Evaluating Endogenous GHB Variation in Hair with a Synthetic Hair Matrix (2024)

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Abstract

Introduction

Drug detection in hair has become more routine as it provides a longer detection window versus blood or urine. Hair analysis is especially useful if the drug has a short elimination half-life, the reported time of consumption has been delayed (e.g., drug-facilitated crimes) or the drug is naturally present in the body. Gamma-hydroxybutyric acid (GHB) is a unique drug, as it fits all three of these scenarios.

GHB is a central nervous system depressant that was originally developed in the 1960s as an anesthetic and then used later to treat narcolepsy and alcohol withdrawal. Due to its amnesic, sedative and euphoric properties, GHB also made its way into the club scene as a recreational drug of abuse and has been implicated in drug-facilitated sexual assaults. A low concentration of GHB is naturally present in humans due to the metabolism of gamma-aminobutyric acid to GHB (1). As a result, it can be difficult to detect a single dose of this drug using conventional methods because of the challenges of differentiating between endogenous and exogenous concentrations of GHB.

There have been several studies that have explored the natural and post-ingestion concentrations of GHB in human hair, many of which have been summarized in a review article by Van Elsué (2). Because of the difficulty in obtaining GHB-free hair, many authors have utilized other matrices in preparing calibrator samples to estimate GHB concentrations. Van Elsué et al. used aqueous-based calibrators and reported a concentration range for endogenous GHB in hair of 0.3–2.0ng/mg, with a mean of 1.1±0.6ng/mg. Both Kintz et al. (3) and Shi et al. (4) used synthetic melanin in suspension as their calibration matrix, but acknowledged that it may not be the ideal substitute for authentic hair. Their studies found endogenous GHB ranges of 0.5–12.0 and 0.28–4.91ng/mg, respectively. Schröck et al. used a laborious approach to generate calibration curves, in which the endogenous GHB in hair was extracted with water by ultrasonication for 2h and the hair fortified with different amounts of GHB (5). Endogenous GHB concentrations were determined to be between 0.1 and 1.3ng/mg for 27 non-GHB users using the GHB-extracted hair as the calibrator. In a study by Bertol et al. (6), hair specimens with GHB concentrations below the limit of detection (LOD) were used to prepare calibrators, but finding a reliable supply of hair samples with such low concentrations of endogenous GHB can be a challenge. Endogenous concentrations from 0 (<LOD) to 5.09ng/mg were reported in the Bertol (6) study using authentic hair as the calibrator. Blood and urine studies often use synthetic matrices (7–12) to combat the issue of endogenous compounds present at low concentrations, but until recently no such synthetic hair matrices were available. Along with the use of different calibration matrices, the wide range of endogenous GHB concentrations reported in human hair could also be attributed to the use of different analytical approaches, to include how the donor hair was prepared, used segmented versus whole, extracted (efficiency) and analyzed.

For this study, a quantitative validation was performed for a liquid–liquid extraction (LLE) and liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the quantitation of GHB in human head hair. A commercial synthetic hair product was used as a matrix to prepare the calibrator samples. This liquid matrix represented many of the same chemicals found in natural hair, but without detectable concentrations of GHB. Further, the synthetic matrix’s availability, batch-to-batch consistency and hom*ogeneity are advantageous compared to authentic hair samples. For these reasons, the synthetic hair matrix was used in preparing both calibrator and quality control (QC) samples. However, for this study, additional QC samples were prepared with authentic hair in order to compare those results to results obtained from QC samples prepared with the synthetic matrix.

An additional study (described later as the replicate study) helped establish if there was significant variation in endogenous GHB concentrations across multiple collection areas of the vertex posterior region of the human head. The potential for endogenous GHB concentration variation by sampling from multiple collection areas on the scalp has not been reported in the literature, even though it is generally acceptable to collect and combine smaller hair samples taken from multiple areas in the vertex posterior region (13).

