Forensic Science International 243 (2014) 144–148
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Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
Post mortem concentrations of endogenous gamma hydroxybutyric acid (GHB) and in vitro formation in stored blood and urine samples§ Francesco Paolo Busardo` a,*, Elisabetta Bertol b, Fabio Vaiano b, Giovanni Baglio c, Angelo Montana d, Nunziata Barbera d, Simona Zaami a, Guido Romano d a
Department of Anatomical, Histological, Forensic and Orthopaedic Sciences, Sapienza University of Rome, Viale Regina Elena 336, 00161, Rome, Italy Department of Health Sciences, Forensic Toxicology Division, University of Florence, Florence, Italy c MSc in Epidemiology, London, UK d Department ‘‘G.F. Ingrassia’’, Laboratory of Forensic Toxicology, University of Catania, Catania, Italy b
A R T I C L E I N F O
A B S T R A C T
Article history: Available online 25 July 2014
Gamma-hydroxybutyrate (GHB) is a central nervous system depressant, primarily used as a recreational drug of abuse with numerous names. It has also been involved in various instances of drug-facilitated sexual assault due to its potential incapacitating effects. The first aim of this paper is to measure the post-mortem concentration of endogenous GHB in whole blood and urine samples of 30 GHB free-users, who have been divided according to the post-mortem interval (PMI) in three groups (first group: 24–36 h; second group: 37–72 h; third group: 73–192 h), trying to evaluate the role of PMI in affecting post mortem levels. Second, the Authors have evaluated the new formation of GHB in vitro in blood and urine samples of the three groups, which have been stored at 20 8C, 4 8C and 20 8C over a period of one month. The concentrations were measured by GC–MS after liquid–liquid extraction according to the method validated and published by Elliot (For. Sci. Int., 2003). For urine samples, GHB concentrations were creatinine-normalized. In the first group the GHB mean concentration measured after autopsy was: 2.14 mg/L (range 0.54– 3.21 mg/L) in blood and 3.90 mg/g (range 0.60–4.81 mg/g) in urine; in the second group it was: 5.13 mg/ L (range 1.11–9.60 mg/L) in blood and 3.93 mg/g (range 0.91–7.25 mg/g) in urine; in the third group it was: 11.8 mg/L (range 3.95–24.12 mg/L) in blood and 9.83 mg/g (range 3.67–21.90 mg/g) in urine. The results obtained in blood and urine samples showed a statistically significant difference among groups (p < 0.001) in the first analysis performed immediately after autopsy. Throughout the period of investigation up to 4 weeks, the comparison of storage temperatures within each group showed in blood and urine samples a mean difference at 20 8C compared to 20 8C not statistically significant at the 10% level. These findings allow us to affirm that the PMI strongly affects the post mortem production of GHB in blood and urine samples. Regarding the new formation of GHB in vitro both in blood and urine samples of the three groups, which have been stored at 20 8C, 4 8C and 20 8C over a period of one month, although there was no significant increases of GHB levels throughout the period of investigation, the lowest increases were found both in blood and urine at 20 8C, therefore we recommend the latter as optimal storage temperature. ß 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Gamma hydroxybutyric acid (GHB) Endogenous concentrations Stored blood and urine samples, In vitro formation
1. Introduction § This paper is part of the special issue entitled ‘‘The 51st Annual Meeting of the International Association of Forensic Toxicologists (TIAFT)’’. September 2–3, 2013, Funchal, Medeira, Portugal. Guest edited by Professor Helena Teixeira, Professor Duarte Nuno Vieira and Professor Francisco Corte Real. * Corresponding author. Tel.: +39 06 49912622; fax: +39 06 4455335. E-mail address: [email protected] (F.P. Busardo`).
http://dx.doi.org/10.1016/j.forsciint.2014.07.019 0379-0738/ß 2014 Elsevier Ireland Ltd. All rights reserved.
