The Detection Of Cocaine In Hair Biology Essay

Cocaine ( coc ) is a compound derived from the Erythroxylon coca tree, by and large grown at height in Peru and Bolivia. The foliages of the tree have been chewed for centuries by South American indigens to cut down weariness and hungriness, frequently assisting to battle the debilitating effects of height. The usage of the cocaine alkaloid derived function did non happen until 1859 when the extraction procedure became possible [ 1 ] . The drug was further popularised by Sir Arthur Conan Doyle who wrote about the usage of cocaine in his fictional books having Sherlock Holmes. Towards the late 1800 ‘s Sigmund Freud undertook a figure of clinical tests on himself and others sing the psychotropic effects of the drug, reasoning cocaine usage increased sexual authority, reduced weariness and eased depression. Freud eventually made remarks about the good effects cocaine ‘s local anesthetic belongingss [ 2 ] . Cocaine presently used as a drug of maltreatment can be found in two signifiers ; the hydrochloride salt or the freebase. The extraction of cocaine from the coca foliage leads to a basal paste normally chemically altered to organize cocaine hydrochloride ( HCL ) salt. Figure 1 demonstrates COC chemical construction.

Figure 1: Cocaine

Cocaine Production

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

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Due to the nature of clandestine COC production it is inherently common to happen contaminations within COC samples, including works alkaloids or fabrication by merchandises [ 3 ] . For this ground the usage of COC as a street drug is a unsafe pattern as the user does non cognize precisely what the merchandise contains, and in many instances will be purchasing something that has been adulterated by any figure of chemicals to increase the output for the trader. Common adulterators include Ethocaine and ethyl aminobenzoate, nevertheless many other family points including amylum or teething pulverization may be added, cyberspace based beginnings sing recent constabulary ictuss suggest that the mean pureness of COC sold on the street has now fallen to an all clip low at 9 % [ 4 ] . Further research of literature suggests a slightly less precise estimate of pureness, which falls within a big scope between 30 % -90 % [ 3 ] .

A­Cocaine ; Prevalence and use

During the early 2000 ‘s the outgrowth of COC into the mainstream was good documented. Prior to this COC gained popularity in the 1980 ‘s when its usage was associated with high earners and famous persons. The increased handiness and decrease in comparative cost of COC has led to a rise in usage from an estimated 1 % of 16-29 twelvemonth olds in 1994 to 5 % in 2000 [ 5 ] . With this 5-fold addition there has been turning force per unit area to be able to analyze samples for the presence of cocaine, a pattern which antecedently relied on the user devouring the compound within a 96 hr period, prior to proving.

Metamorphosis of Cocaine

The metamorphosis of COC is considered to be a speedy procedure, and the drug has a half life of about 30-90 proceedingss depending upon the person and specific conditions [ 1 ] . The half life is the clip taken for the concentration of drug within the organic structure to fall by half ( t1/2 ) [ 6 ] . About all the COC ingested by a user undergoes metamorphosis, through a combination of enzymes located in both the plasma and the liver. Very small COC is excreted unchanged, most undergoes biotransformation. COC metamorphosis is believed to be mediated chiefly by plasma and tissue esterases, taking to elimination in piss. The three chief 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 mode [ 7 ] . The sensing of COC in piss is an established technique nevertheless due to its comparatively short presence within the bodily eliminations more long term methods of sensing were produced.

Hair Testing

The usage of hair for drug proving saw its birth during the 1980 ‘s when the procedure was under treatment, many progresss have occurred since so and in 1995 the Society of Hair Testing ( SOHT ) was set up and is partially responsible for the Quality Control ( QC ) processs and patterned advance that has occurred within the field of hair proving. During the early phases of development the procedure suffered from a deficiency of sensitiveness, delivered by Gas Chromatography ( GC ) , High-Pressure Liquid Chromatography ( HPLC ) and Thin-Layer Chromatography ( TLC ) . The innovation of immunochemical assaies made the analysis of hair possible, by responding antibodies with a high affinity for the drug of involvement, the presence of COC in a hair infusion could be assessed. The technique nevertheless, did non present to the full, as although sensitiveness was improved, immunochemical assaies suffered from a deficiency of specificity due to the fact that substances with similar construction to the mark analyte react with the antibodies [ 8, 9 ] . Presently immunochemical assaies are frequently used as a pre-cursor to Mass Spectrometric ( MS ) techniques as they can be applied to a big figure of samples rapidly, bettering through flow of samples. Subsequent positive immunochemical assaies can so be assessed utilizing MS instrumentality to supply specificity. The debut and development of MS provided the specificity required for positive drug designations and after a figure of old ages the yoke of chromatographic techniques such as GC-MS, HPLC-MS and tandem MS/MS led to refinement of hair testing, supplying both specificity and sensitiveness [ 8, 10 ] .