Experimental

Materials

GHB, deuterated GHB (GHB-d₆) and 1,4-butanediol standards were purchased from Cerilliant Corporation (Round Rock, TX, USA). A synthetic hair matrix, SMx™ hair, was purchased from UTAK® (Valencia, CA, USA) as a frozen matrix and contained the following materials: L-alanine, L-arginine, L-cysteine, L-glycine, L-lysine, L-proline, L-serine, L-threonine, L-valine, cholesterol, linoleic acid, oleic acid, melanin and methyl-β-cyclodextrin. The concentration of the human hair mimic was 20mg hair/mL, and the pH was 1.28. For processing, the matrix was thawed at room temperature and then 0.5mL was aliquoted into 2.0mL Eppendorf™ Safe-Lock™ tubes (Fisher Scientific, Pittsburgh, PA, USA). All of the solution was aliquoted at the same time, and excess tubes with the synthetic hair were refrozen until needed. Ammonium acetate (99.999% trace metal basis), (R)-2-hydroxybutyric acid and 3-hydroxybutyric acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Methanol (sequencing grade peroxide free), ethyl acetate (Optima), acetonitrile (Optima LC/MS), dichloromethane (Optima), acetic acid (Optima LC/MS), formic acid (Optima LC/MS), sulfuric acid (certified ACS plus), sodium hydroxide (ACS reagent) and water (Optima LC/MS) were all purchased from Fisher Scientific.

Hair specimens

Different hair specimens were collected for each study. Bulk hair samples (non-segmented) were collected from 15 non-GHB users to test for interferences in the validation study. For the evaluation of endogenous GHB concentrations across multiple locations in the vertex posterior region of the head (replicate study), hair samples were collected from 11 non-GHB users (six females, five males). A separate set of samples was also collected from 11 other non-GHB users (six females, five males) to evaluate the variation within a single bundle of hair (repeat study). Lastly, for the comparison of the synthetic matrix and authentic hair as calibrators, a hair sample from two non-GHB users (one female, one male) was collected. The same authentic hair was used as QC samples in all studies. This hair was collected from one non-GHB user, ground in bulk and divided into 10-mg portions for analysis. The overall age range for the hair donors was 18–60years.

All hair samples were collected in accordance with the Federal Bureau of Investigation’s Institutional Review Board-approved protocols. Samples and donor information were kept anonymous, identification was through an alphanumeric code and signed consent forms were separately maintained. Sample donation was sought from volunteers that had not used GHB, even for prescribed purposes. Samples were collected following the guidelines recommended by the Society of Hair Testing (13). A pencil-sized thickness of hair was collected from the vertex posterior region of the volunteer’s head, as close to the scalp as possible, for the validation, repeat study and calibrator comparison study. Three smaller amounts of hair (one-third of a pencil’s thickness) were contemporarily collected for the replicate study.

During collection, instead of gathering the bundle of hair tightly into a cone, the hair strands were spread out evenly between the collector’s fingers and cut following the contour of the donor’s head. This technique allowed for a more even cut and minimized the amount of hair left behind on the scalp. The hair strands were aligned with the root end of the sample clearly identified and secured with yarn (or in aluminum foil for shorter bundles) and then stored in paper envelopes at room temperature until processing.

Collected hair samples were segmented into 1-cm sections with the exception of the QC authentic hair and hair interference samples for the validation. Segments were labeled numerically, starting at 1, for each centimeter section of hair cut from the proximal end of the hair and moving along the hair shaft towards the distal end. Each individual segment was intended to represent a discrete and consistent period of time for hair growth and GHB incorporation. For analysis, the target number of hair segments was six per donor. Having a consistent number of segments for both males and females was important for comparison of the data sets. Additional hair segments beyond 6cm were processed if available (up to 12cm), but those data will be included in a later study. Triplicate samples were obtained for most of the segments and the relative standard deviations (RSDs) of their quantitative results were calculated in order to evaluate the variation within and between different areas on the head.

