Drug Testing and Analysis
Research article Received: 27 November 2013
Revised: 2 May 2014
Accepted: 6 May 2014
Published online in Wiley Online Library
(www.drugtestinganalysis.com) DOI 10.1002/dta.1679
Determination of GHB in human hair by HPLC-MS/MS: Development and validation of a method and application to a study group and three possible single exposure cases Elisabetta Bertol,a Francesco Mari,a Fabio Vaiano,a Guido Romano,b Simona Zaami,c Giovanni Baglìod and Francesco Paolo Busardòc* Gamma-hydroxybutyrate (GHB) over the last two decades has generated increased notoriety as a euphoric and disinhibiting drug of abuse in cases of drug-related sexual assault and for this reason it is considered a ‘date rape’ drug. The first aim of this paper was to develop and fully validate a method for the detection of GHB in human hair by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) after liquid-liquid extraction (LLE). The second aim was the application of the method to hair samples of 30 GHB-free users in order to determine the basal level. The results obtained showed no significant differences in endogenous concentrations (p = 0.556) between hair samples of the three groups (black, blonde, and dyed hair) and the age and sex of the subjects did not affect the endogenous levels. Another 12 healthy volunteers, with no previous history of GHB use, were selected and a single dose (25 mg/Kg) was orally administered to all of them and hair samples were collected before the administration of the single dose and other two samples were collected one month and two months later, respectively. The segmental analysis of the latter two samples allowed us to calculate two ratios: 4.45:1 (95% C.I. 3.52–5.63) and 3.35:1 (95% C.I. 2.14–5.18), respectively, which can be recommended as reasonable values for a positive identification of GHB intake. Finally the method was applied to three real cases where a GHB single exposure probably occurred. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: gamma-hydroxybutyrate (GHB); hair; segmental analysis; LC–MS/MS
Introduction
Drug Test. Analysis (2014)
* Correspondence to: Francesco Paolo Busardò, Department of Anatomical, Histological, Medico-legal and Orthopaedic Sciences, Sapienza University of Rome, Viale Regina Elena 336 (00185) Rome, Italy. E-mail: fra.busardo@ libero.it a Department of Health Sciences, Forensic Toxicology Division, University of Florence, Italy b Department ‘G.F. Ingrassia’, Laboratory of Forensic Toxicology, University of Catania, Italy c Department of Anatomical, Histological, Forensic and Orthopaedic Sciences, Sapienza University of Rome, Viale Regina Elena 336, 00161, Rome, Italy d MSc in Medical Statistics, London, UK
Copyright © 2014 John Wiley & Sons, Ltd.
1
Gamma-hydroxybutyrate (GHB) is a central nervous system (CNS) depressant, primarily used as a recreational drug of abuse with numerous names (‘Georgia Home Boy’, ‘Juice’, ‘Liquid Ecstasy’, ‘Mils’, ‘G’, ‘Liquid X’, ‘Liquid G’, and ‘Fantasy’) and it can be taken as a colourless, odourless liquid or white powder, tablet, and capsule forms. [1,2] It is also used as a therapeutic substance both in the USA and Europe with the commercial name of Xyrem for the treatment of narcolepsy with cataplexy in adult patients and only in Europe with the commercial name of Alcover is it used as adjuvant in the control of alcohol withdrawal syndrome.[3,4] Over the last two decades GHB has generated increased notoriety as a euphoric and disinhibiting drug of abuse in particular in certain social backgrounds and in cases of drug-related sexual assault. For this reason it is considered to be a ‘date rape’ drug, because it can easily be added to drinks, most of all when the victim is vulnerable due to concomitant alcohol consumption. In forensic investigations it is usually required to determine if any GHB detected is due to endogenous production or exogenous ingestion. For its rapid metabolism and elimination (within 4–8 h post-ingestion), concentrations both in blood and urine can drop very quickly. Because of this, the use of an alternative matrix, for example hair, may be really useful in order to demonstrate a possible intake even after a long time, however it is
necessary to take into consideration the basal levels of GHB normally found in hair specimens, which are subject to individual variations. [1,5–9] The first aim of this paper is to develop and fully validate a method for the detection of GHB in human hair by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) after liquid-liquid extraction (LLE). The second aim of the paper is the application of this method in hair samples of 30 GHB-free users in order to determine the basal level, taking into account the hair colour and any difference in concentration between dyed and undyed hair. Another 12
Drug Testing and Analysis
E. Bertol et al.
healthy volunteers, with no previous history of GHB use, were selected and a single dose (25 mg/Kg) dissolved in 100 mL of water was orally administered to all of them. Hair samples were collected immediately before the administration of the single dose and the other two samples were collected one month and two months after the single administration, respectively. Finally the method was applied to three real cases where a GHB single exposure probably occurred.
Materials and methods
For preparation of the calibrators, hair samples with GHB under the limit of detection (LOD) were selected. For all cases, hair specimens were collected from the posterior vertex region of the head, close to the scalp and then stored in dry foil at room temperature, according to the Society of Hair Testing (SoHT) guidelines.[10] Each hair sample was carefully divided into segments of 5 mm each, from the origin close to the root to the distal end. Hair samples were decontaminated twice using 5 mL of methylene chloride for 2 min, dried, and milled into a powder. The last washing solution was concentrated and analyzed by HPLC-MS/ MS to check for the absence of GHB.