Gamma-hydroxybutyrate (GHB) is a central nervous system 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’’) in a colourless, odourless liquid or white powder, tablet and capsule forms [1,2]. It
F.P. Busardo` et al. / Forensic Science International 243 (2014) 144–148
is also used as a therapeutic substance both in U.S.A. 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) it is used as adjuvant in the control of alcohol withdrawal syndrome and in the initial phase of multimodal treatment of alcohol dependence [3,4]. GHB is endogenously produced as metabolite and precursor of g-hydroxybutyrate (GABA), the principal inhibitory neurotransmitter and it acts as a neuromodulator in the GABA system. Moreover, growing evidence suggests that GHB affects several neuromodulatory systems in the central nervous system (CNS), such as neurosteroids, dopaminergic, serotonergic and cholinergic systems, growth hormone, opioids etc. [5]. It has also been involved in various instances of drug-facilitated sexual assault (DFSA) due to its potential incapacitating effects. 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 [6–8]. Gamma-butyrolactone (GBL) and 1,4-butanediol (1,4-BD) are sold as solvents prevalently for industrial use; they represents precursors/analogous of GHB and they are converted very quickly and easily into GHB after ingestion, however, they are not present endogenously [9,10]. In forensic investigations it is usually required to determine if any GHB detected is due to endogenous production or exogenous ingestion and it can generate interpretative problems in particular in cadaveric samples due to the post mortem production [11,12]. The first aim of this paper is to measure the post mortem concentration of endogenous GHB in whole blood and urine samples of 30 GHB free-users, who have been divided according to the post-mortem interval (PMI) in three groups, trying to evaluate the role of PMI in affecting post mortem levels. The second aim of this research was to determine the new formation of GHB in vitro in blood and urine samples of the three groups, which have been stored at three different temperatures ( 20 8C, 4 8C and 20 8C) over a period of one month. 2. Materials and methods 2.1. Reagents 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); sulphuric acid (98%) and ethyl acetate were purchased from Merck (Milan, Italy); BSTFA +1% TMCS derivatising agent was purchased Supelco (Bellefonte, USA). 3. Extraction and analysis The analyses were performed following the published method suggested by Elliott [13], which has been adapted and revalidated to the conditions of the present study according to the guidelines of Peters et al. [14]. For the extraction, in a 4 mL capped vial 100 mL blood or urine were added, followed by 50 mL of GHB-D6 internal standard (10 mg/L in 0.005 M H2SO4). After vortex mixing, five hundred microliters of ethyl acetate extraction solvent was added and vortex mixed for 30 s followed by centrifugation at 5000 rpm for 5 min. Five hundred microliters of upper (solvent) layer was transferred to a glass vial and evaporated to dryness at 45 8C under nitrogen flow. Seventy-five microliters of BSTFA 1% TMCS derivatizing agent was added followed by brief vortex mixing and incubation at 90 8C for 5 min in a heating block. After cooling and further brief vortex mixing, the solution was transferred to a GC–MS vial insert ready for injection. The injection volume was 1 mL. The GC/MS analysis was carried out using an Agilent Technologies (AT) model 6890 N GC
145
coupled with an AT mod. 5973 Inert mass selective detector and an AT 7683 Series automatic sampler at the following chromatography conditions: DB5-MS capillary column (30 m, 0.32 mm i.d.) with a temperature gradient starting at 60 8C (for 2 min) and ramping to 180 8C (20 8C/min for 6 min) then ramping to 250 8C (50 8C/min for 1 min) and finally 70 8C post-run. The mass spectrometer operated in the electron impact (EI) mode with ionization energy of 70 eV. The ion source temperature was 230 8C, quadrupole temperature 150 8C and solvent delay 2 min. The following ions were measured in selected ion monitoring (SIM) mode: derivatized GHB (GHB-diTMS): 233-target, 248, 147, 117, 73, IS GHB-D6 (GHB-D6-diTMS): 239-target, 120. Quantification was based on peak area ratios of the target ion relative to the respective internal standard (IS). 