Synthetic SMx™ hair matrix

Due to the presence of endogenous GHB in hair and the need for a large amount of material for the validation and GHB variation study, a synthetic matrix was desirable. The synthetic hair matrix was used to prepare calibrators, positive controls and negative controls. The positive controls were fortified at two concentrations (1.2 and 9.6ng/mg), whereas the negative controls remained unspiked. Authentic hair from non-GHB users was screened to identify candidate samples for preparation of QC samples. An authentic hair sample with an endogenous GHB concentration was identified and used to prepare additional positive and negative controls in each batch. Further, a method blank (extracted sample without hair) was processed with all batches of donor hair to ensure that there was no contamination during sample preparation. GHB-d₆ served as the method’s internal standard and was added at 8ng/mg to all samples in the study.

Multipoint calibration curves were generated using the synthetic hair matrix fortified with GHB at the following concentrations: 0.4, 0.8, 1.2, 2.0, 4.0, 9.6 and 12.0ng/mg. Separately, for comparison purposes, a calibration curve was generated using authentic hair fortified with the same concentrations as above. Endogenous GHB concentrations in two donor hair samples were then determined using the peak area ratios of GHB to GHB-d₆ and the regression equation from the multipoint calibration curves generated from the synthetic hair calibrators, as well as the calibrators prepared in the authentic hair matrix. This allowed for multiple comparisons to be made, including the linear equations of the two calibration curves, the background concentrations of GHB in the synthetic hair matrix versus the authentic hair and the measured endogenous GHB concentrations in the donor hair samples.

Extraction

The extraction method was modified from Jagerdeo et al. (14). In addition to the use of the synthetic hair matrix to prepare the calibrators, other modifications included the use of a single LLE instead of a dual extraction and a reduction in the sample size to 10mg. To evaluate the baseline GHB concentrations, hair collected from non-GHB users was segmented into 1-cm increments and numbered based on proximity to the scalp. The segments were added to Safe-Lock™ tubes and washed with 1.6-mL aliquots of methanol, methylene chloride and methanol again. After drying overnight, two stainless steel grinding beads, 3.2mm in diameter (BioSpec Products, Bartlesville, OK, USA), were added to each tube, and the hair was cryogenically ground into a fine powder using a Retsch® CryoMill (Newtown, PA, USA) with liquid nitrogen. The CryoMill was to set to automatically grind the hair for 6.5min at 25Hz after pre-cooling at 5Hz. A 10-mg sample of the pulverized hair was taken from each segment tube, internal standard added and the sample digested with 500μL of 1M sodium hydroxide for 45min at 75°C. After digestion, the samples were acidified with 750μL of 1N sulfuric acid and extracted in round-bottom glass culture test tubes via LLE with 6mL of ethyl acetate. The organic layer was transferred for evaporation to conical bottom centrifuge tubes. It should be noted that the Kimble® disposable round-bottom culture tubes (16×100 mm) and conical bottom centrifuge tubes (10mL) from Fisher Scientific were cleaned before use by rinsing three times with 18 MΩ water (Barnstead Purification System, Thermo Scientific, Waltham, MA) and heating overnight at 275°C. Extracts were evaporated to dryness on a Cerex® 48 Concentrator (SPEware, Baldwin Park, CA, USA) and reconstituted in 600μL of 50% Mobile phase A: 50% Mobile phase B. Mobile phase A consisted of 55% acetonitrile and 45% aqueous buffer (25mM ammonium acetate adjusted to pH5.49 with acetic acid), whereas Mobile phase B was 100% Optima grade water. The samples were filtered using water-rinsed SUN-SRi™ 0.2-μm polyvinylidene fluoride (PVDF) centrifuge filters purchased from Fisher Scientific and transferred to MicroSolv™ (Eatontown, NJ, USA) reduced surface activity (RSA) glass vials for analysis by LC-MS/MS. The rinsing of supplies was required to reduce background noise and interfering peaks that were observed in many consumables.

