Abstract

The abuse of gamma-hydroxybutyric acid (GHB) and its suspicion in cases of suspected drug-facilitated sexual assault is of keen interest to forensic toxicology laboratories. This paper reports an extraction, separation and detection procedure for GHB in hair utilizing a combination of liquid–liquid extraction and solid-phase extraction using ethyl acetate and Oasis Max® cartridge, respectively, after the hair sample was digested. Analysis was by LC–MS-MS using a gradient separation on an Acclaim® TrinityTM P1 column performing three multiple-reaction monitoring (MRM) transitions each for GHB and its internal standard. The procedure was validated over a range from 0.4 to 50 ng/mg with estimated limit of detection (LOD) of 0.33 and an administratively set limit of quantitation (LOQ) of 1.2 ng/mg. Twenty hair specimens collected from individuals with no known exposure to GHB were analyzed for matrix interferences and to establish initial background levels of GHB. A wide range of endogenous GHB levels were observed in these samples (from less than the LOQ to 4.4 ng/mg). The results suggest the need for additional studies to better establish the full range of endogenous GHB levels in hair and that extreme caution is required in interpreting GHB findings in hair samples.

Introduction

Gamma-hydroxybutyric acid (GHB) is a short-chained fatty acid that occurs naturally in mammalian species, and is thought to have neurotransmitter properties. Sources of GHB have been around since the early 1960s. It was initially used as a general anesthetic and later for the treatment of narcolepsy, as well as alcohol and opiate withdrawal dependence.

Although the benefits of GHB were identified over 50 years ago, it has only been within the last two decades that GHB was identified as a drug of abuse and began appearing in cases of suspected drug-facilitated sexual assault. This abuse of GHB led to the implementation of strict regulatory controls in several countries (1–6). The chemical precursors, gamma butyrolactone (GBL) and 1,4-butanediol (BD) which are rapidly metabolized to GHB are also abused (7, 8).

Toxicological analysis of GHB is commonly performed in traditional biological specimens (e.g., urine or blood/plasma), by headspace gas chromatography/flame ionization (9), gas chromatography/mass spectrometry (GC–MS) (10–15) or liquid chromatography/mass spectrometry (LC–MS) (16–21).

A unique challenge is that a dose of GHB can be totally excreted from the body within a few hours after ingestion, which highlights the importance of quickly obtaining a blood or urine specimen. Interpretation of GHB findings can also be a challenge. For example, a urinary cutoff concentration of 10 µg/mL is typically used to differentiate endogenous levels from those associated with exogenous exposure to GHB (4, 22–25).

With recent improvements in LC–MS sensitivity and in separation chemistry, the analysis of low concentrations of GHB in hair specimens has become possible. Hair has several advantages over traditional biological specimens because: (i) it provides a longer window of detection after exposure; (ii) it can be stored at room temperature; (iii) it can be easily transported; (iv) it is not easily adulterated; (v) it is obtained non-invasively and (vi) hair analysis may complement the results generated from urine/blood analysis (26–28). Unfortunately, there is currently no generally accepted cut-off for GHB in hair to assist in interpreting findings and differentiating endogenous from exogenous GHB. Some have recommended using an individual's hair as its own control to establish a baseline for endogenous GHB in the regions outside of a specific time period of investigation (12, 29). Goulle et al. (12) tested hair from 61 individuals with no known exposure to GHB and found that the samples all contained less than 2 ng/mg GHB. However, in segmental analysis of 12 samples, they reported concentrations as high as 8.4 ng/mg. Similarly, Kintz et al. (2) measured the endogenous GHB content in the hair of 24 individuals and identified a range of 0.5–12 ng/mg of GHB in the hair of these individuals.

Hair analysis involves several critical steps, including sample collection, washing the hair to remove external contamination, extraction of the drug from the hair matrix and selection of an instrumental detection procedure. The procedure detailed in this paper combines multiple techniques to efficiently extract GHB from hair. Further, the method presented here allows for chromatographic separation and LC–MS-MS detection and identification of GHB in hair. Digestion of the hair leads to a very complex solution, and, as a result, a combination of both liquid–liquid (LLE) and solid-phase extraction (SPE) was required to reduce matrix interferences with GHB. Most of the methods published in the literature use a reversed-phase column for the separation of GHB from the other compounds present in hair matrix. Reversed-phase liquid chromatography is commonly used for a wide range of compounds, but fails to significantly retain hydrophilic compounds, such as GHB. Due to the polarity of GHB, retention on a reversed-phase column is not sufficient to separate GHB from most of the matrix interferences. However, ion chromatography (IC) has been utilized with considerable success for the separation of ionic compounds (30). In the present method, a mixed-mode chromatographic approach that combines reversed-phase, hydrophilic interaction liquid chromatography (HILIC) and an ion exchange retention mechanism was successfully used. The advantage of using mixed-mode columns is that selectivity can be optimized by adjusting the mobile phase pH, organic solvent gradient and ionic strength (31–33).

