Forensic Applications of Gas Chromatography - Forensic Applications of Gas Chromatography - page 102

the liquid sample will then be further cleaned up before extraction or extrac-
tion will occur immediately after the protein precipitation stage.
8.3.4.2 Hydrolysis
Many drugs and metabolites will form conjugates with d-glucuronic acid as
glucuronides or with sulphates that will be excreted in the urine. It is impor-
tant in toxicological analyses that appropriate consideration is taken to dif-
ferentiate between 'free' and 'bound' drugs or metabolites. Either selective
(enzymatic) or nonselective (chemical) hydrolysis can be carried out on the
urine sample.
Enzymatic hydrolysis methods produce clean extracts; however, they
take much more time to carry out and are also more expensive than chemical
methods. A number of enzymes can be used and are commercially available.
Typically, overnight incubation with β-glucuronidase and/or arylsulfate is
carried out, but control of pH and temperature is required to achieve opti-
mum cleavage of the conjugate bond. Chemical hydrolysis methods are harsh
and require strong acids or alkalis at elevated temperatures, usually in a pres-
sure cooker or microwave oven. These methods do, however, yield unwanted
by-products and generally require time-consuming cleanup procedures.
8.3.5 Extraction Techniques
Extraction techniques, in particular liquid-liquid extraction (LLE) and solid
phase extraction (SPE), are used in toxicological analysis and some drug
analysis, prior to chromatographic analysis. The process of extraction is used
to extract organic substances, such as drugs, directly from body fluids and
tissues. The two main types of extraction used in these types of analyses are
liquid-liquid extraction and solid phase extraction.
8.3.5.1 Liquid-Liquid Extraction
Liquid-liquid extraction has been discussed in Chapter 7. The following
example outlines the analytical method used for preparing an 'unknown'
blood sample using a combination of pH adjustment and LLE prior to GC-MS
analysis. First, a 1 mL volume of the blood is transferred into a screw-top test
tube. To this, 20 μL of a 0.1 mg/L standard of Proadifen as internal stan-
dard is added. ( Note: Proadifen is used in our laboratory since it is ampho-
teric; this means that it will be extracted in both the acidic and basic layers.)
Typically in a working forensic toxicology laboratory, a deuterated analogue
of the compound being quantified is added as the internal standard.
To extract any acidic compounds present in the blood sample, 1 mL of
0.025 M hydrochloric acid is added to the sealed test tube containing the
blood. To this, 5 mL of diethyl ether is added, the lid replaced and the tube