4. Sensitivity and linearity The lower limit of quantification (LLOQ) for GHB was defined as the lowest standard on the calibration curve and it was 0.5 mg/L, with a peak response at least 10 times (S/N 10) that of the blank response. Two calibration curves were used, each of them have included 7 points. The following GHB calibrators: 0.5, 1, 5, 10, 15, 30 and 40 mg/L were prepared in blank (pre-screened) equine plasma for the analysis of blood samples and in human urine (endogenous GHB concentration determined to be 0.20). A first analysis of GHB concentration was performed in blood (collected from femoral veins for all cases) and urine samples immediately after autopsy and then 1 mL aliquots were transferred in 2 mL Eppendorf capped tubes and all samples were stored at three different temperatures: 20 8C, 4 8C and 20 8C and extracted and analyzed at three days, 1 week, 2 weeks, 3 and 4 weeks in duplicate. No preservatives were added to the specimens. 6. Determination of creatinine concentrations and creatinine normalization procedure In urine samples, the creatinine (CR) concentration was performed by using a Beckman Synchron CX3 Clinical Analyzer. GHB concentrations in urine specimens were creatinine-normalized and the procedure used for CR normalization was based on a previously published method [15], using the following equation:
F.P. Busardo` et al. / Forensic Science International 243 (2014) 144–148
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Table 1 Main features of the three groups divided according to the post-mortem interval. Mean age (years) (S.D.)
Sex
Mean PMI (hours) S.D.(range)
Place of death/discovery
Mean ET (8C) and HR (%) (S.D.)
Cause of death
First group (n = 10)
42.7(14.2)
7 M/3 F
27 3.6(26–36)
Hospital (6)Street (3) Private flat (1)
18.3(5.6)46(7.8)
Second group(n = 10)
49.6(12.5)
6 M/4 F
59 11.7(37–72)
Private flat (5) Basement (4) Car parking (1)
15.9(6.4)51(11.2)
Third group(n = 10)
47.7(15.1)
7 M/3 F
142.4 45.6(73–192)
Private flat (7) Basement (2) Stable (1)
15.2(4.4)49(12.8)
RTA (3) Cardiac diseases (3) PT (2) Hanging (1) Firearm injuries (1) Cardiac diseases (3) PT (2) Stabbing injuries (2) Hanging (2) Strangulation (1) Cardiac diseases (4) Firearm injuries (2) Stroke (2) Stabbing injuries (1) Hanging (1)
ET: environmental temperature; HR: humidity rate; RTA: road traffic accident; PT: pulmonary thromboembolism.
Concentration CR normalized = GHB concentration (CR reference)/(CR specimen). The CR reference utilized in all corrections was 100 mg/dL [15]. 7. Statistical analysis The statistical analysis of data was performed by using the analysis of variance in order to compare the three groups, and ttest (for the comparison of groups two by two). The STATA software 11.0 version was used. 8. Results In the first group the GHB mean concentration measured after autopsy was: 2.14 mg/L (range 0.54–3.21 mg/L) in blood and [(Fig._1)TD$IG]
3.90 mg/g (range 0.60–4.81 mg/g) in urine; in the second group it was: 5.13 mg/L (range 1.11–9.60 mg/L) in blood and 3.93 mg/g (range 0.91–7.25 mg/g) in urine; in the third group it was: 11.8 mg/ L (range 3.95–24.12 mg/L) in blood and 9.83 mg/g (range 3.67– 21.90 mg/g) in urine (see Figs. 1 and 2). For urine samples, GHB concentrations were creatininenormalized, although there was no statistically significant difference (p > 0.20) between concentrations of endogenous GHB before and after creatinine-normalization. The results obtained in blood and urine samples showed a statistically significant difference among groups (p < 0.001) in the first analysis performed immediately after autopsy. In particular, in blood samples the mean difference was +2.99 mg/L (p < 0.01) for the second group and +9.66 mg/L (p < 0.0001) for the third group, both in comparison to the first group. In urine samples, the
Fig. 1. (A) Blood samples (First group, PMI range 24–36 h). (B) Blood samples (Second group, PMI range 37–72 h). (C) Blood samples (Third group, PMI range 73–192 h). (A–C) GHB mean concentration (mg/L) measured after autopsy and mean values obtained at three different storage temperatures: 20 8C, 4 8C and 20 8C and extracted and analyzed at three days, 1 week, 2 weeks, 3 and 4 weeks. (D) Blood samples. In blood samples the mean increase Is 2.99 mg/L (p < 0.01) for the second group and 9.66 mg/L (p < 0.0001) for the third group, both in comparison to the first group.