Experimental

Materials

GHB, GHB-d6, 1,4-butanediol and gamma-butyrolactone, as well as standards used for the interference studies were purchased from Cerilliant Corporation (Round Rock, TX, USA). (R)-2-hydroxybutyric, 3-hydroxybutyric acid, ammonium acetate and formic acid were purchased from Sigma-Aldrich (St Louis, MO, USA). Methanol, acetonitrile, dichloromethane, acetic acid, sulfuric acid, sodium hydroxide, ammonium hydroxide and Optima grade water were purchased from Thermo-Fisher Scientific (Pittsburgh, PA,USA). The analytical column used for the method was an Acclaim® Trinity™ P1, 2.1 mm × 150 mm, 3 µm and was purchased from Dionex (Bannockburn, IL). SPE was performed using Oasis Max 6 cc (500 mg) cartridges purchased from Waters (Milford, MA, USA). All samples were filtered before transferring them to high-performance liquid chromatography (HPLC) vials using Costar Spin-X micro 0.45 µm centrifuge filters purchased from Thermo-Fisher Scientific, Pittsburgh, PA, USA.

Blank hair samples were donated anonymously by FBI employees and their family members who were not taking GHB clinically. The FBI's Institutional Review Board (IRB) approved the anonymous collection of these samples.

Sample preparation

Decontamination, grinding and digestion

Hair samples were initially washed in bulk with methanol (∼40 mL) by vortexing for 1 min, followed by a methylene chloride wash (∼40 mL) for 1 min and finally by a methanol wash (∼40 mL) for 1 min. For routine analysis of samples, this would approximately equate to washing 50 mg of hair with 2 mL of solvent. The hair was allowed to air dry at room temperature overnight to evaporate any residual solvent that remained. Next, the hair samples were cryogenically ground using a SPEX SamplePrep 6,870 Freezer/Mill under liquid nitrogen to form a homogenous powder (Metuchen, NJ, USA). Initially, it was important to screen all the powdered hair samples to determine the endogenous GHB levels present. For the blank matrix used for the standard curve and QC samples, it was critical to obtain a large quantity of hair from individual sources and keep them separated in order to screen for endogenous GHB levels using the current procedure. The samples with the lowest amounts of GHB were then used as the ‘blank’ matrix for calibrator and control preparation in the rest of the study. Hair powder (25 mg) was digested using 500 µL of 1 M sodium hydroxide at 90°C for 30 min in screw-capped extraction tubes. The samples were allowed to cool at room temperature before adding 750 µL of 1 N sulfuric acid and vortexing for 30 s.

Liquid–liquid extraction

The extraction was performed by vortexing with 6 mL of ethyl acetate for 2 min, followed by centrifugation at 3,500 rpm for 10 min. The organic supernatant was then transferred to a 13 × 100 mm culture tubes and dried down under nitrogen using a Cerex® Sample Concentrator (Model System 48) at a temperature of 60°C. Samples were reconstituted with 50 µL of methanol and vortexed for 60 s, followed by the addition of 3.5 mL of water containing 0.03% ammonium hydroxide and another 30 s of vortexing. The pH was verified as >7 with pH paper on a single sample as a check. For the entire study, no adjustment in the pH was made to the samples after the addition of the ammonium hydroxide solution.

Solid-phase extraction

SPE was performed on an Agilent VacElut manifold (Santa Clara, CA, USA). Waters Oasis® Max cartridges were conditioned with 3 mL of methanol, followed by a wash with 3 mL of water. The samples were loaded onto the cartridges and the solution was allowed to flow by gravity. Cartridges were then washed with 3 mL of water containing 0.03% ammonium hydroxide (prepared fresh daily), followed by a wash with 3 mL of methanol. The cartridges were then allowed to dry for 15 min under vacuum (∼15 in. Hg). The analytes were eluted with 3 mL of 5% acetic acid in methanol. The final extracts were dried under nitrogen at 60°C and reconstituted with 600 µL of mobile phase A: mobile phase B (50:50), and vortexed for 1 min [Table I]. Samples were filtered using 0.45 µm centrifuge filters for 2.5 min at 10,000 rpm as a cleanup procedure. The filtered solutions were transferred to HPLC vials.