Instrumentation

As with the extraction method, the LC-MS/MS method parameters were adapted from Jagerdeo et al. (14). The filtered samples obtained after LLE were analyzed on a Spark Holland Symbiosis™ HPLC connected to an AB Sciex™ 5500 Qtrap triple quadrupole mass spectrometer. The ion source was operated in negative electrospray ionization mode. The analytical column used for separation was an Acclaim® Trinity™ P1 column (2.1mm×150mm, 3μm) with a guard column (2.1mm×10mm, 3 micron), which were both purchased from Fisher Scientific. The LC method used the same analysis gradient, but was modified from the original method by placing the equilibration time of 16min at the beginning of the run to ensure consistent retention times. The MS method was modified by monitoring the transition of m/z 103→101 instead of m/z 103→55. The transition to m/z 55 gave a lower response than the one to m/z 101 and was difficult to integrate at low GHB concentrations, which was problematic for determining endogenous GHB in hair. In total, three multiple reaction monitoring (MRM) transitions were monitored for GHB and two were monitored for GHB-d₆ (Table I). The declustering potential, collision energy, and collision exit potential were fine-tuned to improve detection of the analytes. Confirmation of the analytes by MS required the presence of one quantifier peak and at least one qualifier peak for GHB and GHB-d₆, the ion ratio of each qualifier peak to the quantifier peak to be within a tolerance of ±10% absolute and a signal-to-noise ratio (S:N) of at least 5:1 for detected peaks. The first MRM transitions for GHB and GHB-d₆ were used for quantifying endogenous GHB concentrations in hair. If a peak was detected at the correct retention time in the total ion chromatogram and met all MS identification criteria, but the calculated concentration was less than the LOD/LOQ, it was listed as “observed.”

Method validation

The SWGTOX Standard Practices for Method Validation in Forensic Toxicology were followed (15). Additionally, the related American Academy of Forensic Sciences Standards Board (ASB) document on method validation was reviewed to ensure all studies also fulfilled the practices of that document (16).

Accuracy, precision and calibration model were evaluated concurrently. Accuracy was determined using synthetic hair fortified at 1.2, 4.0 and 9.6ng/mg. Analyses were performed by at least two individuals, in triplicate, and over the course of 5days to assess variability. As a reference, an authentic hair sample was fortified at the same concentrations and analyzed in triplicate over five runs. Within-run precision and intermediate precision were assessed using the one-way analysis of variation (ANOVA) approach along with the accuracy data. The within-run precision provided a measure of repeatability for each day, whereas the intermediate precision gave the overall repeatability for the five testing days (combined data). The maximum acceptable accuracy and precision deviations were ±20% at each concentration. A new calibration curve of the synthetic hair was analyzed in conjunction with each sample set over the five validation days. Residual plots were evaluated to check for outliers and hom*oscedasticity (constant variance) across the entire calibration range.

The LOD was defined as the estimate of the lowest GHB concentration that could be reliably differentiated from the background noise of the unspiked synthetic hair. It was evaluated by analyzing triplicates of the synthetic hair fortified with 0.3, 0.4 and 0.5ng/mg GHB over 3days. The LOD was set at a concentration that met detection and identification criteria, which included the following: one quantifier and qualifier peak present for GHB, a S:N above 5 and ion ratios within ±10% absolute. The LOQ was defined as the estimate of the lowest GHB concentration that could be reliably differentiated and quantitated from the synthetic hair. It was also evaluated by analyzing triplicates of synthetic hair fortified with 0.3, 0.4 and 0.5ng/mg GHB over 3days. The LOQ was set at the concentration that met all detection and identification criteria required for the LOD while also maintaining acceptable accuracy and precision.

