Invited critical review
State of the art in hair analysis for detection of drug and alcohol abuse

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Abstract

Hair differs from other materials used for toxicological analysis because of its unique ability to serve as a long-term storage of foreign substances with respect to the temporal appearance in blood. Over the last 20 years, hair testing has gained increasing attention and recognition for the retrospective investigation of chronic drug abuse as well as intentional or unintentional poisoning. In this paper, we review the physiological basics of hair growth, mechanisms of substance incorporation, analytical methods, result interpretation and practical applications of hair analysis for drugs and other organic substances. Improved chromatographic–mass spectrometric techniques with increased selectivity and sensitivity and new methods of sample preparation have improved detection limits from the ng/mg range to below pg/mg. These technical advances have substantially enhanced the ability to detect numerous drugs and other poisons in hair. For example, it was possible to detect previous administration of a single very low dose in drug-facilitated crimes. In addition to its potential application in large scale workplace drug testing and driving ability examination, hair analysis is also used for detection of gestational drug exposure, cases of criminal liability of drug addicts, diagnosis of chronic intoxication and in postmortem toxicology. Hair has only limited relevance in therapy compliance control. Fatty acid ethyl esters and ethyl glucuronide in hair have proven to be suitable markers for alcohol abuse. Hair analysis for drugs is, however, not a simple routine procedure and needs substantial guidelines throughout the testing process, i.e., from sample collection to results interpretation.

Introduction

Hair differs from other human materials used for toxicological analysis such as blood or urine because of its substantially longer detection window (months to years) enabling retrospective investigation of chronic and past consumption. Because of its solid and durable nature, hair analysis can be performed even centuries after growth. Toxic metal ions such as Tl, As, Pb or Hg were the first poisons that could be analyzed in hair to document historic exposure (for reviews see Refs. [1], [2]). Later on, gradual development of analytical technologies, offering methods with sufficient sensitivity, enabled hair analysis for organic substances [3]. In 1979, Baumgartner et al. [4] extracted hair of heroin users and determined opiate content by radioimmunoassay (RIA). Following matrix disintegration with NaOH, the first chromatographic detection of opiates in hair was performed in 1980 by Klug [5]. This study found that the concentration along the hair shaft differed and corresponded with the time course of drug intake. Following these initial reports, two decades of scientific research has led to the development of a standardized hair testing approaches that were documented in consensus papers and official guidelines [6], [7]. Preliminary immunochemical tests for selected drug groups may be accepted as the first step [8], [9]. Although gas chromatography–mass spectrometry (GC–MS) is generally the method of choice [10], various tandem mass spectrometry methods (GC–MS–MS or LC–MS–MS) are also used for targeted analyses of low dose compounds such as fentanyl, buprenorphine, and flunitrazepam [11], [12], [13], [14], [15]. These methods are also useful for detection of some important specific metabolites typically present in hair at trace concentrations such as 11-nor-9-carboxy-delta(9)-tetrahydrocannabinol (THCOOH) [16], [17] or for retrospective detection of drugs administered as single doses [18], [19].

As described above, hair analysis is a complex scientific undertaking. Several books and comprehensive reviews have been published on this topic (e.g., Refs. [20], [21], [22]). Because of the intense interest in this topic, a number of scientific conferences have been held with their findings published as special journal issues [23], [24], [25], [26], [27]. Although the anatomy, physiology, physical and chemical properties of hair have generally been described [28], [29], [30], [31], [32], [33], hair remains an enigmatic structure to date [34], [35]. A hair is not a homogeneous fiber, but consists of keratinized cells glued by the cell membrane complex that together form three concentric structures: cuticle, cortex and medulla (Fig. 1a). The pigmented cortex is responsible for the stretching stability and color composition, whereas the 5–10 layers of shingle-like cells of the non-pigmented cuticle is responsible for high chemical and physical resistance and shine.

Hair originates from the hair follicle (Fig. 1b) located 3–5 mm below the skin surface. The hair follicle is surrounded by a rich capillary system that provides the growing hair with necessary metabolic material. The germination center around the hair bulb papilla is formed by matrix cells (keratinocytes and melanocytes) present on the basement membrane. This association gives rise to the different hair shaft layers including the cuticle, cortex and medulla. The matrix cell cycle is one of the most rapid of all human tissues. Rapid mitosis forces a migration of the upper zones into the direction of the hair root mouth. In the next higher zones the genes for formation of keratine are expressed. It should be noted that cell development occurs differently for the cortex and cuticle. Cortex cells change from spherical shape at the germinative level to a spindle-like form. Protein filaments are synthesized which fill the cell and fuse together. In the zone of hardening, disulfide bonding, resorption and dehydration, all cytoplasmic organelles disappear with cellular residue coupled by membrane structures. Cuticle cells originate from matrix cells of the outer sphere of the papilla. These cells change to a shingle-like structure and contain amorphous protein. The cell membrane complex consists of proteins and a protein–lipid complex originating from previous cell membranes. This part of hair is most vulnerable to chemical and mechanical attack and is the primary diffusion point for incorporation and elimination of drugs. As can be expected lipophilic drugs are preferentially deposited in the cell membrane complex.