Reagents and standards GHB sodium salt in methanol (1.0 mg/mL) and GHB-D6 sodium salt (0.1 mg/mL) were purchased from Cerilliant-Sigma-Aldrich (St Louis, MO, USA); Sodium hydroxide (NaOH), sulfuric acid (H2SO4), formic acid, ethyl acetate and methanol (MeOH) were purchased from Merck Millipore (Darmstadt, Germany).
Hair specimens The hair samples of 30 volunteers were selected for the present study. They were divided according to colour (10 samples of black hair, 10 samples of blonde hair, and 10 samples of hair dyed with different colours). All samples belonged to non-GHB users and they were analyzed in order to determine the endogenous concentration and if there were any variation of concentrations due to the colour (natural or artificial) and sex. For this purpose, except for specimens of dyed hair, which belonged to women in all the cases, the other two groups (black and blonde hair) were each homogeneously composed of 5 men and 5 women. The main features of the three groups are reported in Table 1. Another 12 healthy volunteers, with no previous history of GHB use, were selected and employed in a controlled human study and a single dose (25 mg/Kg) dissolved in 100 mL of water was orally administered to all of them. Hair samples were collected immediately before the administration of the single dose and the other two samples were collected one month and two months after the single administration, respectively. The main features regarding age, sex and hair colour are reported in Figures 1A–1C. The method was applied to three real cases in order to assess a possible single GHB exposure.
Extraction For the extraction, 25 mg of hair was digested with 0.5 mL of 1 M NaOH in the presence of 50 ng I.S. (2 ng/mg) at 90 °C for 20 min. After cooling at room temperature, the sample was added with 0.6 mL of 1 M H2SO4 and 3 mL of ethyl acetate, immediately agitated for 30 s and centrifuged at 1500 g for 5 min. The supernatant organic layer was dried and reconstituted in 50 μL of mobile phase and then 10 μL was injected into the LC-MS/MS. Instrumentation The analysis was performed by an Agilent 1290 Series HPLC system coupled to an Agilent 6460 Triple Quad LC/MS. Chromatography was achieved using a Gemini Reverse-phase LC C18 column (150 mm x 2 mm, 5 μm) held at 30 °C and protected with a Gemini guard column (4 x 2 mm) purchased from Phenomenex. Isocratic elution was performed at 0.3 mL/min with the following mobile phase composition: 90% of 0.1% aqueous formic acid and 10% of methanol. Negative electrospray ionization (ESI) was used at a temperature of 350 °C. Nitrogen was used as collision gas (flow rate, 11 L/min), the nebulizer pressure at 30 psi and capillary 4000 V. The method was run in multiple reaction monitoring (MRM) mode and set to detect the precursor and product ions of GHB and its deuterated internal standards. All details are reported in Table 2. Method validation The lower limit of quantification (LLOQ) for the analyte was defined as the lowest standard on the calibration curve (0.5 ng/mg) with a
Table 1. Main features of the 30 volunteers divided according to their hair colour (black, blonde and dyed hair) and GHB mean concentrations for each group, GHB mean values obtained excluding the first hair segment, and GHB mean concentrations of the first hair segment
2
Black hair group (n = 10) Blonde hair group (n = 10) Dyed hair group n = 10 Colour (OC) Blond (brown 2, grey 1) Brown (blonde 1, grey 1) Auburn (black 1) Multicolour (brown 1) Black (blonde 1, brown 1, grey 1)
Mean age (years) ± S.D. (range)
Sex
Mean length (mm) ± S.D.
GHB mean conc. (ng/mg) ± S.D. (range)
GHB mean conc. st (ng/mg) except 1 segment ± S.D.
GHB mean conc. st (ng/mg) 1 segment ± S.D.
37 ± 9.85 (24 -54) 33 ± 6.2 (21 - 41) 39 ± 12.6
5M / 5F 5M / 5F 10 F
35 ± 3.24 37 ± 4.11 43 ± 5.6
2.11 ± 1.4 (0 – 4.49) 2.25 ± 1.31 (0.58 – 5.09) 2.39 ± 0.97 (0.61 – 4.02)
2.1 ± 1.37 2.21 ± 1.30 2.35 ± 0.97
2.34 ± 1.54 2.51 ± 1.36 2.65 ± 0.98
Legend - OC: Original colour.
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Copyright © 2014 John Wiley & Sons, Ltd.