Carryover was evaluated by running an unspiked synthetic hair sample before and immediately after a high concentration (12 or 24ng/mg) hair sample. This test was repeated three times. In order to pass, no carryover must be observed in an unspiked synthetic hair sample immediately following an injection of the high calibrator. For the interference study, 1-ppm standards of 2-hydroxybutyric acid, 3-hydroxybutyric acid and 1,4-butanediol were analyzed by LC-MS/MS in order to verify that they did not interfere with detection of GHB or GHB-d₆. Fifteen authentic hair samples and one synthetic hair sample, without the addition of GHB-d₆, were also analyzed to evaluate the selectivity of the method. It is recognized that GHB may be detected in these matrices, as it is an endogenous compound in hair. However, the focus was on identifying other peaks that elute near GHB that could affect detection and integration. Stable-isotope internal standard interferences were assessed by spiking the synthetic hair with GHB-d₆ (8ng/mg) and then monitoring the GHB signal. In addition, the synthetic hair was spiked at a high concentration (12.0ng/mg) with GHB and analyzed without GHB-d₆ to evaluate if the unlabeled analyte appeared as isotopically labeled compound ions. Interfering signals from the synthetic hair or authentic hair (not including GHB), as well as from the deuterated internal standard, 2-hydroxybutyric acid, 3-hydroxybutyric acid and 1,4-butanediol, were evaluated. The last three compounds were monitored as interferences because they are GHB-related compounds.

Processed sample stability was investigated using synthetic hair fortified at 1.2 and 9.6ng/mg GHB and extracted via LLE. The goal was to determine the length of time a prepared sample could be maintained before it underwent unacceptable changes, preventing reliable detection or quantitation. Several samples at the same concentration were pooled and then divided into different vials for analysis. The first vials of each concentration were immediately analyzed in triplicate to establish the time zero response. All remaining vials were loaded into the autosampler compartment at ≤ 14°C and analyzed in triplicate at two, seven and eight working days later to assess changes in area ratios. Due to the endogenous nature of GHB in hair and difficulty in finding sources of blank authentic hair, ionization suppression/enhancement experiments were not completed. Instead, the study relied on GHB-d₆ to compensate for any suppression or enhancement that may occur because they co-elute (Supplementary Figure S1).

Repeat and replicate studies

The variation in endogenous GHB concentration across multiple samplings (spatially) on a human head was examined after method validation. Samples were collected sequentially from a total of three random areas within the vertex posterior region of the donor’s head (Figure 1A). There is generally less variability in the growth rate of hair in this region (17). Samples collected by this method are herein referred to as “replicate samples.” After collection, replicate samples were segmented, washed and ground separately prior to weighing aliquots and processing for analysis. The results from the replicate samples were compared to those obtained for hair taken from a single location within the vertex posterior region of the same donor’s head. These later samples are referred to as “repeat samples” in the remainder of this paper. For the repeat samples, the hair was segmented into 1-cm sections, washed and ground, but was then split into three separate 10-mg samples prior to extraction (Figure 1B). The repeat samples required the collection of at least 30mg/segment of hair from each donor, whereas the replicate samples required a minimum of 10mg/segment from each collection spot on the scalp.

Results and Discussion

Validation

Using the synthetic hair as a calibrator matrix, a quantitative validation was performed successfully for the extraction and analysis of endogenous GHB in human hair. To evaluate accuracy and precision, controls of the synthetic matrix were analyzed at 1.2, 4.0 and 9.6ng/mg in triplicate over 5days. The accuracy for the three concentrations was determined to be within ±12% for the three GHB transitions. The within-run precision and intermediate precision were determined to be within ±2% and ±12%, respectively. Both the accuracy and precision fell within the guidelines set forth by SWGTOX and ASB (15, 16). A linear model was obtained for the calibration from 0.4 to 12ng/mg. Predicted values in the residual plots were within ±0.04ng/mg for all 5days ,and no outliers or non-constant variances were observed in the residuals. There was constant variance in each of these plots, which indicated that the linear model adequately fit the data. Additionally, given that the average coefficients of determination for the three GHB MRM transitions were 0.9979, 0.9988 and 0.9976 respectively, a linear model was appropriate.