[(Fig._2)TD$IG]
F.P. Busardo` et al. / Forensic Science International 243 (2014) 144–148
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Fig. 2. (A) Urine samples (First group, PMI range 24–36 h). (B) Urine samples (Second group, PMI range 37–72 h). (C) Urine samples (Third group, PMI range 73–192 h). (A–C) GHB mean concentrations after creatinine normalization (mg/g) measured after autopsy and mean values obtained at three different storage temperatures: 20 8C, 4 8C and 20 8C and extracted and analyzed at three days, 1 week, 2 weeks, 3 and 4 weeks. (D) Urine samples. The mean increase is not significant for the second group (0.03 mg/g; p = 0.98), whereas it is significant for the third group (5.93 mg/L; p < 0.005), both in comparison to the first group.
mean difference was not significant for the second group (+0.03 mg/g; p = 0.98), whereas it was significant for the third group (+5.93 mg/g; p < 0.005), both in comparison to the first group. Throughout the period of investigation up to 4 weeks, the comparison of storage temperatures within each group showed in blood samples a mean difference at 20 8C compared to 20 8C of + 0.29 mg/L (first group at 4 weeks), +0.71 mg/L (second group at 4 weeks) and +1.62 mg/L (third group at 4 weeks); these differences in concentration were not statistically significant at the 10% level. In urine samples a mean difference at 20 8C compared to 20 8C was of +0.62 mg/g (first group at 4 weeks), +0.64 (second group at 4 weeks) and +1.67 (third group at 4 weeks); these increases were not statistically significant at the 10% level. During the period of investigation, the highest mean increase of concentration in blood, was found at 3 weeks in the third group at the storage temperature of 20 8C (2.7 mg/L), which is not significant (p = 0.29). Differently from blood, the highest mean increase of concentration in urine, was found at 4 weeks in the third group at the storage temperature of 20 8C (2.41 mg/L), which is not significant (p = 0.31). 9. Discussion and conclusions Endogenous GHB concentrations in cadaveric blood and urine are often markedly higher than living subjects, showing variable ranges depending on different studies [16,17]; Kintz et al. [17] tested GHB endogenous concentrations in 71 cardiac blood specimens and the results obtained were in the range 0.4– 409 mg/L, with a major distribution in the range 10–40 mg/L and a concentration >50 mg/L was observed only in 14 cases. In five cases Kintz et al. [17] compared the corresponding GHB concentrations in cardiac and femoral blood and the results
obtained showed a mean concentration in femoral blood (30.4 mg/ L) more than four times lower than cardiac blood (133.5 mg/L). In the study here presented, blood specimens (30 cases) were collected always from femoral veins and taking into account the three postmortem intervals the following range of values was obtained: 0.54–24.12 mg/L (blood samples analyzed immediately after autopsy). These values fall within the ranges above reported. Regarding postmortem urine samples different concentrations have been reported in literature; Elliott [18] presented the following range 0–18 mg/L, whereas Moriya et al. [19] reported a mean concentration of 0.6 1.2 mg/L. In our study, taking into account the three postmortem intervals the following range of values was obtained: 0.58–22.13 mg/L (urine samples analyzed immediately after autopsy); the upper value of this range is higher (4.13 mg/L) than the upper value reported by Elliott [18], who measured GHB concentrations in urine samples from 40 fatalities unrelated to GHB, but in that case the PMI ranged from 2 to 62 days. Moreover, GHB concentrations in urine samples after creatinine-normalization did not show a significant