Drug Test. Analysis (2014)
Drug Testing and Analysis
Determination of GHB in human hair Hair samples before GHB administration
(A)
3.5
GHB conc. (ng/mg)
3 2.5 2 Auburn (OC: Black) F 32 Auburn (OC: Blond) F 29 Black M 39 Blond F 24 Brown (OC: Black) F 21 Black M 23 Blond M 28 Black M 26 Brown F 22 Dark Blond F 44 Black M 30 Brown M 34
1.5 1 0.5 0 1
2
3
4
5
6
1
2
3
4
5
6
Brown M 34
0.98
0.78
0.79
0.75
0.75
0.73
Black M 30
1.34
1.21
1.2
1.18
1.15
1.15
Dark Blond F 44
0.59
0
0
0
0
Brown F 22
1.81
1.75
1.72
1.69
1.67
1.62
Black M 26
0.88
0.7
0.71
0.68
0.67
0.64
Blond M 28
1.44
1.32
1.31
1.32
1.29
1.25
Black M 23
1.89
1.75
1.72
1.7
1.71
1.69
Brown (OC: Black) F 21
0.89
0.8
0.78
0.7
0.71
0.68
Blond F 24
3.21
3.02
2.97
2.96
2.96
Black M 39
2.06
1.98
1.95
1.94
1.89
1.83
Auburn (OC: Blond) F 29
1.07
0.93
0.9
0.89
0.9
0.87
Auburn (OC: Black) F 32
1.22
1.13
1.13
1.12
1.1
1.09
0
2.93
Segments (0.5 mm)
Hair samples 1 month after GHB administration
(B)
8
GHB conc. (ng/mg)
7 6 5 4 Auburn (OC: Black) F 32 Auburn (OC: Blond) F 29 Black M 39 Blond F 24 Brown (OC: Black) F 21 Black M 23 Blond M 28 Black M 26 Brown F 22 Dark Blond F 44 Black M 30 Brown M 34
3 2 1 0
1
2
1 Brown M 34
1.08
Black M 30
1.65
Dark Blond F 44
3
4
5
6
7
8
2 4.8
3
4
5
6
7
8
0.92
0.77
0.74
0.7
0.69
0.68
1.22
1.18
1.16
1.13
1.14
6
1.31
0.62
1.4
0.56
0
0
0
0
0
Brown F 22
1.8
6.88
1.98
1.72
1.68
1.65
1.63
1.63
Black M 26
0.97
5.45
1.14
0.71
0.69
0.66
0.63
0.64
Blond M 28
2.15
5.21
1.34
1.32
1.3
1.27
1.22
1.18
Black M 23
1.85
7.12
1.94
1.71
1.69
1.67
1.67
1.68
Brown (OC: Black) F 21
0.92
3.94
1.2
0.78
0.73
0.7
0.7
0.69
Blond F 24
3.15
6.82
3.45
2.98
2.95
2.9
2.88
2.85
Black M 39
3
5.14
2.01
1.96
1.93
1.93
1.9
1.89
Auburn (OC: Blond) F 29
1.1
7.12
1.02
0.95
0.92
0.9
0.89
0.84
Auburn (OC: Black) F 32
1.25
4.28
1.54
1.18
1.15
1.1
1.1
1.08
Segments (0.5 mm)
3
Figure 1. (A) – Segmental analysis of the hair samples of twelve healthy volunteers before the administration of a single dose of GHB. For all subjects are reported the hair colour, age and sex. (B) – Segmental analysis of the hair samples of twelve healthy volunteers collected one month after the administration of a single dose of GHB. The second segment shows for all cases the highest concentration. (C) – Segmental analysis of the hair samples of twelve healthy volunteers collected two months after the administration of a single dose of GHB. The fourth segment shows for all cases the highest concentration.
Drug Test. Analysis (2014)
Copyright © 2014 John Wiley & Sons, Ltd.
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Drug Testing and Analysis
E. Bertol et al.
Hair samples 2 months after GHB administration
(C)
7
GHB conc. (ng/mg)
6 5 4 3 Auburn (OC: Black) F 32 Auburn (OC: Blond) F 29 Black M 39 Blond F 24 Brown (OC: Black) F 21 Black M 23 Blond M 28 Black M 26 Brown F 22 Dark Blond F 44 Black M 30 Brown M 34
2 1 0
1
2
3
4
5
6
7
8
9
10
1
2
3
4
5
6
7
8
9
10
Brown M 34
1.03
0.81
1.54
4.4
0.79
0.71
0.67
0.65
0.66
0.64
Black M 30
1.38
1.29
1.44
5.6
1.22
1.18
1.11
1.1
1.12
1.09
Dark Blond F 44
0.57
0
0
1.28
0.64
0
0
0
0
0
Brown F 22
1.79
1.76
1.7
6.32
1.82
1.67
1.65
1.64
1.61
1.6
Black M 26
0.92
0.75
0.73
4.76
0.88
0.69
0.65
0.67
0.63
0.6
Blond M 28
1.52
1.33
2.49
4.71
1.32
1.28
1.26
1.18
1.18
1.16
Black M 23
1.8
1.74
1.75
6.59
1.8
1.73
1.7
1.69
1.67
1.65
Brown (OC: Black) F 21
0.9
0.81
0.84
3.45
0.96
0.74
0.73
0.74
0.7
0.67
Blond F 24
3.16
3
2.95
5.09
3.16
2.89
2.87
2.86
2.84
2.85
Black M 39
2.15
1.96
2.5
4.11
1.9
1.89
1.88
1.88
1.86
1.85
Auburn (OC: Blond) F 29
1.13
0.96
0.95
6.24
1.1
0.89
0.87
0.85
0.83
0.8
Auburn (OC: Black) F 32
1.27
1.11
1.1
4
1.28
1.13
1.08
1.05
1.03
1
Segments (0.5 mm)
Figure 1. (Continued)
Table 2. GHB precursor and product ions Drug GHB Quantifier GHB Qualifier GHB-d6 Quantifier GHB-d6 Qualifier
Precursor Ion Fragmentor Product Ion CE dwell (m/z) (V) (m/z) (eV) (ms) 103 103 109
80 80 80
57 85 61
9 5 10
200 200 200
109
80
90
7
200
4
peak response at least 10 times (S/N ≥ 10) that of the blank response. The limit of detection (LOD) was calculated at a signal-to-noise ratio of 3 (S/N ≥ 3) by adding decreasing quantities of GHB to 25 mg of hair. Methanolic working standards for GHB and GHB-D6 were used to fortify 25 mg aliquots of hair. A calibration curve included the following standards: 0.5, 1.0, 2.5, 5.0, 10, 20, and 30 ng/mg and for each of them 3 replicates were performed. All samples were added with 5 μL of IS solution (0.1 mg/mL) to give a final GHB-D6 concentration of 2 ng/mg. The hair specimens used for preparation of standards had GHB concentrations less than LOD. Accuracy and precision were determined using three quality controls (QC) at 1.5, 5 and 15 ng/mg. Imprecision, as a degree of repeatability, was estimated as the average of relative standard deviation (%RSD) values calculated for QC samples with 5 replicates. The intra-day and inter-day imprecisions were calculated