No carryover was observed in the unspiked synthetic hair samples following injections of 12 or 24ng/mg GHB. No interfering signals (except endogenous GHB) were observed in the 15 authentic hair extracts from non-GHB users tested in the selectivity study. In addition, GHB was not detected in the synthetic hair fortified with 8ng/mg GHB-d₆. Synthetic hair fortified with 1ppm of 2-hydroxybutyric acid, 3-hydroxybutyric acid or 1,4-butanediol did not show any interferences for GHB or GHB-d₆. The LOD and LOQ of the method were both estimated experimentally to be 0.4ng/mg. The GHB peaks at 0.3ng/mg had a S:N ratio less than 5. The determined LOD concentration is similar to what others have reported in the literature (2). Extracts were determined to be stable up to 8days while being stored at ≤ 14°C, since the average signal (ratio of peak area of analyte to internal standard) did not decrease under 80% or increase above 120% of the average time-zero response. On an absolute signal basis, the peak areas were within stability tolerance on Day 2, but not on Days 7 and 8.

Repeat and replicate studies

After validation, the repeat and replicate studies were completed to evaluate the variation in endogenous GHB concentrations within the vertex posterior region of each individual’s head. The results are summarized in Table II. Over all individual donors and their segments, a wide range of concentrations was observed, from 0.4 to 5.6ng/mg (Supplementary Tables S1 and S2). However, focusing on the individual segments within a donor (representing synchronous time periods), observed results are consistent across the same segment (average RSD < 5% for repeats and replicates). This trend holds true for both the repeat study and the replicate study for all donors. Since the use of the synthetic hair matrix for the calibration curve reliably gave similar results within repeat and replicate segments, it can be concluded that the synthetic matrix worked as a control for authentic hair samples. Measurable differences in endogenous GHB concentrations for individuals were observed along the length of hair for many donors.

In the replicate study involving three samples taken from the vertex posterior region of the individuals’ heads, the total number of segments with detectable concentrations was 185 out of 198. These concentrations ranged from 0.46 to 1.65ng/mg, which fall within concentration ranges reported in the literature for non-GHB users (2). Of the 11 donors, two males (Donors 2 and 11) did not have enough hair in the sixth segments to obtain triplicate 10-mg samples for testing. Males, in all of our studies, generally had shorter lengths and thinner hair than females. One additional male (Donor 5) was missing a replicate in the third segment due to loss during sample preparation. The RSD was still calculated for segments with only duplicates. If the estimated concentration was less than the method’s estimated LOD/LOQ, GHB’s presence was listed as “observed” and these values did not contribute to the averages or RSD values for reported GHB concentrations. This was the case for one of the female donors in the replicate study (Donor 10), in which two of her segments had calculated GHB concentrations below 0.4ng/mg. Considering all of the donors in the replicate study, the average and median variation across the segments were 3.0 and 2.9%, respectively. As these values are below 5%, it suggests that there is minimal variability in endogenous GHB concentrations across the vertex posterior region of the head. The maximum RSD for the 11 donors was 7.1%. This is not surprising considering the fact that samples were taken from three different areas on the head, and each bundle was independently segmented, washed, ground and weighed out separately before processing. Despite this higher variation for one donor, it does not contradict the conclusion that a single bundle of hair collected from the vertex region of the head is representative of an individual when determining endogenous GHB concentrations.

Similar to the replicate study, 2 of the 11 donors did not have enough hair in the sixth segment to obtain three 10-mg samples for the comparison of multiple samples from the same collection area (i.e., repeat study). One male (Donor 13) did not have enough hair in the sixth segment for any analyses, while another male (Donor 15) only had enough hair within the segment to obtain two 10-mg samples. Additionally, one of the female donors had non-detects (did not meet the qualitative detection criteria) for all of her segments and were therefore not reported (see Supplementary Table S2). As a result, the total number of segments with detectable GHB concentrations was 176 out of 198. The average and median variation for those segments with concentrations were 1.8 and 1.5%, respectively. The average RSD is much lower in the repeat study because the hair came from a single location on the head and the repeats were obtained after the bundle was segmented, washed and ground. This indicates that the samples are hom*ogenous after grinding and goes to the reproducibility of the method. The average endogenous GHB concentration measured for the repeat study was 1.08ng/mg, but the range of concentrations extended from 0.41 to 5.60ng/mg. The highest endogenous GHB concentration was observed in the first segment of a donor, which others have also observed (3, 6, 18–19).

