Although it has been the subject of much scrutiny, the washing procedure which has been developed through the years is actually quite good. There are many techniques used in washing a hair sample prior to testing for the presence of cocaine. These can include washing with shampoos, aqueous solutions like buffers or water, and organic solvents like methylene chloride, surfacants, and acetone. Care must be taken when washing a hair sample to ensure that ingested (or internal) cocaine is not washed out, but that external contamination is removed.[5,13] Aqueous buffers and organic solvents can solubilize drugs from hair and make concentrations in hair appear lower than they actually are, and therefore, are not recommended.

There are three domains in hair called the surface domain, the accessible domain, and the inaccessible domain. The surface domain is the surface of the hair and is where most external contamination occurs. The surface domain can be washed with non-swelling agents like ethanol for sufficient removal of external contamination. The accessible domain is the middle portion of the hair shaft and it is slightly vulnerable to external contamination. The accessible domain may be freed of external contamination using swelling solvents which open up the outside matrix of the hair enough to get some of the external contamination in the accessible domain out. Water is a swelling solvent which is often used for this purpose. The inaccessible domain is by far the largest domain and is the portion of hair in the middle of the hair shaft. It is nearly impossible for drugs to contaminate this part of the hair externally, although soaking hair in a drug solution has been shown to cause minimal contamination of the inaccessible domain. Neither ethanol nor water disturbs the inaccessible domain of the hair, so ingested cocaine remains in the hair while external contamination is removed.[13]

The delicate balance between removing all of the external contamination and not removing ingested cocaine is reached through two procedures. The procedures are called truncated washing and extended washing. The truncated washing procedure is used in screening and in work place testing because it is much less time consuming and more practical. The number and duration of the washes is fixed for all hair samples at a value which cleans the average sample sufficiently. The procedure includes one fifteen minute wash in ethanol followed by three thirty minute washes with phosphate buffer. The extended wash is used in criminal cases and other cases where accurate results carry much importance. The extended procedure involves taking samples of each washing and testing them for drug levels using radioimmuno assay until the washing level reaches a plateau when the hair is assumed to be washed completely. The plateau is determined by the ratio of the total amount of drug in the hair wash to the total amount removed by washings. If the ratio is greater than 0.25 the washing is complete. The results of the extended wash procedure are best seen by graphing the concentrations of cocaine after each wash. In Figure 1,

(Click on the image to see an enlarged Figure in a separate browser window.)

curve C1 refers to a sample which was contaminated only on the surface domain. C2 shows more severe external contamination in which the accessible interior domain was affected by the contamination. C3 is a sample which was so severely contaminated externally that the inaccessible interior domain was moderately penetrated. It is believed that exposure as severe as that in C3 would result in a positive urine test as well. The curves labeled U in the figure represent positive samples, sample with a ratio lower than the cut-off level. The differences in the curves results from differences in porosity and thickness of the hair used in the testing. The curve U1 is of a very thick and non-porous sample where U3 is a very porous sample due to chemical cosmetic treatment. Curve U4 is a very rare case of extremely damaged hair which may skew results because internal contamination is removed during the washes. This can be remedied by using less harsh solvents during the washing.[13]

Different solvents are used in these procedures depending upon the type of hair in the sample. Porous hair is treated with ethanol and water while nonporous hair is treated with phosphate buffer. The porosity of the sample can be determined through staining with methylene blue. This staining involves an extra rinsing step and examination with a microscope. Porosity can be caused by cosmetic treatment, race differences, or bad preservation of hair. The conclusions reached from many studies on the washing procedure have determined that washing is necessary and have suggested examples for standardization.[13]

After the hair sample is washed, it is necessary to extract the drugs and their metabolites from the hair sample. The sample must be put through these procedures without being degraded, hydrolyzed, or otherwise altered. Suggested methods of treatment for extracting hair are: treatment with alkali, acids, enzymes, or other organic solvents. Some specific substances used in these extractions are tris buffer, dithiothreitol, sodium dodecyl sulfate, proteinase K, and phosphate buffers. If the extraction occurs near a pH value of 6.2, the cocaine is safe from conversions into hydrolysis products.[13] The treatment with enzymes seems to be the safest way to extract hair using wet chemistry because it does not compromise the sample in any way.[14] The acid and base extractions are known to hydrolyze cocaine. Extraction also removes melanin which is a major culprit of race biases in hair testing.