difference (p > 0.20). These results are consistent with data obtained by LeBeau et al. [20], who evaluated intra and interindividual variations in urinary concentrations of endogenous GHB in living subjects. However, this does not suggest that creatinine-normalization is not beneficial. Such an endeavor removes the effect that urine dilution has on the interpretation of endogenous concentrations [20]. Because numerous factors can affect post mortem GHB production, in particular the length of time after death and the temperature at which cadavers are placed [21,22], different mechanisms are thought to be involved, e.g. residual enzyme activities and putrefactive bacteria, especially during the early postmortem period [11,23,24]; for this reason the Authors in order
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F.P. Busardo` et al. / Forensic Science International 243 (2014) 144–148
to evaluate the role of PMI for GHB production both in blood and urine specimens, have selected three groups with no statistical significant differences (p > 0.20) in terms of age, sex, exposure to environmental temperature and humidity. The results obtained, allow us to affirm that the PMI strongly affects the post mortem production of GHB in blood and urine samples, taking into consideration the statistically significant difference among groups (p < 0.001) in the first analysis performed after autopsy. It is interesting to note that in blood samples the mean difference of concentrations in the second and third group, both in comparison to the first group was statistically significant (+2.99 mg/L; p < 0.01 and 9.66 mg/L; p < 0.0001, respectively) whereas, in urine samples only the mean difference of the third group in comparison to the first group was significant (+5.93 mg/g; p < 0.005). Taking into consideration that the estimation of PMI is a crucial step in forensic investigation and sometimes this estimation can be very difficult to achieve [25], the three ranges of PMI here reported, according to which the 30 GHB-free users have been divided, may effectively help in the interpretation of post-mortem GHB levels in order to distinguish between endogenous production and exogenous administration. Regarding the new formation of GHB in vitro both in blood and urine samples of the three groups, which have been stored at three different temperatures ( 20 8C, 4 8C and 20 8C) over a period of one month, it showed no statistically significant increases at the 10% level. During the period of investigation, the highest mean increase of concentration in blood, was found at 3 weeks in the third group at the storage temperature of 20 8C (2.7 mg/L), which is not significant (p = 0.29), whereas the highest mean increase of concentration in urine, was found at 4 weeks in the third group at the storage temperature of 20 8C (2.41 mg/L), which is not significant (p = 0.31). Although there was no significant increases of GHB in vitro both in blood and urine samples throughout the period of investigation (4 weeks) at three different temperatures ( 20 8C, 4 8C and 20 8C) the lowest increases were found both in blood and urine at 20 8C (see Figs. 1–2), therefore we recommend the latter ( 20 8C) as optimal storage temperature. This study, although preliminary, for the small number of cases analyzed (n = 30), may provide a valid support in addressing open issues related to the behavior of gamma-hydroxybutyrate in postmortem blood and urine and certainly further investigations will be performed in order to increase current evidences. Conflict of interest statement The authors confirm that this article content has no conflicts of interest. Acknowledgement This work was funded by the Deutsche Forschungsgemeinschaft, grant no. 1208/6.