Table I

Chromatographic parameters

Time (Min) Flow (mL/min) Pump AaPump Bb
0.01 0.3 50 50 
2.00 0.3 50 50 
2.10 0.3 100 
16.00 0.3 100 
16.01 0.3 50 50 
30.00 0.3 50 50 
Time (Min) Flow (mL/min) Pump AaPump Bb
0.01 0.3 50 50 
2.00 0.3 50 50 
2.10 0.3 100 
16.00 0.3 100 
16.01 0.3 50 50 
30.00 0.3 50 50 

aMixture of 55% (100% acetonitrile)/45% (25 mM ammonium acetate, pH 5.49 with acetic acid).

b100% water.

Wash solvents for needle, loop and valves

i. Line 1: 50% mobile phase A: 50% mobile phase B.

ii. Line 2: 100% acetonitrile/0.1% formic acid.

iii. Line 3: 100% acetonitrile.

Procedure

Nine-point calibration curves (0.4, 0.8, 1.2, 2, 8, 16, 32, 40, 50 ng/mg) were prepared by spiking GHB solutions (1 µg/mL and 10 µg/mL in methanol) onto 25 mg of blank hair powder. Independent solutions of 1 µg/mL and 10 µg/mL were prepared daily from a quality control (QC) stock of 1,000 µg/mL. From the intermediate QC solutions, QC samples at 1.2, 16 and 32 ng/mg were prepared in triplicate by spiking the appropriate amount of the intermediate QC solution on to 25 mg of blank hair. Samples were spiked with 40 µL of 10 µg/mL GHB-d6 in methanol (16 ng/mg final concentration) followed by digestion. Blank hair samples were analyzed with and without an internal standard added for each run. The samples were digested using the procedure described under the Sample Preparation section.

Instrumentation

High-performance liquid chromatography

A Spark Holland Symbiosis (Emmen, The Netherlands) was operated in the HPLC mode. The system is comprised of a refrigerated storage compartment (maintained at 14°C), an autosampler and two HPLC pumps with high pressure gradient mixing. The analytical column used for the method was a Dionex Acclaim® Trinity™ P1, 2.1 mm × 150 mm, 3 µm. The gradient started at 50% mobile phase A: 50% mobile phase B, changing to 100% mobile phase A at 2.1 min, then back to the initial conditions at 16.01 min with a total runtime of 30 min (Table I).

Mass spectrometry

The mass spectrometer was an AB Sciex 5,000 triple quadrupole with a Turboionspray® (electrospray) source operated in the negative mode. GHB and GHB-d6 ionized to produce [M-H]+ ions at m/z 103 and 109, respectively. Three multiple reaction monitoring (MRM) transitions were monitored for GHB and its internal standard. Although only one transition was required for the internal standard, three were monitored in case a matrix interference was encountered with one of the internal standard transitions. Figure 1 shows the transitions used for GHB and its internal standard for the quantitative validation. Quantitations were carried out using the first transition of each analyte as listed in Table II. For confirmation of the analyte identity, the percent ratio of each additional transition to the quantitation transition was calculated and monitored. An acceptable tolerance of 20%, absolute, was used for the quantifier ion. The ion ratios for the QC samples used for the validation were calculated and determined to be within the tolerance set for the study at all concentrations analyzed. The observed MRM transitions and ion source parameters are listed in Table II. The data were collected using Analyst software version 1.5 and the data processing was performed using AB Sciex MultiQuant software version 2.1.

Table II

Mass spectrometer parameters

Scan mode Turbo spray
 
Polarity
 
Negative 
Resolution Unit
 
Scan type
 
MRMd 
Curtain gas Nitrogen (35) Ionspray voltage
 
−4,500 
Source temperature 675°C Nebulizer gas
 
Nitrogen (50) 
Dwell time 75 ms
 
Entrance potential
 
−10 V 
Collision gas 8 (Nitrogen) Turbo gas
 
Nitrogen (50) 
Analyte Q1 Mass (m/zQ3 Mass (m/zDP (v)a CE (v)b CXP (v)c 
GHB-1 (MRM-1) 103 57 −60 −18 −21 
GHB-2 (MRM-2) 103 85 −60 −14 −9 
GHB-3 (MRM-3) 103 55 −60 −28 −21 
GHB-1-d6 (MRM-1) 109 61 −40 −20 −9 
GHB-2-d6 (MRM-2) 109 90 −40 −14 −5 
GHB-3-d6 (MRM-3) 109 65 −40 −14 −7 
Scan mode Turbo spray
 
