ISTDs if these reduce precision
Understand the clinical pharmacology, Hazardous solvents and, if possible, toxic/
metabolism, and toxicology of the analyte hazardous reagents
Use the smallest possible sample Using more sample than is needed
compatible with the required
sensitivity/selectivity
Increase sample size/injection volume to Using an extraction solvent that is too
increase sensitivity, if needed 'powerful' for the application
Use external standards in color tests, UV/ Using unnecessary extremes of heat or pH
Visible spectrophotometry, and TLC
Use ISTDs in GC, LC, and MS
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Modes of Sample Preparation
(a) Solids/tissues
• Physical disruption (homogenization, sonication, heat/microwave) •
Chemical disruption (enzymes, acid/base treatment)
(b) Liquids
• Direct analysis after (filtration, ISTD addition, protein
precipitation)
• Direct 'on-line' analysis
• Headspace (HS) analysis
• Liquid–liquid extraction (LLE)
• Solid phase extraction (SPE)
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Extraction
The process of extraction is the first step in the
qualitative and quantitative analysis of drugs and
poisons from complex biological specimens.
The isolation of the compounds of interest from the
biological matrix is essential for their successful
detection, identification and quantification.
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Liquid–liquid extraction
• Advantages
a. Rapid b.
Inexpensive c.
Efficient.
• Type of sample
oProtein-free samples (such as urine) or oLiquid
samples with low protein content (such as serum or
plasma) need to be extracted
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Steps of liquid–liquid extraction
• Extracting the biological sample with an inert, water- immiscible organic
solvent at an appropriate Ph. • Mechanical mixing of the aqueous
and organic phases is normally necessary, vortex-mixing is the quickest
and the most efficient method for relatively small volumes.
• After mixing, the phases should be separated by centrifugation (eg using
bench-top centrifuge attaining speeds of 2000–3000 rpm).
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Steps of liquid–liquid extraction
• After centrifugation, the extract is usually transferred to a clean vessel
by pipetting. • Once the extract has
been transferred, the extraction solvent is often vaporized under a stream
of either compressed air, or nitrogen.
• Reconstitution of the dried extract in a suitable volume of an appropriate
solvent.
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1. Good 'extracting power'
2. Low solubility in water
3. Moderate volatility to facilitate
Properties of Removal by evaporation, but not so
volatile as to evaporate during sample
the preparation
'ideal' 4. Stable/inert
5. Low flammability
extraction
6. Low toxicity (not only by inhalation, but
solvent also via dermal absorption)
7. Inexpensive and readily available in
required purity
8. No response/deleterious effects on GC detectors
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• Dichloromethane •
Some Ethyl acetate
commonly •Cyclohexane •
used Methyl tert-butyl ether •
extraction Heptane
solvents •Chloroform •
Methanol
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Solid phase extraction
Advantages a.
High extraction efficiency
b. Very small sample sizes are sufficient c. Reducing
solvent consumption. d. No emulsion
formation.
e. Its compatibility with automation.
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Steps of solid phase extraction
The analyzes are
isolated from the
aqueous sample by Followed by washing and
adsorption onto a solid sorbent elution
eg non- modified
silica
