The Detection Of Cocaine In Hair Biology Essay

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Cocaine (coc) is a compound derived from the Erythroxylon coca tree, generally grown at altitude in Peru and Bolivia. The leaves of the tree have been chewed for centuries by South American natives to reduce fatigue and hunger, often helping to combat the debilitating effects of altitude. The use of the cocaine alkaloid derivative did not occur until 1859 when the extraction process became possible [1]. The drug was further popularised by Sir Arthur Conan Doyle who wrote about the use of cocaine in his fictional books featuring Sherlock Holmes. Towards the late 1800's Sigmund Freud undertook a number of clinical trials on himself and others regarding the psychoactive effects of the drug, concluding cocaine use increased sexual potency, reduced fatigue and alleviated depression. Freud finally made comments about the beneficial effects cocaine's local anaesthetic properties [2]. Cocaine currently used as a drug of abuse can be found in two forms; the hydrochloride salt or the freebase. The extraction of cocaine from the coca leaf leads to a base paste usually chemically altered to form cocaine hydrochloride (HCL) salt. Figure 1 demonstrates COC chemical structure.

Figure 1: Cocaine

Cocaine Production

There are three basic steps that are required for the extraction of COC from the leaf. (1) Extraction of crude coca paste from the coca leaf; (2) Purification of the coca paste to COC base; (3) Conversion of the COC base to hydrochloride [3].

Due to the nature of clandestine COC production it is inherently common to find contaminants within COC samples, including plant alkaloids or manufacturing by products [3]. For this reason the use of COC as a street drug is a dangerous practice as the user does not know exactly what the product contains, and in many cases will be buying something that has been adulterated by any number of chemicals to increase the yield for the dealer. Common adulterants include procaine and benzocaine, however many other household items including starch or teething powder may be added, internet based sources regarding recent police seizures suggest that the average purity of COC sold on the street has now fallen to an all time low at 9% [4]. Further research of literature suggests a somewhat less precise approximation of purity, which falls within a large range between 30%-90% [3].

­Cocaine; Prevalence and usage

During the early 2000's the emergence of COC into the mainstream was well documented. Prior to this COC gained popularity in the 1980's when its use was associated with high earners and celebrities. The increased availability and reduction in relative cost of COC has led to a rise in use from an estimated 1% of 16-29 year olds in 1994 to 5% in 2000 [5]. With this 5-fold increase there has been growing pressure to be able to analyse samples for the presence of cocaine, a practice which previously relied on the user consuming the compound within a 96 hour period, prior to testing.

Metabolism of Cocaine

The metabolism of COC is considered to be a quick process, and the drug has a half-life of approximately 30-90 minutes depending upon the individual and specific conditions [1]. The half-life is the time taken for the concentration of drug within the body to fall by half (t1/2) [6]. Nearly all the COC ingested by a user undergoes metabolism, through a combination of enzymes located in both the plasma and the liver. Very little COC is excreted unchanged, most undergoes biotransformation. COC metabolism is believed to be mediated mainly by plasma and tissue esterases, leading to excretion in urine. The three main metabolites formed by these enzymes are ecgonine methyl ester, benzoylecgonine and ecgonine. It is thought that up to 90% of the COC ingested is excreted through the urine due to detoxification in this manner [7]. The detection of COC in urine is an established technique however due to its relatively short presence within the bodily excretions more long term methods of detection were produced.

Hair Testing

The use of hair for drug testing saw its birth during the 1980's when the process was under discussion, many advances have occurred since then and in 1995 the Society of Hair Testing (SOHT) was set up and is partly responsible for the Quality Control (QC) procedures and progression that has occurred within the field of hair testing. During the early stages of development the process suffered from a lack of sensitivity, delivered by Gas Chromatography (GC), High-Pressure Liquid Chromatography (HPLC) and Thin-Layer Chromatography (TLC). The invention of immunoassays made the analysis of hair possible, by reacting antibodies with a high affinity for the drug of interest, the presence of COC in a hair extract could be assessed. The technique however, did not deliver fully, as although sensitivity was improved, immunoassays suffered from a lack of specificity due to the fact that substances with similar structure to the target analyte react with the antibodies [8, 9]. Presently immunoassays are often used as a pre-cursor to Mass Spectrometric (MS) techniques as they can be applied to a large number of samples quickly, improving through flow of samples. Subsequent positive immunoassays can then be assessed using MS instrumentation to provide specificity. The introduction and development of MS provided the specificity required for positive drug identifications and after a number of years the coupling of chromatographic techniques such as GC-MS, HPLC-MS and tandem MS/MS led to refinement of hair testing, providing both specificity and sensitivity [8, 10].