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in the same day and in 5 different days during a month, respectively. Accuracy (n = 25) was estimated at the following concentrations: 1.5, 5 and 15 ng/mg, whereas recovery was calculated by the comparison of peak areas of GHB and its IS added before and after the extraction, for 3 replicates at the following concentrations: 1.5, 5 and 15.0 ng/mg GHB and 2 ng/mg IS. Matrix effects (ion suppression or enhancement) were evaluated by post-column infusion of the analyte (at 2 and 10 ng/mg GHB). A constant signal at the detector was generated. Matrix effects were determined by injecting extracts from GHB-free hair samples and monitoring the detector response. Case samples Three cases were selected in order to assess a possible single GHB exposure. Case 1: A 29-year-old male was brought to the emergency department (ED) for an acute intoxication due to alcohol and GHB, which he had taken for the first time. The blood analysis collected approximately 4 h after consumption highlighted the following concentrations: 1.77 g/L for alcohol and 39 mg/L for GHB. A hair sample was collected in order to determine the endogenous levels and at a distance of approximately one month (32 days) and three months (84 days) he provided further two samples. The samples were collected from the vertex region of the head and they showed the following length: 2.2, 3 and 4.6 cm, respectively.
Copyright © 2014 John Wiley & Sons, Ltd.
Drug Test. Analysis (2014)
Drug Testing and Analysis
Determination of GHB in human hair
regression analysis to assess the association between the following three variable (sex, age, hair colour) and GHB mean concentrations. The STATA software 11.0 version was used.
Case 2: A 26-year-old female, during a party consumed with a male (ex-boyfriend) a few alcoholic drinks and shortly after began to feel sleepy and her ex-boyfriend tried to approach her insistently. The girl managed to reach her car where she fell asleep and the following morning she went to a nearby hospital because she suspected that the boy had added something to her drink without her knowledge. The girl reported that she had never taken GHB or other drugs, but she suspected a similar episode at her ex boyfriend’s apartment about two and a half months before. The research of drugs in blood and urine was negative but the following GHB concentrations were detected: 3.32 mg/L in blood and 4.21 mg/L in urine. Both concentrations fell within the endogenous range, [11–13] for this reason taking into account the rapid metabolism of GHB it wasn’t possible to discriminate between the endogenous production and external administration. The girl provided a hair sample in order to determine the endogenous levels and a possible previous consumption and at a distance of one month, another hair sample was taken. Both samples were collected from the vertex region of the head and they showed the following length: 5.9 and 7 cm, respectively. Case 3: A 20-year-old male, an occasional cocaine user, during a medical examination for suspension of his driving license, reported that he had taken in the past GHB, among other substances, once with alcohol approximately three months earlier, but he hadn’t consumed any drugs for the last two months. He provided a hair sample collected from the vertex region of 3.6 cm in length. In the three cases reported a segmental analysis of hair samples was performed using the above method. Each hair sample was carefully divided into segments of 5 mm each, from the origin close to the root to the distal end.
Results Validation results The calibration curves, including seven non-zero calibrators, were linear over the range: 0.5–30.0 ng/mg. From five calibrations, the correlation coefficients were ≥ 0.99. The LLOQ and LOD were 0.5 and 0.3 ng/mg, respectively. Imprecision and inaccuracy (% MRE) were always lower than 17 % and 8%, respectively and all details are reported in Table 3. The extraction recovery (n = 3) at three different concentrations is shown in Table 3. An MRM chromatogram for GHB and GHB-d6 in a QC spiked at 0.6 ng/mg is presented in Figure 2. A high signal-to-noise ratio was obtained and there were no interferences at the retention time of the analyte. Determination of matrix effect was performed using four hair extracts with GHB concentrations lower than the LOD. Ion suppression or enhancement at 2 ng/mg and 10 ng/mg was negligible. The analysis of the last washing solution showed GHB values lower than the LOD.