Due to the similarity of results and low RSD values seen in the repeat and replicate studies, it was determined that the variation across the scalp was negligible and that collecting a sample from a single location was sufficient for the rest of the study. Furthermore, the amount of hair collected from that single location could also be reduced since the variation was low for repeat samples taken from the same area. This allowed a 10-mg/segment sample from a single location on the scalp to be used for each donor. One advantage of collecting a smaller mass of hair for processing is that it is often easier to conceal the site of collection once the hair is cut, which increases the willingness of donors to provide samples.

Comparison of synthetic matrix and authentic hair calibrators

All of the results from the validation, repeat study and replicate study were generated using the synthetic hair as the calibration matrix. However, for comparison purposes, the measured endogenous GHB concentrations for two donors were also evaluated using calibration curves prepared with authentic hair. The linear equations of the two calibration curves (Supplementary Figure S2) had the same slope, but different y-intercepts. The y-intercept of the authentic hair calibration equation was about 6× greater than the intercept for the synthetic hair, which indicated that the authentic hair had a detectable concentration of background GHB and could lead to an underestimation of GHB in donor samples. This underestimation was confirmed when the authentic hair matrix calibration curve was applied to two donor hair samples. In the first donor sample, all of the male’s first six segments (6×3 repeats) returned negative concentration values (Supplementary Table S3) when calculated with the authentic hair’s calibration curve. Using the synthetic hair as the calibration matrix, the first six segments of the same male donor provided an average endogenous GHB concentration and average RSD of 0.63ng/mg and 2.5%, respectively. The second sample, a female donor, provided positive concentrations for all of the repeats in the first six segments, but approximately half of them fell below the method’s estimated LOD/LOQ when using the authentic hair as the calibration matrix. The average GHB concentration for her hair segments that had measureable endogenous GHB above the estimated LOD/LOQ was 1.01ng/mg with RSD values > 4%. Using the synthetic hair as the calibration matrix, these same segments were determined to have an average GHB concentration and average RSD of 1.74ng/mg and 2.6%, respectively.

To further demonstrate the suitability of the synthetic hair as a calibration matrix and explain the underestimation of endogenous GHB in donor samples with authentic hair, the implied GHB concentrations in both matrices were compared by two different methods. First, the y-intercepts were set to zero (no response for GHB) and the background concentrations (x) calculated at the intercepts using the linear fit equations of the calibration curves. Using the respective calibration equations, the calculated GHB concentrations were 0.20ng/mg for the synthetic hair (below the method’s LOD) and 1.19ng/mg for the authentic hair. The second method used for evaluating the background concentrations in the unspiked authentic hair (negative control) and lowest synthetic hair calibrator (0.4ng/mg) involved calculation of the concentration of each based on the calibration curves generated from their opposite matrices. The authentic hair negative control’s GHB concentration was calculated using the synthetic hair calibration curve and estimated to be 1.23ng/mg. This concentration is within 3.5% of the concentration calculated by the first method. Conversely, a negative concentration was calculated for the synthetic hair lowest calibrator (−0.61ng/mg) using the authentic hair calibration equation. This discrepancy, along with the donor hair results above using the human hair calibration matrix, confirms that the authentic matrix is likely to underestimate GHB concentrations. As a result, it is recommended that caution be taken when using authentic hair as a calibration matrix because it could have a significant impact on the measured concentrations of GHB in hair samples, which is particularly important for criminal cases involving exogenous administration.