Drug Incorporation

The detection of cocaine in hair is made possible due to the drugs incorporation into the hair matrix sometime after ingestion. There are traditionally three recognised routes of incorporation:

Drugs are incorporated into the hair shaft as a result of the hair follicles good blood supply. The drugs are therefore transported to the hair follicle where by they enter the hair matrix cells. The passive diffusion model assumes the incorporation of drug within the hair shaft as a result of passive diffusion of the drugs across the membrane into newly created hair cells within the follicle. The hair subsequently dies forming the hair shaft and as a result the drugs are "trapped" within the stable structure. It is suggested that this passive diffusion model is facilitated by the hair binding to components within the hair such as melanin. However this may not be the case as drug incorporation occurs in albino test subjects. An alternative model suggests that the drugs bind to sulfhydryl cross-links abundant in hair as a result of their amino-acid content [8, 11].

Drugs are incorporated into the hair as a result of the surrounding secretions that occur through the skin from sweat glands and sebaceous glands. It is well documented that drugs and their metabolites are excreted through the skin suspended within sweat. With this in mind the hair, as it grows, is bathed in a solution containing the drug, which diffuse into the hair. Similar studies have been produced whereby the incorporation of cortisol and cortisone are believed to enter the hair matrix as a result of their presence in sweat not the bloodstream [12]. Conflicting studies also suggest the deposition of a range of drugs, from sebum onto the hair is actually insignificant in many instances. However more lipophilic substances demonstrate a greater ability to incorporate themselves, as they can cross the cell membrane more easily [13]. Finally any drugs incorporated into the hair from sweat or sebaceous secretions are representative of the user's drug in take but may in fact complicate results as their presence on the hair is likely to broaden the positive results of segmental analysis [8].

Finally, drugs are incorporated through external contamination. This occurs in a similar manner to the previous method, however does not represent usage of a specific drug but contamination. The removal of risk of external contamination is completed by washing the hair in sequential solvent and bath phases with all wash solutions kept for analysis of the presence of drugs. In reality the risk of external contamination with consideration for COC is highly unlikely unless the suspect is exposed to COC regularly, such as during the clandestine production of the drug [8]. There is some literature suggesting that COC in contact with the surface of hair for more than one day can not be distinguished from cocaine present through usage [14]. However the circumstances under which such conditions may arise are somewhat questionable.

A brief review of literature has demonstrated that the incorporation of drugs into the hair matrix may not be a simple process, with anyone mechanism leading to the incorporation of drugs. Rather, incorporation may be as a result of a number of factors, representing that the process may still require some research. Whatever the mechanism of drug incorporation, the following paper is based on the assumption that any external contamination is removed prior to analysis.


There are a number of common techniques used to extract the drug from within the hair matrix. All of which are preceded by an initial wash step. Most techniques used for washing the hair involve sequential washes, usually conducted three times in solvent and deionised water. Barrosso et al (2008) precedes extraction with washes in dichloromethane, deionised water and methanol. Each step is conducted sequentially and conducted three times with the wash solutions being stored for analysis to eliminate the possibility of external contaminants [15]. Huang et al (2009) use a similar technique but choose to use only one solvent type, dichloromethane, with the hair being sequentially washed as above [16]. Finally Uhl (2000) proposes the use of N-hexane followed by acetonitrile to remove external contaminants [17]. Many other techniques have been applied; some are considerably more complicated including the use of phosphate buffers. The significance of these steps has however come under scrutiny in recent years and may be over complicating the procedure unnecessarily [8, 18].