Black, blonde, and dyed hair groups The segmental analysis of hair samples showed the following concentrations: for the black hair group the mean concentration was 2.11 ± 1.40 ng/mg with a range from 0 (
Research article Received: 27 November 2013
Revised: 2 May 2014
Accepted: 6 May 2014
Published online in Wiley Online Library
(www.drugtestinganalysis.com) DOI 10.1002/dta.1679
Determination of GHB in human hair by HPLC-MS/MS: Development and validation of a method and application to a study group and three possible single exposure cases Elisabetta Bertol,a Francesco Mari,a Fabio Vaiano,a Guido Romano,b Simona Zaami,c Giovanni Baglìod and Francesco Paolo Busardòc* Gamma-hydroxybutyrate (GHB) over the last two decades has generated increased notoriety as a euphoric and disinhibiting drug of abuse in cases of drug-related sexual assault and for this reason it is considered a ‘date rape’ drug. The first aim of this paper was to develop and fully validate a method for the detection of GHB in human hair by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) after liquid-liquid extraction (LLE). The second aim was the application of the method to hair samples of 30 GHB-free users in order to determine the basal level. The results obtained showed no significant differences in endogenous concentrations (p = 0.556) between hair samples of the three groups (black, blonde, and dyed hair) and the age and sex of the subjects did not affect the endogenous levels. Another 12 healthy volunteers, with no previous history of GHB use, were selected and a single dose (25 mg/Kg) was orally administered to all of them and hair samples were collected before the administration of the single dose and other two samples were collected one month and two months later, respectively. The segmental analysis of the latter two samples allowed us to calculate two ratios: 4.45:1 (95% C.I. 3.52–5.63) and 3.35:1 (95% C.I. 2.14–5.18), respectively, which can be recommended as reasonable values for a positive identification of GHB intake. Finally the method was applied to three real cases where a GHB single exposure probably occurred. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: gamma-hydroxybutyrate (GHB); hair; segmental analysis; LC–MS/MS
Introduction
Drug Test. Analysis (2014)
* Correspondence to: Francesco Paolo Busardò, Department of Anatomical, Histological, Medico-legal and Orthopaedic Sciences, Sapienza University of Rome, Viale Regina Elena 336 (00185) Rome, Italy. E-mail: fra.busardo@ libero.it a Department of Health Sciences, Forensic Toxicology Division, University of Florence, Italy b Department ‘G.F. Ingrassia’, Laboratory of Forensic Toxicology, University of Catania, Italy c Department of Anatomical, Histological, Forensic and Orthopaedic Sciences, Sapienza University of Rome, Viale Regina Elena 336, 00161, Rome, Italy d MSc in Medical Statistics, London, UK
Copyright © 2014 John Wiley & Sons, Ltd.
1
Gamma-hydroxybutyrate (GHB) is a central nervous system (CNS) depressant, primarily used as a recreational drug of abuse with numerous names (‘Georgia Home Boy’, ‘Juice’, ‘Liquid Ecstasy’, ‘Mils’, ‘G’, ‘Liquid X’, ‘Liquid G’, and ‘Fantasy’) and it can be taken as a colourless, odourless liquid or white powder, tablet, and capsule forms. [1,2] It is also used as a therapeutic substance both in the USA and Europe with the commercial name of Xyrem for the treatment of narcolepsy with cataplexy in adult patients and only in Europe with the commercial name of Alcover is it used as adjuvant in the control of alcohol withdrawal syndrome.[3,4] Over the last two decades GHB has generated increased notoriety as a euphoric and disinhibiting drug of abuse in particular in certain social backgrounds and in cases of drug-related sexual assault. For this reason it is considered to be a ‘date rape’ drug, because it can easily be added to drinks, most of all when the victim is vulnerable due to concomitant alcohol consumption. In forensic investigations it is usually required to determine if any GHB detected is due to endogenous production or exogenous ingestion. For its rapid metabolism and elimination (within 4–8 h post-ingestion), concentrations both in blood and urine can drop very quickly. Because of this, the use of an alternative matrix, for example hair, may be really useful in order to demonstrate a possible intake even after a long time, however it is
necessary to take into consideration the basal levels of GHB normally found in hair specimens, which are subject to individual variations. [1,5–9] The first aim of this paper is to develop and fully validate a method for the detection of GHB in human hair by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) after liquid-liquid extraction (LLE). The second aim of the paper is the application of this method in hair samples of 30 GHB-free users in order to determine the basal level, taking into account the hair colour and any difference in concentration between dyed and undyed hair. Another 12
Drug Testing and Analysis
E. Bertol et al.
healthy volunteers, with no previous history of GHB use, were selected and a single dose (25 mg/Kg) dissolved in 100 mL of water was orally administered to all of them. Hair samples were collected immediately before the administration of the single dose and the other two samples were collected one month and two months after the single administration, respectively. Finally the method was applied to three real cases where a GHB single exposure probably occurred.
Materials and methods
For preparation of the calibrators, hair samples with GHB under the limit of detection (LOD) were selected. For all cases, hair specimens were collected from the posterior vertex region of the head, close to the scalp and then stored in dry foil at room temperature, according to the Society of Hair Testing (SoHT) guidelines.[10] Each hair sample was carefully divided into segments of 5 mm each, from the origin close to the root to the distal end. Hair samples were decontaminated twice using 5 mL of methylene chloride for 2 min, dried, and milled into a powder. The last washing solution was concentrated and analyzed by HPLC-MS/ MS to check for the absence of GHB.