Hair color is produced by melanocytes located in the basal layer in contact with the basal membrane (Fig. 1c). They are mostly found in the basal layer of the cortex in a ratio of about 1:5. Melanocytes produce melanin pigments in melanosoms and have long dendrites that penetrate neighboring keratinocytes and discharge vesicles with the melanosoms. Vesicular membranes and melanosoms are digested by phagocytosis with the remaining pigments imbedded in keratinocytes. Melanocytes and pigmentation play an important role in the incorporation of basic drugs into hair.

Each hair can be erected by muscular action. Each hair also belongs to a sebaceous gland with the duct leading to the upper part of the root to ensure that the mature hair is bathed in sebum for two to three days prior to reaching the skin surface. The eccrine sweat glands are nearby but separated from the hair root. The sweat glands wet the hair shaft and can contribute to the incorporation of hydrophilic drugs. Interestingly the hair shaft can incorporate up to 30% water by radial swelling. In contrast, apocrine sweat glands are only found in hair of the armpit and at other intimate sites. These emerge into hair follicles superficial to sebaceous gland ducts.

Hair grows in a cycle composed of the anagen (active growing), catagen (transition) and telogen (resting) stages. The individual length of hair depends on stage duration and growth rate. The average values for these stages are 4–8 years, a few weeks, and 4–6 months, respectively. Scalp hair growth ranges 0.6–1.4 cm per month in general [22], [36], [37], [38]. It should be noted, however, that there are significant differences both in the proportions anagen/telogen hair and in growth rate from various anatomical sites. Both parameters are dependent on race, sex, age and state of health (Table 1). At any one time, approximately 85% of adult scalp hair is in the growing phase (anagen) with the remaining 15% in the resting phase (telogen). The significant consequence of cyclic growth is hair age heterogeneity with respect to distance from the skin.

Drug of abuse testing in hair has become routine practice in forensic toxicological laboratories. This practice has now been extended to therapeutic drugs and alcohol metabolites. In this paper, we review practical concepts and experimental principles of hair testing methods.

Section snippets

Incorporation and elimination of drugs in hair

The precise mechanisms involved in the incorporation of drugs into hair remain unclear requiring further investigation. Incorporation models typically assume that drugs or chemicals enter hair by passive diffusion from blood capillaries into growing cells over a length of 1.2 to 1.5 mm between the level of matrix cells and end of the keratinization zone of the hair follicle. This period would correspond to a timetable of drug exposure of about three days. Experimental data, however, indicates

Performance of hair analysis

Hair analysis for drugs is preferentially performed in forensic cases. Because of its serious consequences, the analyst assumes a high responsibility for obtaining a correct result. Therefore, the whole process from sampling to result interpretation must be well organized and precisely performed to avoid any potential error. For example hair sampling and analysis are not allowed in locations where the drugs themselves are handled. Practical steps of hair analysis are outlined (Fig. 5).

Interpretation of analytical results

After hair analysis, answers to the following questions are usually expected:

  • Did the individual use drugs?

  • Which drugs were used?

  • Was it single, occasional, regular or excessive use?

  • When were the drugs used?

These answers, however, are not typically derived on the sole basis of analytical results. In general, these issues require expert and critical examination of the case history, variability of hair growth (cf. Section 2) and the hair sample itself, drug pharmacology and thorough review of

Human performance toxicology

Drugs seriously interfere with human physical and/or psychological performance. These effects have serious negative consequences for many common activities ranging from industrial safety to driving ability. Recently, drug use has been highlighted as a means to enhance sports performance. Because of its potential as a long-term index of drug use history, hair analysis provides a mechanism to monitor and control abuse in all these cases.

Conclusions and future prospects in hair analysis

Hair as a medium for diagnosis of previous and chronic drug exposure has received increased attention because of a wider detection time frame, less embarrassing circumstances of collection, and greater stability versus body fluids or other tissues. Improved analytical technology has resulted in improved sensitivity and accuracy thus providing better scientific understanding and test interpretation. These advances will further promote the use of hair analysis as a useful and objective tool of

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