Toxicological hair analysis: Pre-analytical,analytical and interpretive aspects

Abstract

Background and aims

Hair analysis for drug detection is one of the widely accepted imperative techniques in the field of forensic toxicology. The current study was designed to investigate the efficacy of chromatography for detection of drugs of abuse in hair.

Method

A comprehensive review of articles from last two decades on hair analyses via PubMed and similar resources was performed. Issues concerning collection, decontamination and analytical techniques are summarised. Physiochemical nature of hair, mechanism of drug incorporation and its stability in hair are briefly discussed. Furthermore, various factors affecting results and interpretation are elucidated.

Result

A hair sample is chosen over traditional biological samples such blood, urine, saliva or tissues due to its inimitable ability to provide a longer time frame for drug detection. Its collection is almost non-invasive, less cumbersome and does not involve any specialised training/expertise. Recent advances in analytical technology have resulted in better sensitivity, reproducibility and accuracy, thus providing a new arena of scientific understanding and test interpretation.

Conclusion

Though recent studies have yielded many insights into drug binding and drug incorporation in hair, the major challenge in hair analysis lies in the interpretation of results, which may be affected by external contamination and thus lead to false-positives. Therefore, there is a need for more sensitive and selective analysis methods to be developed in order to minimise factors that induce the effect of melanin, age and so on, and this would certainly provide a new dimension to hair analysis and its applications.

Keywords

Hair analysis drug detection dose–concentration relationship

Background

Drugs have been used and abused now for centuries. In India, there have been three predominant reasons for drug use: medicinal, religious and recreational. In ancient times, drugs were primarily used either during religious ceremonies or for treating ailments. There were times when not only was using drugs one of the status symbols amongst the wealthy, it was also a mode of seeking pleasure. Drugs may modify one or more bodily function in living organism. The number of drug-facilitated crimes has increased in recent years. As per the data provided by National Crime Record Bureau (NCRB, India), the highest number of cases pertaining to cannabis derivatives have been registered under NDPS Act.¹

Various methods have been developed to detect prior drug abuse. Once incorporated into growing hair, drugs can be detected long after they have been eliminated from conventional samples such as blood and urine.^2–6 Hair samples are extensively being used for drug detection for forensic analysis purposes, as hair provides a longer detection window.^7–13 Accordingly, hair analysis has found applications in drug treatment programmes, workplace testing, criminal justice cases and child custody disputes.^14–17

Hair anatomy and physiology

Hair is a complex epidermal outgrowth, synthesised in the hair follicle. It is composed of proteins (65–95%), lipids (1–9%) and pigments (0.1–5%; melanin), as well as small amounts of trace elements, polysaccharides and water.¹⁸ Human hair contains two types of cells. The cuticle is composed of overlapping scale cells, and the cortex contains spindle-shaped cortical cells. In the core of the cortex, there may be condensed cells forming the medulla, which might be continuous or interspersed with air spaces.¹⁹ The hair follicle is an appendage of the skin that develops from the human epidermal layer around the end of the third gestational month in repeated cycles. Each of these cycles can be divided into three different phases.

The anagen or growth phase

At any given time, approximately 85% of all hairs are in the growing phase. The anagen phase can last from two to six years. On average, a human hair grows approximately 10 cm per annum, and it is unlikely for an individual hair to grow >1 m in length.

The catagen or transitional phase

This phase succeeds the anagen phase in the hair-growth cycle and lasts approximately one to two weeks. During this phase, the hair follicle shrinks to about one-sixth of its length, subsequently resulting in the destruction of the lower part that leads the dermal papilla to break away and rest below.

The telogen or resting phase

The telogen phase is the last phase of the hair-growth cycle and lasts about five to six weeks. At any given time, approximately 10–15% of all hairs are in this phase. During this phase, whilst the dermal papilla stays in a resting phase below, the hair does not grow staying attached to the follicle. As the resting phase approaches its end, the hair-growth cycle begins with the anagen or growth phase once again joining the dermal papilla and the base of the follicle, in turn leading to the formation of new hair. If the old hair has not already been shed, the new hair pushes it out, starting the growth cycle all over again.^20,21

Drug incorporation into the hair

Over the years, several studies have been conducted to understand the mechanism of the incorporation of drugs into the hair from the bloodstream. However, the mechanism of incorporation of drugs into the hair remains unclear and requires further investigation.^22–27

The following three models have been proposed to explain the mechanism of incorporation of drugs into the hair:

Active or passive diffusion from the capillaries that feed the dermal papilla,

Diffusion from biological secretions such as sweat that bathe the developing or developed hair or

External drug deposition caused by vapours or powders that diffuse into the fully grown hair.

