Michael I Schaffer and Virginia A Hill


Compounds trapped in hair during growth collect and remain in the mature hair strand. Defined lengths of the hair strand can be analyzed to provide information on ingestion of a substance during the window of time corresponding to the growth period of the segment of hair analyzed. Both screening (immunoassay) and confirmation (liquid chromatography-tandem mass spectrometry, gas chromatography-mass spec-trometry, gass chromatography-tandem mass spectrometry) methods for drugs in hair require methods of liquefaction and/or extraction of the solid hair fiber. Extensive washing of hair samples to remove external contamination and/or drugs from sweat prior to analysis is integral to a meaningful hair result, particularly to distinguish use from contamination and to utilize the hair's ability to reflect dose. Some results of drugs-of-abuse analysis in washed hair of proven drug users ranged (in ng/10 mg hair) from the cut-offs to 2270 (cocaine), 559 (morphine), 79 (methamphetamine), and 150 (phencyclidine). The metabolite of cannabis use, carboxy-tetrahydrocannabinol, was present in users' hair samples in amounts up to 76 pg/10 mg hair. Hair analysis for drugs of abuse is most widely used for pre-employment and workplace testing, but has also shown utility in criminal justice settings, for diagnostic and monitoring purposes in rehabilitation programs, in determining prenatal drug exposure, and other arenas.

1. Introduction

Over the past 25 yr, analysis of hair to examine retrospectively a subject's drug ingestion has become an accepted and even routine procedure. This

From: Forensic Science and Medicine: Drugs of Abuse: Body Fluid Testing Edited by R. C. Wong and H. Y. Tse © Humana Press Inc., Totowa, NJ

acceptance is reflected in a bibliography of publications that includes over 750 citations worldwide from 1984 to 2002 (1). Although the most common use of hair testing in the United States may be workplace testing, especially pre-employment testing for drugs of abuse, other applications include maternal prenatal testing, post-partum testing, epidemiology, criminal justice, drug rehabilitation, and anthropological studies (2).

Hair analysis is unique in that it contains retrospective information, as compared with point-in-time information provided by other body fluids or tissues. This stems from the unique property that trace amounts of a substance from blood circulation can be trapped in a segment of hair as it develops in the hair follicle—that is, once a hair segment emerges from the follicle and becomes keratinized, it carries with it the substance it has trapped in the hair follicle. This trapping allows analysis of a sample that may be of a few months' growth to detect ingestion of a substance back in time when this hair segment was still growing in the follicle (3). In addition, relative to urine in workplace testing, hair has the advantages of ease of collection, the capacity for full observation over the course of the process, and fewer privacy issues. Handling, shipping, and storage of samples require no special packaging, preservatives, or refrigeration. Whereas a urine or blood sample represents a single point in time that can never be re-sampled, a second hair sample taken some days later can produce results similar to the first. Furthermore, hair is not amenable to adulteration, a common problem with urine.

With its inclusion in the 2004 Proposed Federal Workplace Testing Guidelines (4), hair testing has achieved recognition as a valid matrix for monitoring use of drugs of abuse. Advances have been made in many aspects of hair analysis, such as the mechanisms of substance incorporation, methods of dissolving and/or extracting substances from hair, and technologies for quick screening of large numbers of samples for the presence of substances. Improvement has also been made in chromatographic/mass spectrometric identification and quantita-tion of drugs at ever lower levels. It is also possible to distinguish external contamination from metabolically deposited drug.

2. Hair Structure and Mechanisms of Drug Incorporation

Hair follicles are surrounded by a dense network of capillaries, which nourish the rapidly growing hair follicles and moderate body temperature. A hair strand consists of a root or bulb, which lies below the surface of the skin, and a keratinized hair shaft, which projects above the skin surface (43). Drugs and their metabolites present in blood circulation diffuse into rapidly growing hair follicles during histogenesis, and are deposited into the hair follicle in keratin structures called microfibrils. Microfibrils are bundled and predominate in the cortex of the hair structure, surrounded by the cuticle. There are three phases of hair growth: anagen, the growth phase; catagen, the resting or transition stage; and telogen, the phase where the hair is readied for removal from the surface of the skin. This is followed by a return to anagen or active growth phase, during which time the dermal papilla forms the hair matrix, resulting in new hair growth. Hair growth rates differ at various body sites; in brief, head hair taken at the vertex grows about 0.51 in per month. Hair from the axilla grows at an average rate of 0.31 in per month, chest hair at an average of 0.47 in per month, and beard hair at an average rate of 0.31 in per month (5). These variations, in addition to dormancy periods, may become significant when interpreting hair analysis results as they relate to a time period of drug use (3,5).

3. Collection and Handling of Hair Samples

When appropriate, as in all forensic testing including workplace drug testing, a hair sample is collected under a chain of custody. The preferred sample is one taken from the posterior vertex of the head, an anatomical site where the majority of the hair population is in the anagen growth phase, with fewer dormant hairs. Normally, the sample is cut with scissors, as close to the skin as possible. A sufficient sample is usually about 50 mg (about the thickness of a shoe-lace tip), depending on the capabilities of the laboratory. The sample is placed in a collection device with the root end identified. If desired, the collection procedure can be viewed by the donor using a mirror. In the laboratory, the strand of hair is cut to a desired length and the segment of interest weighed, as hair results are expressed as mass of drug per weight of hair.

Making a liquid sample of a hair specimen is a primary challenge of hair analysis. Three main approaches to sample preparation for screening assays as well as for chromatographic/mass spectrometric analyses have been described and are currently in use in hair-testing laboratories: enzymatic digestion (6-8), treatment with acid (9) or strong base (9-11), and organic solvent extraction, most often with methanol (8,9,12). Enzymatic digestion has the advantages of dissolving the sample, releasing all of the drug from the hair, and allowing the melanin fraction to be removed by centrifugation. A solvent extraction method, if it is to be reliable, must be tested for equivalent efficiency and completeness, or at least for uniform results among different samples. This is especially critical at lower levels of drug, where incomplete extraction may result in misidentifying a sample containing drug as negative simply because the drug was not extracted.

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