Drug Incorporation

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

Drugs are incorporated into the hair shaft as a consequence of the hair follicles good blood supply. The drugs are hence transported to the hair follicle where by they enter the hair matrix cells. The inactive diffusion theoretical account assumes the incorporation of drug within the hair shaft as a consequence of inactive diffusion of the drugs across the membrane into freshly created hair cells within the follicle. The hair later dies organizing the hair shaft and as a consequence the drugs are “ at bay ” within the stable construction. It is suggested that this inactive diffusion theoretical account is facilitated by the hair adhering to constituents within the hair such as melanin. However this may non be the instance as drug incorporation occurs in albino trial topics. An alternate theoretical account suggests that the drugs bind to sulfhydryl cross-links abundant in hair as a consequence of their amino-acid content [ 8, 11 ] .

Drugs are incorporated into the hair as a consequence of the environing secernments that occur through the tegument from perspiration secretory organs and greasy secretory organs. It is good documented that drugs and their metabolites are excreted through the tegument suspended within perspiration. With this in head the hair, as it grows, is bathed in a solution incorporating the drug, which diffuse into the hair. Similar surveies have been produced whereby the incorporation of hydrocortisone and Cortone Acetate are believed to come in the hair matrix as a consequence of their presence in perspiration non the blood stream [ 12 ] . Conflicting surveies besides suggest the deposition of a scope of drugs, from sebum onto the hair is really undistinguished in many cases. However more lipotropic substances demonstrate a greater ability to integrate themselves, as they can traverse the cell membrane more easy [ 13 ] . Finally any drugs incorporated into the hair from perspiration or greasy secernments are representative of the user ‘s drug in return but may in fact complicate consequences as their presence on the hair is likely to broaden the positive consequences of segmental analysis [ 8 ] .

Finally, drugs are incorporated through external taint. This occurs in a similar mode to the old method, nevertheless does non represent use of a specific drug but taint. The remotion of hazard of external taint is completed by rinsing the hair in consecutive dissolver and bath stages with all wash solutions kept for analysis of the presence of drugs. In world the hazard of external taint with consideration for COC is extremely improbable unless the suspect is exposed to COC on a regular basis, such as during the cloak-and-dagger production of the drug [ 8 ] . There is some literature proposing that COC in contact with the surface of hair for more than one twenty-four hours can non be distinguished from cocaine nowadays through usage [ 14 ] . However the fortunes under which such conditions may originate are slightly questionable.

A brief reappraisal of literature has demonstrated that the incorporation of drugs into the hair matrix may non be a simple procedure, with anyone mechanism taking to the incorporation of drugs. Rather, incorporation may be as a consequence of a figure of factors, stand foring that the procedure may still necessitate some research. Whatever the mechanism of drug incorporation, the undermentioned paper is based on the premise that any external taint is removed prior to analysis.


There are a figure of common techniques used to pull out the drug from within the hair matrix. All of which are preceded by an initial wash measure. Most techniques used for rinsing the hair involve consecutive washes, normally conducted three times in dissolver and deionised H2O. Barrosso et Al ( 2008 ) precedes extraction with washes in methylene chloride, deionised H2O and methyl alcohol. Each measure is conducted consecutive and conducted three times with the wash solutions being stored for analysis to extinguish the possibility of external contaminations [ 15 ] . Huang et Al ( 2009 ) utilize a similar technique but choose to utilize merely one dissolver type, methylene chloride, with the hair being consecutive washed as above [ 16 ] . Finally Uhl ( 2000 ) proposes the usage of N-hexane followed by acetonitrile to take external contaminations [ 17 ] . Many other techniques have been applied ; some are well more complicated including the usage of phosphate buffers. The significance of these stairss has nevertheless come under examination in recent old ages and may be over perplexing the process unnecessarily [ 8, 18 ] .