The Extraction Process

As with the wash phase there are many techniques proposed for the extraction of COC from within the hair matrix. Most common are procedures involving extraction with mildly acidic solutions and solvents, incubated for varying periods of time. Alternative techniques include incubation with enzymes or buffers. Romolo et al (2003) utilises a buffer extraction, incubating hair with a phosphate buffer 0.1N at pH5 for 18 hours at 45°C [19]. Enzymatic treatment has been used effectively. Cairns et al (2004) utilised an unnamed enzyme to extract COC from within the hair matrix, digesting hair for 2 hours in a solution at pH9.5 [20]. One issue that often deters from the use of enzymatic digestion is the cost of the enzymes required for digestion [8]. Furthermore Clauwaert et al (1998) concluded that the use of enzymes including proteinase and pronase, caused interference with analyte peaks when utilised in conjunction with HPLC fluorescence detection [21].

Barroso et al (2008) undertook a relatively extensive project into extraction optimisation, concluding that soaking the hair in methanol with hydrochloric acid 0.1m (3:1), for 3 hours at 65°C was the most efficient technique [15]. Graph 1 demonstrates Barroso et al's (2008) findings with regards to recovery yields achieved using different solvent types and graph 2 demonstrates findings with regards to incubation period using the most efficient solvent methanol and HCL 0.1m (3:1).

Graph 1: Demonstrating recovery yield (peak area) of cocaine using different extraction fluids

Image taken from [15]

Graph 2: Demonstrating recovery yield of cocaine after extractions in MeOH:HCL of differing time.

Image taken from [15]

Purification/Sample Clean-up

As with all stages of this process there are a various options available when considering how the extraction fluids are cleaned up prior to analysis. Most techniques employ Solid Phase Extraction (SPE) cartridges. Although, initially Liquid-Liquid Extraction (LLE) was investigated as a means of purifying the hair isolate with a number of laboratories still employing such techniques [8]. The use of SPE cartridges see's a number of advantages over LLE, not just for drug extraction, but when considering organic compounds in general [8]. These advantages include increased specificity, reproducibility and cleaner extracts, an important advantage when considering the analysis of samples by hyphenated MS techniques [8]. SPE cartridges range in size and packing but all work using as a result of interactions between the compounds in a solution and the packing material within the cartridge.

There is an extensive range of branded SPE cartridges available. Isolute â„¢ cartridges have been utilised by Schaffer et al (2002) [22], Isolute â„¢ Confirm HCX have been utilised by Barroso et al (2008) and Cognard et al (2005) [15, 23]. Finally Clean Screen â„¢ cartridges have been utilised by Bourland et al (2000) [24].

Although the above techniques have been used successfully the most commonly used cartridge SPE in literature is the Bond Elut Certify â„¢ [8]. Romolo et al (2003) propose a technique for the purification of a hair extraction utilising a non defined pH adjusted solution [19]. Montagna et al (2000) specify a similar technique with step by step instructions of the procedure undertaken [25].

Sample Analysis

In commercial laboratories routine samples are often placed through a screening mechanism prior to quantitative analytical techniques being employed. This is completed to save time and money, improving the efficiency of commercial outfits. In essence a large number of samples can be quickly screened, with any negative results not being placed through subsequent analysis. However it is important the validity and sensitivity of prior screening techniques are well tested, with sensitivities as low as 1pg/mg required [8]. A range of screening techniques can be employed however as mentioned previously the most common is the immunoassay. The sample solution is subject to a solution containing antibodies that have specificity for the drug and/or metabolites. Two well documented techniques include the radioimmunoassay and the coated plate ELISA test, with the latter often being preferred due to lack of radioactively labelled components [8]. Segura et al (1999) confirmed ELISA was not only an effective pre-analysis screening technique, but also provided some degree of quantitation [26].



Conclude Incorporation

Literature of available techniques for extraction

Analysis literature gc, gc-ms, HPLC-MS

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Rapid Communications in Mass Spectrometry, 2008. 22: p. 3320-3326.

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