Hair Sample Preparation, Extraction, and Screening Procedures for Drugs of Abuse and Pharmaceuticals

Robert Kronstrand1,2, Malin Forsman1 and Tor Seldén1,2,    1National Board of Forensic Medicine, Department of Forensic Genetics and Forensic Toxicology, Linköping, Sweden,    2Division of Drug Research, Linköping University, Linköping, Sweden

The aims of this chapter are to give the reader a good insight into the practical preanalytical aspects of hair testing and also to inform about the different screening strategies that can be used. The chain of events leading up to the accurate interpretation of results starts already at the sampling site but this chapter will focus on the preparation of hair samples, performed at the laboratory, to ensure a valid result. This includes segmentation, homogenization, and the extraction of drugs from the hair matrix. After extraction, screening of drugs and medications is usually performed and if presumptive positive a confirmatory analysis is performed. Common strategies include immunoassays that are quick, sensitive, and cost-effective. However, chromatographic techniques are used more and more particularly by forensic laboratories where the positivity rate is much higher.

Keywords

Hair; segmentation; extraction; screening; immunoassay; chromatography

2.1 Introduction

The aims of this chapter are to give the reader a good insight into the practical preanalytical aspects of hair testing and also to inform about the different screening strategies that can be used. The chain of events leading up to the accurate interpretation of results starts already at the sampling site but this chapter will focus on the preparation of hair samples, performed at the laboratory, to ensure a valid result. This includes segmentation, homogenization, and the extraction of drugs from the hair matrix. After extraction, screening of drugs and medications is usually performed and if presumptive positive a confirmatory analysis is performed. Depending on the problem trying to be solved, the strategies for screening will vary. Common strategies include immunoassays that are quick, sensitive, and cost-effective, especially when the proportion of positive samples is low. However, chromatographic techniques are used more and more particularly by forensic laboratories where the positivity rate is much higher. Sometimes, the chromatographic screening methods are used as the end point even though, in contrast to forensic best practice, only one analysis has been performed. The reason is the high power of identification of specific analytes as well as accurate determination of their concentration that can be retrieved from such methods in combination with an increased security of sample integrity using barcodes and scanners throughout the laboratory. Also, the use of time-of-flight mass spectrometric methodology enables the analytical toxicologist to further explore many analytes in the same run even though true untargeted screening methods are rare. However, the analytical work begins with obtaining an appropriate and representative aliquot of the hair to be used for extraction and analysis.

2.2 Sample Preparation

2.2.1 Segmentation

Even though analysis may be performed on the whole hair, it is very common to use one or more segments of the sample. A commonly used segment length is the 3-cm proximal portion representing hair grown during the last 3 months. However, depending on the case, different strategies for segmentation may apply. Several short segments can provide a much more detailed profile of an individual’s drug exposure than a single segment. The accuracy of such segmental analysis depends on both the sampling and the segmentation procedure at the laboratory. Poor sampling procedures with unparallel hair strands may severely lower the segmental resolution.

When given a single dose of a drug or medication one would assume that only one segment of hair, grown at the time after administration, would be positive. However, even under controlled study conditions the drug may be found in more than one segment [1]. This is illustrated in Figure 2.1 depicting data from a study with single intake of flunitrazepam. The subject received a single 2 mg dose of flunitrazepam and hair samples were obtained after 2 and 4 months. The samples were segmented in 5 mm segments and analyzed with liquid chromatography tandem mass spectrometry (LC-MS/MS). In the 2-month sample the concentration of the metabolite, 7-aminoflunitrazepam, is high in S3 corresponding to the time of intake whereas the surrounding segments have much less drug. In the 4-month sample, segment S7 should represent the time of intake but both segments S7 and S8 have similar concentrations, significantly lower than those in the previous sample. This illustrates how the results are obscured over time when hair from different growth cycles is included in the measurement or if hair has shifted during sampling, transport, and segmentation. It is important to understand that when investigating a single intake, the use of large segments will dilute the incorporated drug and cause false negative results. Figure 2.2 illustrates in a schematic way, how the resolution in time is increasing with shorter segments and also that the measured concentrations increases because of less dilution from drug-free hair. The Society of Hair Testing (SoHT) recommends that only hair cut from the scalp with the root end identified should be subjected to segmental analysis. One should also be aware of the possibility that the hair is not cut right at the scalp, as recommended. Indeed, LeBeau et al. showed this in a study where they instructed experienced and unexperienced samplers to obtain hair samples [2]. The mean length of hair remaining on the scalp was 0.8 cm. Therefore, one should be careful when extrapolating the hair length used to a particular time period using the sampling date as baseline.

Segmental hair analysis can also provide detailed temporal information about medication intake or drug abuse pattern. If the purpose is to evaluate the possible tolerance or nontolerance to certain drugs or groups of drugs, the more recent hair segments are most relevant to analyze. For opioid drugs, tolerance develops gradually, as does the loss of tolerance. The time frame for these processes are poorly understood, and complicated by the fact that tolerance to the euphoric effects may be different from that to respiratory depression. A period of abstinence of 7–14 days is however likely to result in a marked reduction in tolerance to opioid drugs, implying that hair segments should be rather short to capture this time period accurately. Positive detections in hair segments of 1 cm or longer may not discriminate between intakes that occurred during a few days before the demise from exposure about 4 weeks back. Segmentation into 5 mm long segments seems to be relevant in opioid overdose cases to separate cases with a recent abstinence from cases with continuous use [3]. To accurately follow the distribution of drugs in the hair over time washing is important. The washing steps should be performed after the segmentation to avoid contamination along the hair shaft during the washing process.

2.2.2 Washing

Since hair is growing on the body surface there is a risk of contamination from outer sources. (This is described in Chapter 3 of this book.) Washing of the hair before analysis is therefore an important step. The washing has two main purposes: first, to remove hair care products, sweat, sebum, or surface material that may interfere with the analysis or that may reduce extraction recovery and second, to remove potential external contamination of drugs from the environment. However, there are probably as many washing procedures as there are laboratories and the debate about the correct way to perform and interpret washing results is still ongoing. The SoHT recommends that laboratories should have a procedure for washing hair samples prior to analysis, that the procedure should include washing steps with both organic solvent and aqueous solutions, and that the laboratory should investigate to what extent their wash procedure removes surface contamination [4].

The use of a first step with an organic solvent that does not swell the hair or penetrate the inner structure of hair will remove hair care products and surface contamination only whereas aqueous solutions also will extract drugs from the hair matrix. Today, it is accepted that drugs deposited into hair are from several sources including the blood stream, sweat, sebum, and other secretions. This means that a drug user or patient, in addition to incorporating drugs through the bloodstream also exposes the hair to an “internal external” contamination that can be very difficult to differentiate from...