The Extraction Process

As with the wash stage there are many techniques proposed for the extraction of COC from within the hair matrix. Most common are processs affecting extraction with mildly acidic solutions and dissolvers, incubated for changing periods of clip. Alternate 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 45A°C [ 19 ] . Enzymatic intervention has been used efficaciously. Cairns et Al ( 2004 ) utilised an nameless enzyme to pull out COC from within the hair matrix, digesting hair for 2 hours in a solution at pH9.5 [ 20 ] . One issue that frequently deters from the usage of enzymatic digestion is the cost of the enzymes required for digestion [ 8 ] . Furthermore Clauwaert et Al ( 1998 ) concluded that the usage of enzymes including protease and pronase, caused intervention with analyte extremums when utilised in concurrence with HPLC fluorescence sensing [ 21 ] .

Barroso et Al ( 2008 ) undertook a comparatively extended undertaking into extraction optimization, reasoning that soaking the hair in methyl alcohol with hydrochloric acid 0.1m ( 3:1 ) , for 3 hours at 65A°C was the most efficient technique [ 15 ] . Graph 1 demonstrates Barroso et Al ‘s ( 2008 ) findings with respects to recovery outputs achieved utilizing different solvent types and graph 2 demonstrates findings with respects to incubation period utilizing the most efficient dissolver methyl alcohol and HCL 0.1m ( 3:1 ) .

Graph 1: Demonstrating recovery output ( peak country ) of cocaine utilizing different extraction fluids

Image taken from [ 15 ]

Graph 2: Demonstrating recovery output of cocaine after extractions in MeOH: HCL of differing clip.

Image taken from [ 15 ]

Purification/Sample Clean-up

As with all phases of this procedure there are a assorted options available when sing how the extraction fluids are cleaned up prior to analysis. Most techniques employ Solid Phase Extraction ( SPE ) cartridges. Although, ab initio Liquid-Liquid Extraction ( LLE ) was investigated as a agency of sublimating the hair isolate with a figure of research labs still using such techniques [ 8 ] . The usage of SPE cartridges see ‘s a figure of advantages over LLE, non merely for drug extraction, but when sing organic compounds in general [ 8 ] . These advantages include increased specificity, duplicability and cleansing agent infusions, an of import advantage when sing the analysis of samples by hyphenated MS techniques [ 8 ] . SPE cartridges scope in size and wadding but all work utilizing as a consequence of interactions between the compounds in a solution and the packing stuff within the cartridge.

There is an extended scope of branded SPE cartridges available. Isolute a„? cartridges have been utilised by Schaffer et Al ( 2002 ) [ 22 ] , Isolute a„? Confirm HCX have been utilised by Barroso et Al ( 2008 ) and Cognard et Al ( 2005 ) [ 15, 23 ] . Finally Clean Screen a„? cartridges have been utilised by Bourland et Al ( 2000 ) [ 24 ] .

Although the above techniques have been used successfully the most normally used cartridge SPE in literature is the Bond Elut Certify a„? [ 8 ] . Romolo et Al ( 2003 ) suggest a technique for the purification of a hair extraction using a non defined pH adjusted solution [ 19 ] . Montagna et Al ( 2000 ) stipulate a similar technique with measure by measure instructions of the process undertaken [ 25 ] .

Sample Analysis

In commercial research labs everyday samples are frequently placed through a showing mechanism prior to quantitative analytical techniques being employed. This is completed to salvage clip and money, bettering the efficiency of commercial outfits. In kernel a big figure of samples can be rapidly screened, with any negative consequences non being placed through subsequent analysis. However it is of import the cogency and sensitiveness of anterior showing techniques are good tested, with sensitivenesss every bit low as 1pg/mg required [ 8 ] . A scope of testing techniques can be employed nevertheless as mentioned antecedently the most common is the immunochemical assay. The sample solution is capable to a solution incorporating antibodies that have specificity for the drug and/or metabolites. Two good documented techniques include the radioimmunoassay and the coated home base ELISA trial, with the latter frequently being preferred due to miss of radioactively labeled constituents [ 8 ] . Segura et Al ( 1999 ) confirmed ELISA was non merely an effectual pre-analysis showing technique, but besides provided some grade of quantitation [ 26 ] .



Conclude Incorporation

Literature of available techniques for extraction

Analysis literature gigahertz, gc-ms, HPLC-MS

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3. Karch, S.B. , Drug maltreatment enchiridion. 2nd erectile dysfunction. 2007, Boca Raton: CRC Press/Taylor & A ; Francis. 1267 P.