Polarity
 
Negative 
Resolution Unit
 
Scan type
 
MRMd 
Curtain gas Nitrogen (35) Ionspray voltage
 
−4,500 
Source temperature 675°C Nebulizer gas
 
Nitrogen (50) 
Dwell time 75 ms
 
Entrance potential
 
−10 V 
Collision gas 8 (Nitrogen) Turbo gas
 
Nitrogen (50) 
Analyte Q1 Mass (m/zQ3 Mass (m/zDP (v)a CE (v)b CXP (v)c 
GHB-1 (MRM-1) 103 57 −60 −18 −21 
GHB-2 (MRM-2) 103 85 −60 −14 −9 
GHB-3 (MRM-3) 103 55 −60 −28 −21 
GHB-1-d6 (MRM-1) 109 61 −40 −20 −9 
GHB-2-d6 (MRM-2) 109 90 −40 −14 −5 
GHB-3-d6 (MRM-3) 109 65 −40 −14 −7 

aDeclustering potential.

bCollision energy.

cCollision exit potential.

dMultiple reaction monitoring.

Figure 1.

MRM transitions observed (highlighted) from a 2 ng/mg GHB extracted from hair were m/z 103→ 56 (A), m/z 103→ 85 (B) and m/z 103→ 55 (C), and 16 ng/mg GHB-d6 transition observed at m/z 109→ 61 (D).

Figure 1.

MRM transitions observed (highlighted) from a 2 ng/mg GHB extracted from hair were m/z 103→ 56 (A), m/z 103→ 85 (B) and m/z 103→ 55 (C), and 16 ng/mg GHB-d6 transition observed at m/z 109→ 61 (D).

Results and discussion

In developing this procedure, several challenges had to be overcome. These included the selection of the matrix lots for the standard curve, the extraction procedure and the chromatographic separation. For validation, large amounts of hair from multiple donors had to be screened for endogenous levels of GHB. The objective was to use one pool of hair from a single source that had a low level of GHB for the entire study, so as to minimize the variability that endogenous GHB in the blank hair contributes to the standard curve and QC samples. The pools of hair that showed levels of endogenous levels of GHB less than the lowest point of the standard curve were thus used as the matrix for the study. The initial objective was to keep the extraction procedure simple, and the focus was therefore centered on an LLE after digestion of the sample. However, this extraction procedure alone was inefficient in removing the interferences. As a result, a SPE was implemented after the LLE and this combined extraction proved satisfactory for successfully validating the procedure.

As previously stated, most of the analytical columns used for the LC/MS analysis of GHB are reversed-phase. GHB's polarity and resulting lack of retention on nonpolar phases results in poor separation from matrix interferences. Therefore, a mixed-mode column was selected for this procedure because it allowed for better retention of GHB permitting it to be separated from these interferences. Furthermore, the chromatography for the peak of interest had to have reasonable peak shape, peak width and resolution. Several mobile phase combinations using ammonium acetate and ammonium formate buffer with acetonitrile were evaluated during the method development phase, but to keep the mobile phase simple the combination listed in Table I was used. Ammonium acetate was chosen over ammonium formate because of its buffering range. Multiple combinations of buffer/acetonitrile were evaluated for optimal conditions. For the mass spectrometer ionization, both atmospheric pressure chemical ionization (APCI) and electrospray spray ionization (ESI) were thoroughly evaluated with ESI in negative mode providing the best response with minimal interferences. The data generated in this paper has some similarity to the results produced by Stout et al. (17). However, there are significant difference in the choice of ionization technique, extraction and the chromatographic procedures.