Reagents and standards GHB sodium salt in methanol (1.0 mg/mL) and GHB-D6 sodium salt (0.1 mg/mL) were purchased from Cerilliant-Sigma-Aldrich (St Louis, MO, USA); Sodium hydroxide (NaOH), sulfuric acid (H2SO4), formic acid, ethyl acetate and methanol (MeOH) were purchased from Merck Millipore (Darmstadt, Germany).
Hair specimens The hair samples of 30 volunteers were selected for the present study. They were divided according to colour (10 samples of black hair, 10 samples of blonde hair, and 10 samples of hair dyed with different colours). All samples belonged to non-GHB users and they were analyzed in order to determine the endogenous concentration and if there were any variation of concentrations due to the colour (natural or artificial) and sex. For this purpose, except for specimens of dyed hair, which belonged to women in all the cases, the other two groups (black and blonde hair) were each homogeneously composed of 5 men and 5 women. The main features of the three groups are reported in Table 1. Another 12 healthy volunteers, with no previous history of GHB use, were selected and employed in a controlled human study and a single dose (25 mg/Kg) dissolved in 100 mL of water was orally administered to all of them. Hair samples were collected immediately before the administration of the single dose and the other two samples were collected one month and two months after the single administration, respectively. The main features regarding age, sex and hair colour are reported in Figures 1A–1C. The method was applied to three real cases in order to assess a possible single GHB exposure.
Extraction For the extraction, 25 mg of hair was digested with 0.5 mL of 1 M NaOH in the presence of 50 ng I.S. (2 ng/mg) at 90 °C for 20 min. After cooling at room temperature, the sample was added with 0.6 mL of 1 M H2SO4 and 3 mL of ethyl acetate, immediately agitated for 30 s and centrifuged at 1500 g for 5 min. The supernatant organic layer was dried and reconstituted in 50 μL of mobile phase and then 10 μL was injected into the LC-MS/MS. Instrumentation The analysis was performed by an Agilent 1290 Series HPLC system coupled to an Agilent 6460 Triple Quad LC/MS. Chromatography was achieved using a Gemini Reverse-phase LC C18 column (150 mm x 2 mm, 5 μm) held at 30 °C and protected with a Gemini guard column (4 x 2 mm) purchased from Phenomenex. Isocratic elution was performed at 0.3 mL/min with the following mobile phase composition: 90% of 0.1% aqueous formic acid and 10% of methanol. Negative electrospray ionization (ESI) was used at a temperature of 350 °C. Nitrogen was used as collision gas (flow rate, 11 L/min), the nebulizer pressure at 30 psi and capillary 4000 V. The method was run in multiple reaction monitoring (MRM) mode and set to detect the precursor and product ions of GHB and its deuterated internal standards. All details are reported in Table 2. Method validation The lower limit of quantification (LLOQ) for the analyte was defined as the lowest standard on the calibration curve (0.5 ng/mg) with a
Table 1. Main features of the 30 volunteers divided according to their hair colour (black, blonde and dyed hair) and GHB mean concentrations for each group, GHB mean values obtained excluding the first hair segment, and GHB mean concentrations of the first hair segment
2
Black hair group (n = 10) Blonde hair group (n = 10) Dyed hair group n = 10 Colour (OC) Blond (brown 2, grey 1) Brown (blonde 1, grey 1) Auburn (black 1) Multicolour (brown 1) Black (blonde 1, brown 1, grey 1)
Mean age (years) ± S.D. (range)
Sex
Mean length (mm) ± S.D.
GHB mean conc. (ng/mg) ± S.D. (range)
GHB mean conc. st (ng/mg) except 1 segment ± S.D.
GHB mean conc. st (ng/mg) 1 segment ± S.D.
37 ± 9.85 (24 -54) 33 ± 6.2 (21 - 41) 39 ± 12.6
5M / 5F 5M / 5F 10 F
35 ± 3.24 37 ± 4.11 43 ± 5.6
2.11 ± 1.4 (0 – 4.49) 2.25 ± 1.31 (0.58 – 5.09) 2.39 ± 0.97 (0.61 – 4.02)
2.1 ± 1.37 2.21 ± 1.30 2.35 ± 0.97
2.34 ± 1.54 2.51 ± 1.36 2.65 ± 0.98
Legend - OC: Original colour.