4. BBC, M.E. ( 2009 ) World Cocaine Market in “ retreat ” . The BBC News Channel.

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7. Pellinen, P. , et al. , Cocaine N-demethylation and the metabolism-related hepatotoxicity can be prevented by cytochrome P450 3A inhibitors. Eur J Pharmacol, 1994. 270 ( 1 ) : p. 35-43.

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10. Gross, J.H. , Mass Spectrometry – a text edition. Vol. 1. 2004, Berlin: Springer.

11. Henderson, G.L. , Mechanisms of drug incorporation into hair. Forensic Science International, 1993. 63 ( 1-3 ) : p. 19-29.

12. Raul, J.-S. , et al. , Detection of physiological concentrations of hydrocortisone and Cortone Acetate in human hair. Clinical Biochemistry, 2004. 37 ( 12 ) : p. 1105-1111.

13. Stout, P.R. and J.A. Ruth, Deposition of [ 3H ] cocaine, [ 3H ] nicotine, and [ 3H ] flunitrazepam in mouse hair melanosomes after systemic disposal. Drug Metab Dispos, 1999. 27 ( 6 ) : p. 731-5.

14. Romano, G. , N. Barbera, and I. Lombardo, Hair proving for drugs of maltreatment: rating of external cocaine taint and hazard of false positives. Forensic Science International, 2001. 123 ( 2-3 ) : p. 119-129.

15. Barroso, M. , et al. , Development and proof of an analytical method for the coincident finding of cocaine and its chief metabolite, benzoylecgonine, in human hair by gas chromatography/mass spectroscopy

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

16. Da-Kong Huang, et al. , Coincident finding of morphia, codeine, 6-acetylmorphine, cocaine and benzoylecgonine in hair by liquid chromatography/electrospray ionisation tandem mass spectroscopy

Rapid Communications in Mass Spectrometry, 2009. 23: p. 957-962.

17. Uhl, M. , Tandem mass spectroscopy: a helpful tool in hair analysis for the forensic expert. Forensic Science International, 2000. 107 ( 1-3 ) : p. 169-179.

18. Wang, W.L. and E.J. Cone, Testing human hair for drugs of maltreatment. IV. Environmental cocaine taint and rinsing effects. Forensic Science International, 1995. 70 ( 1-3 ) : p. 39-51.

19. Romolo, F.S. , et al. , Optimized conditions for coincident finding of opiates, cocaine and benzoylecgonine in hair samples by GC-MS. Forensic Science International, 2003. 138 ( 1-3 ) : p. 17-26.

20. Cairns, T. , et al. , Levels of cocaine and its metabolites in washed hair of demonstrated cocaine users and workplace topics. Forensic Science International, 2004. 145 ( 2-3 ) : p. 175-181.

21. Karine M. Clauwaert, et al. , Narrow-Bore HPLC in Combination with Fluorescence and Electrospray Mass Spectrometric Detection for the Analysis of Cocaine and Metabolites in Human Hair

Anal. Chem. , 1998. 70: p. 2336-2344.

22. Schaffer, M.I. , W.L. Wang, and J. Irving, An rating of two wash processs for the distinction of external taint versus consumption in the analysis of human hair samples for cocaine. J Anal Toxicol, 2002. 26 ( 7 ) : p. 485-8.

23. Cognard, E. , et al. , Analysis of cocaine and three of its metabolites in hair by gas chromatography-mass spectroscopy utilizing ion-trap sensing for CI/MS/MS. Journal of Chromatography B, 2005. 826 ( 1-2 ) : p. 17-25.

24. Bourland, J.A. , et al. , Quantitation of cocaine, benzoylecgonine, cocaethylene, methylecgonine, and norcocaine in human hair by positive ion chemical ionisation ( PICI ) gas chromatography-tandem mass spectroscopy. J Anal Toxicol, 2000. 24 ( 7 ) : p. 489-95.

25. Montagna, M. , et al. , Simultaneous hair proving for opiates, cocaine, and metabolites by GC-MS: a study of appliers for driving licences with a history of drug usage. Forensic Science International, 2000. 107 ( 1-3 ) : p. 157-167.

26. Segura, J. , et al. , Immunological showing of drugs of maltreatment and gas chromatographic-mass spectrometric verification of opiates and cocaine in hair. Journal of Chromatography B: Biomedical Sciences and Applications, 1999. 724 ( 1 ) : p. 9-21.

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