In validating this procedure, the Scientific Working Group for Forensic Toxicology (SWGTOX) Standard Practices for Method Validation was used as a guide (34). The method was validated over a range of 0.4–50 ng/mg, and an average correlation of determination was >0.99 for all transitions (Table III). Residual plots were evaluated to confirm the model used was appropriate. This concentration range was chosen based on results previously published in the literature (12, 29). The standard curves were plots of the ratio of the analyte/internal standard response (peak area) as a function of the analyte concentration. The data were fitted to a linear least-squares regression curve with a weighting index of 1/x2. The limit of detection (LOD) was determined statistically to be 0.33 ng/mg using the equation: 3.3 × standard deviation of the mean of the y intercept/slope for five calibration curves generated on 5 days. Inaccuracy was observed at the low end of the calibration curve, in part, due to endogenous GHB in the negative control hair samples. If the negative control hair samples used to prepare the calibrator and control samples could be consistently obtained with no detectable GHB, a lower LOQ might be reliably achieved. The LOQ was administratively set to 1.2 ng/mg, as it was observed that this level could be consistently quantitated with accuracy from batch to batch. The imprecision was determined using a simple analysis of variance (ANOVA: single factor). For the experiments to obtain the data for the bias and imprecision, controls at 1.2, 16 and 32 ng/mL were analyzed at three levels in triplicate over 5 days. The results demonstrated that the bias was <7%, while the imprecision was determined to be <10%. For the interference study, blank hair and hair samples were spiked in the range from 25 to 250 ng/mg with the compounds listed in Table IV, extracted and analyzed. Except fentanyl that was spiked at 25 ng/mg, the rest of the analytes were spiked in the range from 200 to 250 ng/mL. No interferences were observed in any of the analytes or internal standard channels.

Table III

Standard curve stability evaluation

Analyte (MRM) Transitions Average correlation of determination Average slope Average intercept 
GHB-1 (103/57) >0.998 0.05675 ± 0.00159 0.2249 ± 0.0057 
GHB-2 (103/85) >0.998 0.05518 ± 0.07764 0.2151 ± 0.3059 
GHB-3 (103/55) >0.995 0.00305 ± 0.00434 0.0118 ± 0.0171 
Analyte (MRM) Transitions Average correlation of determination Average slope Average intercept 
GHB-1 (103/57) >0.998 0.05675 ± 0.00159 0.2249 ± 0.0057 
GHB-2 (103/85) >0.998 0.05518 ± 0.07764 0.2151 ± 0.3059 
GHB-3 (103/55) >0.995 0.00305 ± 0.00434 0.0118 ± 0.0171 

n = 5.

Average line equation y = average slope (±3SD) X + average intercept (±3SD). Data were fit to a linear least-squares regression curve with a weighing index of 1/x2.

Table IV

Compounds used in the interference study

Solution 1 (R)-2-hydroxybutyric 
Solution 2 3-Hydroxybutyric acid 
Solution 3 1,4-Butanediol 
Solution 4 Gamma-aminobutyric acid 
Solution 5 Gamma-valerolactone 
Solution 6 Ephedrine, amphetamine, methamphetamine, phentermine, MDA, MDMA, MDEA 
Solution 7 Codeine, 6-acetylmorphine, morphine, methadone, tramadol, meperidine, oxycodone, oxymorphone, hydrocodone, hydromorphone, fentanyl 
Solution 8 Alprazolam, clonazepam, diazepam, flunitrazepam, lorazepam, nitrazepam, oxazepam, temazepam 
Solution 9 Tetrahydrocannabinol, hydroxy-tetrahydrocannabinol, carboxy-tetrahydrocannabinol, cannabinol, cannabidol 
Solution 10 Cocaine, benzoylecgonine, cocaethylene and ecgonine methyl ester 
Solution 1 (R)-2-hydroxybutyric 
Solution 2 3-Hydroxybutyric acid 
Solution 3 1,4-Butanediol 
Solution 4 Gamma-aminobutyric acid 
Solution 5 Gamma-valerolactone 
Solution 6 Ephedrine, amphetamine, methamphetamine, phentermine, MDA, MDMA, MDEA 
Solution 7 Codeine, 6-acetylmorphine, morphine, methadone, tramadol, meperidine, oxycodone, oxymorphone, hydrocodone, hydromorphone, fentanyl 
Solution 8 Alprazolam, clonazepam, diazepam, flunitrazepam, lorazepam, nitrazepam, oxazepam, temazepam 
Solution 9 Tetrahydrocannabinol, hydroxy-tetrahydrocannabinol, carboxy-tetrahydrocannabinol, cannabinol, cannabidol 
Solution 10 Cocaine, benzoylecgonine, cocaethylene and ecgonine methyl ester 

Ionization suppression/enhancement was investigated using an approach where two different sets of samples are prepared and the analyte peak areas are compared to evaluate the ionization suppression/enhancement effects. The first set consisted of the GHB standards at an equivalent concentration of 1.2 and 32 ng/mg. The second set was made up of a minimum of five samples extracted from different matrix sources fortified with GHB after extraction at both the low and high control concentrations. The average areas of each set were used to estimate the suppression/enhancement effect at each level using the equation: [(average area of set 2)/(average area set 1)] × 100. The results showed that suppression was present at each level at an average of 20%. For ionization suppression/enhancement evaluation, it is recommended that at least 10 different sources of matrix be evaluated (34). However, due to the difficulty in obtaining blank hair with very low endogenous GHB, five different matrix sources were used for this evaluation.