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Drug Test. Analysis (2014)
Drug Testing and Analysis
Determination of GHB in human hair Hair samples before GHB administration
(A)
3.5
GHB conc. (ng/mg)
3 2.5 2 Auburn (OC: Black) F 32 Auburn (OC: Blond) F 29 Black M 39 Blond F 24 Brown (OC: Black) F 21 Black M 23 Blond M 28 Black M 26 Brown F 22 Dark Blond F 44 Black M 30 Brown M 34
1.5 1 0.5 0 1
2
3
4
5
6
1
2
3
4
5
6
Brown M 34
0.98
0.78
0.79
0.75
0.75
0.73
Black M 30
1.34
1.21
1.2
1.18
1.15
1.15
Dark Blond F 44
0.59
0
0
0
0
Brown F 22
1.81
1.75
1.72
1.69
1.67
1.62
Black M 26
0.88
0.7
0.71
0.68
0.67
0.64
Blond M 28
1.44
1.32
1.31
1.32
1.29
1.25
Black M 23
1.89
1.75
1.72
1.7
1.71
1.69
Brown (OC: Black) F 21
0.89
0.8
0.78
0.7
0.71
0.68
Blond F 24
3.21
3.02
2.97
2.96
2.96
Black M 39
2.06
1.98
1.95
1.94
1.89
1.83
Auburn (OC: Blond) F 29
1.07
0.93
0.9
0.89
0.9
0.87
Auburn (OC: Black) F 32
1.22
1.13
1.13
1.12
1.1
1.09
0
2.93
Segments (0.5 mm)
Hair samples 1 month after GHB administration
(B)
8
GHB conc. (ng/mg)
7 6 5 4 Auburn (OC: Black) F 32 Auburn (OC: Blond) F 29 Black M 39 Blond F 24 Brown (OC: Black) F 21 Black M 23 Blond M 28 Black M 26 Brown F 22 Dark Blond F 44 Black M 30 Brown M 34
3 2 1 0
1
2
1 Brown M 34
1.08
Black M 30
1.65
Dark Blond F 44
3
4
5
6
7
8
2 4.8
3
4
5
6
7
8
0.92
0.77
0.74
0.7
0.69
0.68
1.22
1.18
1.16
1.13
1.14
6
1.31
0.62
1.4
0.56
0
0
0
0
0
Brown F 22
1.8
6.88
1.98
1.72
1.68
1.65
1.63
1.63
Black M 26
0.97
5.45
1.14
0.71
0.69
0.66
0.63
0.64
Blond M 28
2.15
5.21
1.34
1.32
1.3
1.27
1.22
1.18
Black M 23
1.85
7.12
1.94
1.71
1.69
1.67
1.67
1.68
Brown (OC: Black) F 21
0.92
3.94
1.2
0.78
0.73
0.7
0.7
0.69
Blond F 24
3.15
6.82
3.45
2.98
2.95
2.9
2.88
2.85
Black M 39
3
5.14
2.01
1.96
1.93
1.93
1.9
1.89
Auburn (OC: Blond) F 29
1.1
7.12
1.02
0.95
0.92
0.9
0.89
0.84
Auburn (OC: Black) F 32
1.25
4.28
1.54
1.18
1.15
1.1
1.1
1.08
Segments (0.5 mm)
3
Figure 1. (A) – Segmental analysis of the hair samples of twelve healthy volunteers before the administration of a single dose of GHB. For all subjects are reported the hair colour, age and sex. (B) – Segmental analysis of the hair samples of twelve healthy volunteers collected one month after the administration of a single dose of GHB. The second segment shows for all cases the highest concentration. (C) – Segmental analysis of the hair samples of twelve healthy volunteers collected two months after the administration of a single dose of GHB. The fourth segment shows for all cases the highest concentration.
Drug Test. Analysis (2014)
Copyright © 2014 John Wiley & Sons, Ltd.
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Drug Testing and Analysis
E. Bertol et al.
Hair samples 2 months after GHB administration
(C)
7
GHB conc. (ng/mg)
6 5 4 3 Auburn (OC: Black) F 32 Auburn (OC: Blond) F 29 Black M 39 Blond F 24 Brown (OC: Black) F 21 Black M 23 Blond M 28 Black M 26 Brown F 22 Dark Blond F 44 Black M 30 Brown M 34
2 1 0
1
2
3
4
5
6
7
8
9
10
1
2
3
4
5
6
7
8
9
10
Brown M 34
1.03
0.81
1.54
4.4
0.79
0.71
0.67
0.65
0.66
0.64
Black M 30
1.38
1.29
1.44
5.6
1.22
1.18
1.11
1.1
1.12
1.09
Dark Blond F 44
0.57
0
0
1.28
0.64
0
0
0
0
0
Brown F 22
1.79
1.76
1.7
6.32
1.82
1.67
1.65
1.64
1.61
1.6
Black M 26
0.92
0.75
0.73
4.76
0.88
0.69
0.65
0.67
0.63
0.6
Blond M 28
1.52
1.33
2.49
4.71
1.32
1.28
1.26
1.18
1.18
1.16
Black M 23
1.8
1.74
1.75
6.59
1.8
1.73
1.7
1.69
1.67
1.65
Brown (OC: Black) F 21
0.9
0.81
0.84
3.45
0.96
0.74
0.73
0.74
0.7
0.67
Blond F 24
3.16
3
2.95
5.09
3.16
2.89
2.87
2.86
2.84
2.85
Black M 39
2.15
1.96
2.5
4.11
1.9
1.89
1.88
1.88
1.86
1.85
Auburn (OC: Blond) F 29
1.13
0.96
0.95
6.24
1.1
0.89
0.87
0.85
0.83
0.8
Auburn (OC: Black) F 32
1.27
1.11
1.1
4
1.28
1.13
1.08
1.05
1.03
1
Segments (0.5 mm)
Figure 1. (Continued)
Table 2. GHB precursor and product ions Drug GHB Quantifier GHB Qualifier GHB-d6 Quantifier GHB-d6 Qualifier
Precursor Ion Fragmentor Product Ion CE dwell (m/z) (V) (m/z) (eV) (ms) 103 103 109
80 80 80
57 85 61
9 5 10
200 200 200
109
80
90
7
200
4
peak response at least 10 times (S/N ≥ 10) that of the blank response. The limit of detection (LOD) was calculated at a signal-to-noise ratio of 3 (S/N ≥ 3) by adding decreasing quantities of GHB to 25 mg of hair. Methanolic working standards for GHB and GHB-D6 were used to fortify 25 mg aliquots of hair. A calibration curve included the following standards: 0.5, 1.0, 2.5, 5.0, 10, 20, and 30 ng/mg and for each of them 3 replicates were performed. All samples were added with 5 μL of IS solution (0.1 mg/mL) to give a final GHB-D6 concentration of 2 ng/mg. The hair specimens used for preparation of standards had GHB concentrations less than LOD. Accuracy and precision were determined using three quality controls (QC) at 1.5, 5 and 15 ng/mg. Imprecision, as a degree of repeatability, was estimated as the average of relative standard deviation (%RSD) values calculated for QC samples with 5 replicates. The intra-day and inter-day imprecisions were calculated