Although a combination of the above-mentioned models may be the most realistic model for explaining the mechanism of drug incorporation into the hair, the relative importance of other routes or models is not yet clear and hence cannot be dismissed.^28–31

Various components of the hair have been suggested as the potential molecular locations for the binding and interaction of drugs. Out of these components, the ones that have been analysed in detail to assess the binding mechanisms are keratin and melanin. A report about the binding of drugs to proteins was first published more than five decades ago.³² Since then, a considerable amount of research has been conducted on various drugs and their physicochemical properties to evaluate the binding mechanism. These studies have highlighted the efficacy of melanin as an absorber of drugs, as it can bind to both charged and neutral species. Several in vitro and in vivo studies have been carried out to understand thoroughly the process of binding of a selection of drugs and inorganic cations to melanin.^24,33,34

The process of drug–melanin surface binding was also demonstrated by Bridelli et al.³⁵ They investigated the binding of three different drugs: gentamicin (MW = 462, water soluble, basic), methotrexate (MW = 454, almost insoluble in water, acidic) and chlorpromazine (MW = 319, water solubility = 0.4 g/mL, pKa 9.3).

In 1991, the scalp hair of eight Chilean mummies with ages ranging from 2000 BC to 1500 BC were positively tested for presence of benzoylecognine in a very stable form. Because of the exceptional stability demonstrated by hair over such a longer period, its analysis may be regarded as extremely advantageous for the detection of various drugs.³⁶

Collection, preparation and analysis of hair

The most common site for collection of hair remains the vertex of the head due to uniformity regarding growth, age and sex, although non-scalp hairs lack substantial growth in comparison to scalp hair and pose challenges regarding invasiveness.^37–41 Hair grows approximately 1 cm per month. Therefore, its segmental analysis can help in detection of past exposure to drugs, which can be used as a ‘calendar’.^42–44 Hair samples should be collected approximately one month after exposure, especially in Drug facilitated assault (DFA) cases.⁴⁴ Sample size may vary from anything between 50 and 200 mg of hair, which is sufficient for screening and drug confirmation.^45–47 The Society of Hair Testing (SoHT) issued the following guidelines and recommendations for the collection of hair samples:⁴⁸

The legal, ethical and human rights of the subject should be respected;

The sample should be collected by a trained individual, not necessarily a physician;

The environment should be free from drug contamination;

Collection should be done from the vertex region and close to the scalp; and

The sample should be wrapped in aluminium foil and stored in a dry place to avoid contamination.

The fundamental part of hair analysis, which will significantly affect the data quality, is sample preparation. Hair analysis requires long procedures due to its complex matrix. The preparation and analysis of hair include the following steps.

Sample decontamination

To achieve precision and accuracy in hair testing and to improve analytical performance, it is important to remove the residue of hair care products, dust, oils and lipids, sweat and so on from the hair sample by washing.^49–51 Drugs may bind to the hair or hair matrix due to passive environment exposure, though this depends upon the porosity of the hair.^52–59 Decontamination procedures help to remove these loosely bound drugs and avoid false-positive results. Decontamination procedures include washing the hair with methanol, acetone, sodium dodecyl sulphate, dichloromethane, other organic solvents, detergents and phosphate buffers.⁶⁰

Digestion or extraction from the hair sample

Bound drugs can be extracted from the hair matrix by three different methods (described in Table 1):

Alkaline digestion, which involve long incubation of samples in alkaline solutions such as sodium hydroxide followed by extraction procedures such as solid phase extraction (SPE) and liquid–liquid extraction (LLE);

Acid extraction, which involves long incubation of samples in acidic solutions such as sulphuric acid followed by extraction procedures such as SPE and LLE; and

Enzymatic digestion, which involves the use of enzymes such as β-glucoronidase/arylsulfatase to release the drug from the hair.

Table 1.

Chromatography-based methods for drug determination form hair samples.