Samples from 20 individuals with no known exposure to GHB were analyzed. As expected, endogenous GHB was often detected. Levels in these samples ranged from less than the LOQ to as high as 4.4 ng/mg (Table V). For all other samples, a peak for GHB was observed that was lower than the statistically calculated LOD of the method; these samples are labeled as ‘observed’ in Table V. No other endogenous compounds were detected that demonstrated interferences into the area of GHB elution. At present, there is no consensus for a GHB reporting limit in hair. However, it is noted that the range of endogenous concentrations observed in our study overlaps with the GHB concentrations reported for hair samples obtained from individuals suspected of having been exposed to exogenous sources of GHB (12, 28).

Table V

Hair samples from individuals with no known exposure to GHB

Sample ID Concentration Detected (ng/mg) 
Sample 1 Positive, <1.2b 
Sample 2 1.4 
Sample 3 2.4 
Sample 4 Observedc 
Sample 5 4.4a 
Sample 6 1.2 
Sample 7 Observedc 
Sample 8 Observedc 
Sample 9 2.4 
Sample 10 Observedc 
Sample 11 Observedc 
Sample 12 Observedc 
Sample 13 Positive, <1.2b 
Sample 14 Observedc 
Sample 15 Observedc 
Sample 5 4.4a 
Sample 16 Observedc 
Sample 17 Observedc 
Sample 18 Observedc 
Sample 19 Observedc 
Sample 20 Observedc 
Sample ID Concentration Detected (ng/mg) 
Sample 1 Positive, <1.2b 
Sample 2 1.4 
Sample 3 2.4 
Sample 4 Observedc 
Sample 5 4.4a 
Sample 6 1.2 
Sample 7 Observedc 
Sample 8 Observedc 
Sample 9 2.4 
Sample 10 Observedc 
Sample 11 Observedc 
Sample 12 Observedc 
Sample 13 Positive, <1.2b 
Sample 14 Observedc 
Sample 15 Observedc 
Sample 5 4.4a 
Sample 16 Observedc 
Sample 17 Observedc 
Sample 18 Observedc 
Sample 19 Observedc 
Sample 20 Observedc 

aThis sample was reanalyzed on a second day to verify the concentration.

bThe values were greater than the LOD, but less than LOQ.

cA peak for GHB was observed that was lower than the statistically calculated LOD for the method.

Processed sample stability was performed on Days 1, 2 and 8. Complete standard curves, including QC samples were run on each of the days. Day 2 and Day 8 samples were stored refrigerated at 14°C, and when analyzed, the peak area for GHB and its internal standard were compared to the Day 1 samples. The results show that the average signal changes from Day 1 to Day 8 for GHB and GHB-d6 were 3 and 4%, respectively.

Conclusion

The purpose of this method was to develop a robust extraction, coupled with adequate separation and detection of GHB in hair with the intention of using it in a forensic toxicology setting. This paper describes an analytical procedure that can be used for a broader survey of hair specimens from individuals with no known exposure to exogenous GHB in order to determine if a GHB decision point can be established to clearly differentiate endogenous from exogenous concentrations in hair. Given our findings of endogenous GHB concentrations that overlap those declared as exogenous concentrations in other studies, further research to clarify these issues is encouraged. The additional studies should encompass segmental analysis of hair from individuals exposed to GHB and a control group to determine if individual changes in hair GHB concentrations can be ascribed to GHB ingestion and, if so, whether the process of using each individual's hair as their own negative control is reliable. The accuracy across the calibration range, especially at the low end, should also be further evaluated.

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Author notes

This is publication 14–18 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 endorsement 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 the U.S. Government. This work was prepared as part of their official duties. Title 17 U.S.C. 105 provides that ‘Copyright protection under this title is not available for any work of the United States Government’. Title 17 U.S.C. 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.