There are other approaches for quantitating endogenous substances, but they can be matrix- and method-specific or consume a significant amount of time and resources. One recently published approach by Desharnais et al. (20) involves the use of standard addition to estimate the endogenous concentration of beta-hydroxybutyric acid (BHB) in whole blood, which is added to the nominal concentrations of the calibration standards to correct for the endogenous presence of BHB in the matrix. Their automatic correction approach is not ideal for endogenous GHB in hair analysis because the target concentrations are often low (2) and may fall outside the range of the corrected calibration curve unless the authentic hair serving as the calibration matrix has a very low endogenous concentration. Many of the observed concentrations fell below the calibration range (1.25–13.25ng/mg) when the correction approach was used to estimate GHB in a donor sample (Supplementary Table S3). It was important in this work to use a blank matrix in order to measure low concentrations of GHB, which is in contrast to the BHB study (20) where the relevant measurement was critical at high concentrations. The stability and quantity of the authentic hair calibration matrix is also a concern; these are less of an issue with the synthetic hair matrix because it is commercially available and stable for 2years.

Conclusion

The research presented here documents the first use of a synthetic SMx™ hair matrix for the determination of endogenous GHB concentrations, utilized to overcome the challenge of GHB’s natural presence in human head hair. Using this hair mimic for controls, the extraction and analysis method was validated after modifications to a previously published method. The validation results suggested that the synthetic hair matrix was a suitable substitute for human head hair for preparing calibrators and controls due to its reproducible nature, stability and non-detectable concentrations of GHB. In addition, the synthetic hair had several benefits over other matrices such as water, melanin and decontaminated hair. It was more appropriate to use the synthetic matrix instead of water or melanin because it better represented whole hair, as it contained 14 chemicals commonly found in human hair (21). The synthetic hair was also easy to prepare in comparison to decontaminated hair, as it was commercially available and only required a few minutes to thaw when needed for analysis. Depending on the native GHB concentration in the authentic hair and the effectiveness of the decontamination procedure, the result will likely mean an underestimation of GHB concentrations when using authentic hair as the calibration matrix. This was observed in the endogenous GHB concentrations measured in two donor hair samples. Therefore, one must be cautious when reporting GHB concentrations in hair samples if using authentic hair as the calibrator and should consider the synthetic hair matrix as a better alternative.

The results of the repeat and replicate studies demonstrated that the modified method was successful for determining endogenous GHB concentrations in authentic hair samples. Minimal variation in GHB concentration was observed in human head hair when comparing samples taken from multiple locations across the vertex posterior region. This supports the recommendations from the Society of Hair Testing that smaller hair samples can be collected from multiple locations in the vertex posterior region and combined for testing, which is beneficial for cases involving children or individuals with baldness or thinning hair. The study also showed that the variation within samples from a single collection area was minimal, if properly hom*ogenized. It was determined that a 10-mg sample could be collected from a single area to represent endogenous GHB concentrations in human head hair. This is important for other studies being conducted to evaluate the variation in GHB concentrations along the length of an individual’s hair.

Disclaimer

This publication is number 19-13 of the Laboratory Division of the Federal Bureau of Investigation (FBI). Names of commercial manufacturers are provided for identification purposes only, and inclusion does not imply endorsem*nt by the FBI. The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the FBI or US Government. One or more of the authors is a US Government employee and prepared this work as part of that person’s official duties. Title 17 United States Code (U.S.C.) Section 105 provides that “Copyright protection under this title is not available for any work of the United States Government.” Title 17 U.S.C. Section 101 defines a United States Government work as a “work prepared by an employee of the United States Government as part of that person’s official duties.”

Acknowledgements

This research was supported in part by an appointment to the Visiting Scientist Program at the Federal Bureau of Investigation Laboratory Division, administered by the Oak Ridge Institute for Science and Education, through an interagency agreement between the US Department of Energy and the FBI. Parts of this work have been presented at the American Academy of Forensic Sciences 70^th Annual Scientific Meeting in Seattle, WA, on 23 February 2018 and as part of a webinar series on Novel Forensic Chemistry Research for early-career scientists on 19 September 2018.

References

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