Washing	Digestion/extraction	Detection	LOD/LOQ(ng/mg)	Substance	Ref.
Isopropanol, water	MeOH/acetonitrile (ACN)/ammonium formate pH 5.3 (overnight, 37°C) filtration through a PTFE filter	UPLC–ESI–MS/MS	2.0–5.0	APs, BZDs, opiates, hallucinogens, antihistamines, antidepressants, antipsychotics, barbiturates	⁶¹
n-hexane, acetone	MeOH (1 h, 50°C)	LC–APCI–MS/MS	0.05–2.0	MOR, COD, 6-AM, AP, MP, BZE, COE, COC, MDA, MDMA, 3,4-methylendioxyethylamphetamine (MDEA)	⁶²

DCM	MeOH, 0.1 M HCl (overnight, 45°C)	Column-switching LC–ESI–MS/MS	0.012–0.05	COC, BZE, COE	⁶³
0.1% Tween 80, water	MAE with MeOH (9 min, 60°C)	HPLC–DAD	0.200	COC, BZE, COE, MOR, 6-AM, COD	⁶⁴
DCM (2 mL, 3 min, twice)	MeOH (15 h, 55°C)	UHPLC–ESI–MS/MS	0.06–0.027	MOR, 6-AM, COD, AP, MP, MDA, MDMA, MDEA, BZE, COC, THC, MTD, buprenorphine	⁶⁵
DCM (1 mL, 3 times)	MeOH (4 h, 40°C)	LC–ESI–MS/MS	0.001–0.010	MOR, COD, 6-AM, COC, BZE	⁶⁶
10% SDS, water, acetone	Micropulverized extraction with water/ACN/1 M TFA (80:10;10, v/v/v), (10 min) Filtration using an Acrodisc with GHP membrane, 13 mm disposable syringe filter	HPLC–ESI–HRMS	0.030–0.150	Opiates, APs, COC, BZDs, antidepressants, hallucinogens	⁶⁷
0.1% SDS, water, DCM	MeOH/25% NaOH (20:1), (sonicated 1 h and left at room temperature overnight) MISPE	LC–ESI–MS/MS	0.1	KET, NKET	⁶⁸
DCM, isopropanol, acetone	0.1 M HCl (overnight, 50°C) SPE (mixed-mode cation exchange, Bakerbond Narc-2) two-step derivatization with MBTFA and MSTFA + 1% TCMS (80°C, 30 min)	GC–EI/MS	0.1–0.2	MOR, 6-AM, COD, hydrocodeine (HCOD), COC, BZE, EME, COE, DZP, nordiazepam (NDZP), MP, MDMA, MDA	⁶⁹
DCM	MeOH (overnight, 56°C) SPE (mixed-mode cation exchange, Oasis MCX) Derivatization with BSTFA–MSTFA + 1% TMCS HS-SPME (PDMS, 100 mm)	GC–EI/MS	0.13–0.20	AP, MP, MDA, MDMA, COD, 6-AM, COC, BZE, COE, NCOC, MTD, oxycodone, oxymorphone, hydrocodone, meperidine, hydromorphone	⁷⁰
Diluted soap solution at physiological pH, water	MSPD-enzymatic hydrolysis, SPE (reversed-phase cartridge, Oasis HLB), Derivatization with BSTFA + 1% TMCS (20 min, 100°C)	GC–EI/MS	0.04–0.18	COC, BZE, COD, MOR, 6-AM	⁷¹
Water, acetone, petroleum ether	1 N NaOH (10 min, 95°C) LLE with n-hexane/ethyl acetate (9:1, v/v) derivatisation with MSTFA	GC–EI/MS	0.02–0.05	THCA-A, THC	⁷
Water	Incubation in MeOH (16 h, 38°C), SPE (mixed-mode, Clean Screen1), Derivatisation with MSTFA (20 min, 90°C)	GC–EI/MS	0.16–0.33	Diazepam, lorazepam, midazolam, zolpidem	⁷²
Water, acetone	Ultrasonication in 0.25 M HCl (1 h, 50°C), derivatization with TFAA (30 min, 70°C)	GC–EI/MS	0.002–0.240	AP, phentermine, MP, cathinones, methcathinone, fenfluramine, desmethylselegiline, MDA, MDMA, MDEA, NKET, mescaline, 4-bromo-2,5-dimethoxyphenethylamine	⁷³
DCM (1 mL, 3 times)	0.1 M HCl (overnight, 37°C), SPE (mixed-mode cation exchange, Bond Elut Certify) derivatization with MTBSTFA + 1% TBMCS	2D GC– TOF–MS	–	COD, MOR, 6-AM, AP, MP, MDA, MDMA, MTD, BZP	⁷⁴