The extraction procedure is the step in which the drug is released from the hair matrix, purified, and concentrated. A good extraction method must remove all of the drug from the hair and not degrade the sample.[18] Methanol extraction, acid/base extraction, and enzymatic extraction have all been studied. Methanol extraction is surprisingly good for many (but not all) drugs, while acids and bases may cause hydrolysis, the products of which may be identical to the metabolites. Enzymatic extraction works well with good recoveries, but involves the use of unpleasant thiols.[14] Extraction by the liquid methods is labor intensive, time-consuming, and is therefore expensive. An extraction technique which is popular and seems a likely candidate for standardization is supercritical fluid extraction using CO2. Supercritical fluid extraction is easy, fast, mild, and does not degrade the hair sample. The extraction conditions using supercitical CO2 can be changed easily by changing the pressure, temperature, or modifier. The hair sample is first "rinsed" with supercritical CO2 which does not remove the cocaine from the hair matrix. A modifier solution is then added to the CO2 to allow for extraction of the cocaine from the hair matrix. A good modifier has been found to be CO2/MeOH/Et3N/H2O.[19] The desorption of cocaine from the matrix is the rate limiting step in the extraction, so the extraction is very dependent on the hair matrix. The modifier works because the triethylammonium cation competes with cocaine for the negative binding sites in the hair. The addition of water helps the extraction by facilitating formation of the triethylammonium cation and by swelling the hair and making the binding sites more accessible to the solvent. Supercritical fluid extraction is not a good technique to recover benzoylecgonine because benzoylecgonine has additional "binding sites" that may also be changed. Since this compound is present in very small amounts in the first place, a good extraction technique for benzoylecgonine is essential if the metabolite is to be used as an internal contamination marker.

Supercritical fluid extraction gives excellent recoveries of cocaine and its metabolites, is simple, rapid, environmentally sound, and can be applied to many types of drugs.[12] Supercritical fluid extraction can also be easily coupled on line with many chromatographic techniques to avoid contamination and to give better detection.[14] Liquid-liquid and solid phase extraction have been investigated significantly, but have been found inferior to supercritical fluid extraction.

A variety of detection methods are also used in drug analysis in hair. Most of these techniques are chromatographic, because chromatography gives the required sensitivity and selectivity. The first technique used though was not a chromatographic technique. It was radioimmuno assy, which is still used in some cases for screening because it meets the needs for sensitivity, throughput, and simplicity needed in these instances.[5] All results from this technique must be verified through GC-MS testing though and this technique involves using radioactive reagents. Immunoassay can only screen for one class of drugs at a time which often makes multiple runs necessary.[16] The specificity of the radioimmuno assay technique is limited to classes of drugs and does not allow specific drug identification. Quantification is not possible using this technique either.[6]

More popular techniques include TLC, HPLC, LC, GC, and GC-MS. Thin layer chromatography (TLC) techniques have limited mass sensitivity and are improved by using high performance thin layer chromatography (HPTLC) with dansyl derivation. TLC shows good mass and quantitation limits. Flourescence detection is usually used with TLC detection of cocaine.[5] High performance liquid chromatography (HPLC) is also useful for detection of cocaine in hair samples. It is more suitable than other chromatographic techniques for biological extracts. Sensitivity and selectivity can be excellent with electrochemical and flourescence detectors. It can handle high work loads and is easily automated. It shows good correlation to results from radioimmunological assay testing.[5] It is rarely used in hair analysis though because it uses too much solvent and mass sensitivity could be improved by using other techniques.[9] Liquid chromatography (LC) has played a very minor role in cocaine analysis and warrants only a brief mention. The last purely chromatographic technique used is gas chromatography (GC). GC is less useful than TLC or HPLC. Many products are found and the spectra are hard to interpret. GC shows poor sensitivity and poor selectivity. Unless coupled with a mass spectrometer, GC is not a viable tool in drug detection in hair.[6,5]

Gas chromatography-mass spectrometry (GC-MS) is considered the gold standard in hair testing. Almost every positive result found is confirmed by GC-MS and it is usually the only test which is accepted in the courts. GC-MS is the tool of choice because it has good sensitivity, selectivity, specificity, a high degree of standardization, sample throughput, and instrument ruggedness. The GC-MS is usually run in the electron ionization-single ion monitoring (EI-SIMs) mode, because this is a good compromise between selectivity and sensitivity. The two ion mode can be used to looking for multiple drugs at the same time. Chemical ionization (CI) is more sensitive than EI and is becoming more popular, but EI is still the preferred ionization method.[5] Advantages of GC-MS over other techniques include a minimal amount of sample use and the ability to screen for the seven most important drugs during one run.[16] The one criticism of GC-MS is that it requires derivitization of the extracted compounds and therefore introduces an additional step with the liabilities associated.[9]