Specimen collection and procedures
Drug solubilization
To determine the amount of a drug that remains in hair after washing, it is necessary to solubilise the drugs in the hair. Solubilisation must be such that the analytes are not altered or lost. Care is necessary to prevent the conversion of cocaine to benzoylecgonine or 6-monoacetylmorphine (6-MAM) to morphine, for example. The hair sample can be pulverised in a ball mill prior to testing, cut into segments or the entire hair dissolved. The preparation techniques are generally based on one of the following procedures:
• incubation in an aqueous buffer and analysis using immunological techniques, mostly RIA
• incubation in an acidic or basic solution followed by liquid–liquid extraction or solid-phase extraction and analysis with chromatographic techniques, mostly GC-MS
• incubation in an organic solvent (generally methanol with or without hydrochloric acid), liquid–liquid extraction or solid-phase extraction and analysis with chromatographic techniques, mostly GC-MS
• digestion in an enzymatic solution, liquid–liquid extraction or solid-phase extraction and analysis with chromatographic techniques, mostly GC-MS.
The protein matrix is destroyed completely by incubating the hair sample in sodium hydroxide. The parameters to be determined are the molarity of sodium hydroxide, the time of incubation and the temperature of incubation. The alkaline hydrolysis of hair is not suitable for the extraction of chemically unstable compounds such as cocaine, 6-MAM, benzodiazepines or esters of anabolic steroids, which are hydrolysed under strong alkaline treatment. Several authors have proposed acid hydrolysis to enable the extraction of cocaine or 6-MAM from hair. The samples can be incubated in 0.1 M hydrochloric acid overnight at room temperature, at 45C or at 56C, or in 0.6 M hydrochloric acid for 30 min at 120C. The organic solvent incubation method is the simplest. It involves placing hair samples in methanol or in ethanol and then in an ultrasound bath at 45C for several hours. GC-MS analysis can be carried out directly following evaporation of the organic solvent. With this method, it is possible to detect diacetylmorphine in the hair of heroin addicts. In addition to chemical hydrolysis and direct extraction of hair with organic solvents, various types of enzymatic digestion of keratin matrix have been proposed. Hair samples can be treated with pronase solution, glucuronidase arylsulfatase or proteinase K. The enzymes act on the keratin without altering the chemical structure of the analytes present in the hair. This is particularly important when the substances being analysed are chemically unstable, such as heroin or cocaine (Sachs and Kintz 1998). An interesting extraction procedure has been developed using supercritical fluid extraction with carbon dioxide. Adding polar modifiers like water, methanol or triethylamine leads to a subcritical fluid with high extractive properties. The high speed of the extraction (30 min) and the potential to connect on-line with GC-MS are advantages that have to be balanced against the high instrumental costs compared with solid phase or liquid–liquid extraction. However, only small amounts of non-halogenated organic solvents are needed, which keeps environmental pollution low (Edder et al. 1994).