2D GC– TOF–MS: Two-Dimensional Gas Chromatography with Time-of-Flight Mass Spectrometric analyses; 6-AM: 6-Acetylmorphine; AP: Amphetamines; BSTFA: N,O-bis[trimethylsilyl] trifluoroacetamide; BZD: Benzodiazepines; BZE: Benzoylecgonine; BZP: Benzylpiperazine; COC: Cocaine; COD: Codeine; COE: Cocaethylene; DCM: Dichloromethane; DZP: Diazepam; EME: Ecgonin Methylester; GC–EI/MS: Gas Chromatograph–Electron Impact-Mass Spectrometer; GHP: GH Polypro (GHP) Membrane; HCl: Hydrochloric Acid; HLB: Hydrophilic-Lipophilic-Balanced; HPLC–DAD: High-Performance Liquid Chromatography with Diode-Array Detection; HPLC–ESI–HRMS: High Performance Liquid Chromatography-Electrospray Ionization-High Resolution Tandem Mass Spectrometry; HS-SPME: Headspace Solid Phase Micro-extraction; KET: Ketamine; LC–APCI–MS/MS: Liquid Chromatography–Atmospheric Pressure Chemical Ionization-Tandem Mass Spectrometry; LC–ESI–MS/MS: Liquid Chromatography– Electrospray Ionization -Tandem Mass Spectrometry; LLE: Liquid Liquid Extraction; LOD: Limit of Detection; LOQ: Limit of Quantification; MAE: Microwave-Assisted Extraction; MBTFA: N-methyl-bis-triflouroacetamide; MCX: Mixed-mode Cation-eXchange; MDA: 3,4-Methylenedioxyamphetamine; MDMA: 3,4-Methylenedioxymethamphetamine; MeOH: Methanol; MISPE: Molecularly Imprinted Solid-Phase Extraction; MOR: Morphine; MP: Mercaptopurine; MSPD: Matrix Solid Phase Dispersion; MSTFA: N-methyl-N (trimethylsilyl) trifluoroacetamide; MTBSTFA: N-Methyl-N-tert-butyldimethylsilyltrifluoroacetamide; MTD: Methadone; NaOH: Sodium Hydroxide; NCOC: Norcocaine; NKET: Norketamine; PDMS: Polydimethylsiloxane; PTFE: Polytetrafluoroethylene; SDS: Sodium Dodecyl Sulfate; SPE: Solid Phase Extraction; TBMCS: Tert-Butyldimethylchlorosilane; TMCS: Trimethylchlorosilane; TFA: Trifluoroacetic Acid; TFAA: Trifluoroacetic Anhydride; THC: Cannabis; THCA-A: Tetrahydrocannabinolic Acid; TMCS: Trimethylchlorosilane; UHPLC–ESI–MS/MS: Ultrahigh Performance Liquid Chromatography Coupled with Electrospray Ionization Tandem Mass Spectrometry; UPLC–ESI–MS/MS: Ultraperformance Liquid Chromatography Coupled with Electrospray Ionization Tandem Mass Spectrometry.

Some of the recently developed techniques such as ultrasonic-assisted extraction and microwave assisted extraction has accelerated and improved the efficiency of hair analysis.^64,75 Miniaturised techniques such as headspace solid-phase micro extraction, hollow-fibre liquid-phase micro-extraction and micro-extraction by packed sorbent dramatically reduce the quantity of organic solvents used and toxic residue generated during the clean-up processes.^3,76–78

Quantification of the various analytes

Chromatographic methods are widely accepted and frequently used for hair analysis (Table 1). Various analytical methods have been developed for quantification of opiates, amphetamines, cocaine, diazepam and nordiazepam from hair using gas chromatography–electron impact/mass spectrometry (GC-EI/MS).⁶⁹ GC-EI/MS-based methods for simultaneous quantification of several phenylalkylamine derivatives in hair specimens have been developed and validated.⁷³ Segmental hair analysis has been carried out for the evaluation of drug abuse, including opioids, cocainics and amphetamines using liquid chromatography atmospheric pressure chemical ionisation tandem mass spectrometry. The investigators were able to distinguish between the concentration and origin of heroin.⁶² A solid-phase micro extraction coupled with gas chromatography–mass spectrometry was developed to detect long-term exposure of 17 drugs in the hair.⁷⁰ Montesano et al. validated a method for screening and quantification of 96 drugs, including opiates, amphetamines, hallucinogens, benzodiazepines, antihistamines, antidepressants, antipsychotics, barbiturates and other sedatives, muscle relaxants and so on in the hair using ultra-performance liquid chromatography–tandem mass spectrometry.⁶¹