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in the same day and in 5 different days during a month, respectively. Accuracy (n = 25) was estimated at the following concentrations: 1.5, 5 and 15 ng/mg, whereas recovery was calculated by the comparison of peak areas of GHB and its IS added before and after the extraction, for 3 replicates at the following concentrations: 1.5, 5 and 15.0 ng/mg GHB and 2 ng/mg IS. Matrix effects (ion suppression or enhancement) were evaluated by post-column infusion of the analyte (at 2 and 10 ng/mg GHB). A constant signal at the detector was generated. Matrix effects were determined by injecting extracts from GHB-free hair samples and monitoring the detector response. Case samples Three cases were selected in order to assess a possible single GHB exposure. Case 1: A 29-year-old male was brought to the emergency department (ED) for an acute intoxication due to alcohol and GHB, which he had taken for the first time. The blood analysis collected approximately 4 h after consumption highlighted the following concentrations: 1.77 g/L for alcohol and 39 mg/L for GHB. A hair sample was collected in order to determine the endogenous levels and at a distance of approximately one month (32 days) and three months (84 days) he provided further two samples. The samples were collected from the vertex region of the head and they showed the following length: 2.2, 3 and 4.6 cm, respectively.
Copyright © 2014 John Wiley & Sons, Ltd.
Drug Test. Analysis (2014)
Drug Testing and Analysis
Determination of GHB in human hair
regression analysis to assess the association between the following three variable (sex, age, hair colour) and GHB mean concentrations. The STATA software 11.0 version was used.
Case 2: A 26-year-old female, during a party consumed with a male (ex-boyfriend) a few alcoholic drinks and shortly after began to feel sleepy and her ex-boyfriend tried to approach her insistently. The girl managed to reach her car where she fell asleep and the following morning she went to a nearby hospital because she suspected that the boy had added something to her drink without her knowledge. The girl reported that she had never taken GHB or other drugs, but she suspected a similar episode at her ex boyfriend’s apartment about two and a half months before. The research of drugs in blood and urine was negative but the following GHB concentrations were detected: 3.32 mg/L in blood and 4.21 mg/L in urine. Both concentrations fell within the endogenous range, [11–13] for this reason taking into account the rapid metabolism of GHB it wasn’t possible to discriminate between the endogenous production and external administration. The girl provided a hair sample in order to determine the endogenous levels and a possible previous consumption and at a distance of one month, another hair sample was taken. Both samples were collected from the vertex region of the head and they showed the following length: 5.9 and 7 cm, respectively. Case 3: A 20-year-old male, an occasional cocaine user, during a medical examination for suspension of his driving license, reported that he had taken in the past GHB, among other substances, once with alcohol approximately three months earlier, but he hadn’t consumed any drugs for the last two months. He provided a hair sample collected from the vertex region of 3.6 cm in length. In the three cases reported a segmental analysis of hair samples was performed using the above method. Each hair sample was carefully divided into segments of 5 mm each, from the origin close to the root to the distal end.
Results Validation results The calibration curves, including seven non-zero calibrators, were linear over the range: 0.5–30.0 ng/mg. From five calibrations, the correlation coefficients were ≥ 0.99. The LLOQ and LOD were 0.5 and 0.3 ng/mg, respectively. Imprecision and inaccuracy (% MRE) were always lower than 17 % and 8%, respectively and all details are reported in Table 3. The extraction recovery (n = 3) at three different concentrations is shown in Table 3. An MRM chromatogram for GHB and GHB-d6 in a QC spiked at 0.6 ng/mg is presented in Figure 2. A high signal-to-noise ratio was obtained and there were no interferences at the retention time of the analyte. Determination of matrix effect was performed using four hair extracts with GHB concentrations lower than the LOD. Ion suppression or enhancement at 2 ng/mg and 10 ng/mg was negligible. The analysis of the last washing solution showed GHB values lower than the LOD.
Black, blonde, and dyed hair groups The segmental analysis of hair samples showed the following concentrations: for the black hair group the mean concentration was 2.11 ± 1.40 ng/mg with a range from 0 (