Factors affecting hair analysis

Hair analysis has found applications in proving chronic intoxication in an individual, helping to solve drug-facilitated crimes and child custody cases, conducting post-mortem drug screening, workplace drug testing and so on, but its scope depends upon the detection of drugs and their metabolites followed by their quantification in the hair matrix. Several factors influence or affect the accumulation of drugs in the hair.⁷⁴

Contamination

The availability of extensive literature on hair analysis makes hair a non-controversial biological sample, but the most important issue – false-positive results – always detracts it from its benefits..^41,79 There is always a question mark on the efficiency of the de-contamination procedure. There is no agreement on the fact that after washing of hair, externally deposited drugs are completely removed.^47,80–84 Hair samples have been treated with methanol and isopropanol/phosphate buffer with three to five washes to remove drugs externally deposited on the hair.^49,85–88 Several studies have reported that washing procedures can remove >90% of externally deposited drugs from hair. Therefore, the criteria for the concentration ratio between the last wash and hair sample were established.^87,89,90

Detection of endogenous metabolites of drugs may also minimise false-positive results.^91–94 Organisations such as SoHT and the Substance Abuse and Mental Health Services Administration established metabolite cut-off levels such as for a cocaine-positive sample: COC ≥500 pg/mg and at least one metabolites of COC ≥50 pg/mg.^48,95 Drug markers were also used to determine active and passive exposure.⁹⁶

Hair colour

The mechanism of a drug binding to melanin and pheomelanin pigments has also been elucidated by several studies.^33,97–99 Darker hair has more melanin which leads to a greater accumulation of drugs compared to light hair. However, studies are still being carried out to determine the binding mechanism between different types of drugs (acid and base) and melanin.^22,100 Researchers have not ruled out the possibility of inheritance playing an important role in melanin concentration which influences drug incorporation into the hair.¹⁰¹

Cosmetic treatments

Cosmetic treatment history of hair should always be taken into consideration while interpreting the results, as studies have indicated that low concentration of drugs may not be detected in hair that has undergone chemical treatment, whereas studies have also reported that chemical treatment damages the hair, making it external drug contamination more likely.^{91,98,102,103}

Dose–concentration relationship

Though some studies have established a weak relationship between the dose and hair concentration, it is very important to find out whether the detected quantity can indicate the ingested amount.^104,105 Hair samples collected from subjects in a case-control study did not yield significant information on dose and concentration of opiates.¹⁰⁶

In view of the various findings and modern techniques mentioned above, hair analysis interpretation is a very complex subject with various biases and pitfalls. A single, unique and accurate method of interpretation is still not available to date. There are several aspects which may influence the detection of drugs such as the purity of the drug, the mechanism of drug incorporation into the hair, the stability of the drug in the hair, the frequency of abuse, cosmetic treatment, hair colour, the section of hair tested and so on.^107,108

Conclusion

The crucial part of any toxicological analysis is selection of an appropriate sample or specimen. The collection and preservation of hair samples is cost-effective, as less training/expertise is needed in comparison to the collection of body fluid for analysis. In conjunction with the biological specimens, hair analysis provides tangible information on previous drug exposure of a subject due to a wider detection time frame and greater stability versus body fluids or other tissues. Hair analysis has found applications in human performance toxicology, chronic intoxications, post-mortem toxicology, criminal assaults to modify human behaviour, drug-facilitated crimes, workplace testing, criminal justice cases and child custody disputes, drug abuse in sports and so on. Though recent studies have yielded many insights into drug binding and drug incorporation in the hair, the major challenge in case of hair analysis is interpreting the results, which may be affected by external contamination leading to false-positives. Recent advances in analytical technology have resulted in better sensitivity, reproducibility and accuracy, thus providing a new arena for scientific understanding and test interpretation. Current generations of chromatographic techniques such as gas chromatography–tandem mass spectrometry and liquid chromatography–tandem mass spectrometry technologies may be standardised for toxicological laboratories. These improved methods will further promote the use of hair analysis as a useful and objective tool of evidence. Further research is required to develop sensitive and selective methods of analysis that minimise factors such as melanin, age and so on that affect the determination of drugs in hair. Moreover, studies should focus more on the mechanism of drug incorporation with factors affecting it such as age, sex and so on which will ultimately help in identifying the dose–concentration relationship. Modernisation and advancement of analytical techniques will provide accurate detection of drugs at lower limits, thus avoiding misinterpretations. Furthermore, hair analysis may play a crucial role in the examination of harmful substances not measurable by present techniques, thus enabling its application in large-scale testing and more general use.

Footnotes

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship and/or publication of this article.

Funding

The authors received no financial support for the research, authorship and/or publication of this article.

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