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Contents lists available at ScienceDirect
Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
Post mortem concentrations of endogenous gamma hydroxybutyric acid (GHB) and in vitro formation in stored blood and urine samples§ Francesco Paolo Busardo` a,*, Elisabetta Bertol b, Fabio Vaiano b, Giovanni Baglio c, Angelo Montana d, Nunziata Barbera d, Simona Zaami a, Guido Romano d a
Department of Anatomical, Histological, Forensic and Orthopaedic Sciences, Sapienza University of Rome, Viale Regina Elena 336, 00161, Rome, Italy Department of Health Sciences, Forensic Toxicology Division, University of Florence, Florence, Italy c MSc in Epidemiology, London, UK d Department ‘‘G.F. Ingrassia’’, Laboratory of Forensic Toxicology, University of Catania, Catania, Italy b
A R T I C L E I N F O
A B S T R A C T
Article history: Available online 25 July 2014
Gamma-hydroxybutyrate (GHB) is a central nervous system depressant, primarily used as a recreational drug of abuse with numerous names. It has also been involved in various instances of drug-facilitated sexual assault due to its potential incapacitating effects. The first aim of this paper is to measure the post-mortem concentration of endogenous GHB in whole blood and urine samples of 30 GHB free-users, who have been divided according to the post-mortem interval (PMI) in three groups (first group: 24–36 h; second group: 37–72 h; third group: 73–192 h), trying to evaluate the role of PMI in affecting post mortem levels. Second, the Authors have evaluated the new formation of GHB in vitro in blood and urine samples of the three groups, which have been stored at 20 8C, 4 8C and 20 8C over a period of one month. The concentrations were measured by GC–MS after liquid–liquid extraction according to the method validated and published by Elliot (For. Sci. Int., 2003). For urine samples, GHB concentrations were creatinine-normalized. In the first group the GHB mean concentration measured after autopsy was: 2.14 mg/L (range 0.54– 3.21 mg/L) in blood and 3.90 mg/g (range 0.60–4.81 mg/g) in urine; in the second group it was: 5.13 mg/ L (range 1.11–9.60 mg/L) in blood and 3.93 mg/g (range 0.91–7.25 mg/g) in urine; in the third group it was: 11.8 mg/L (range 3.95–24.12 mg/L) in blood and 9.83 mg/g (range 3.67–21.90 mg/g) in urine. The results obtained in blood and urine samples showed a statistically significant difference among groups (p < 0.001) in the first analysis performed immediately after autopsy. Throughout the period of investigation up to 4 weeks, the comparison of storage temperatures within each group showed in blood and urine samples a mean difference at 20 8C compared to 20 8C not statistically significant at the 10% level. These findings allow us to affirm that the PMI strongly affects the post mortem production of GHB in blood and urine samples. Regarding the new formation of GHB in vitro both in blood and urine samples of the three groups, which have been stored at 20 8C, 4 8C and 20 8C over a period of one month, although there was no significant increases of GHB levels throughout the period of investigation, the lowest increases were found both in blood and urine at 20 8C, therefore we recommend the latter as optimal storage temperature. ß 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Gamma hydroxybutyric acid (GHB) Endogenous concentrations Stored blood and urine samples, In vitro formation
1. Introduction § This paper is part of the special issue entitled ‘‘The 51st Annual Meeting of the International Association of Forensic Toxicologists (TIAFT)’’. September 2–3, 2013, Funchal, Medeira, Portugal. Guest edited by Professor Helena Teixeira, Professor Duarte Nuno Vieira and Professor Francisco Corte Real. * Corresponding author. Tel.: +39 06 49912622; fax: +39 06 4455335. E-mail address: [email protected] (F.P. Busardo`).
http://dx.doi.org/10.1016/j.forsciint.2014.07.019 0379-0738/ß 2014 Elsevier Ireland Ltd. All rights reserved.
Gamma-hydroxybutyrate (GHB) is a central nervous system 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’’) in a colourless, odourless liquid or white powder, tablet and capsule forms [1,2]. It
F.P. Busardo` et al. / Forensic Science International 243 (2014) 144–148
is also used as a therapeutic substance both in U.S.A. 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) it is used as adjuvant in the control of alcohol withdrawal syndrome and in the initial phase of multimodal treatment of alcohol dependence [3,4]. GHB is endogenously produced as metabolite and precursor of g-hydroxybutyrate (GABA), the principal inhibitory neurotransmitter and it acts as a neuromodulator in the GABA system. Moreover, growing evidence suggests that GHB affects several neuromodulatory systems in the central nervous system (CNS), such as neurosteroids, dopaminergic, serotonergic and cholinergic systems, growth hormone, opioids etc. [5]. It has also been involved in various instances of drug-facilitated sexual assault (DFSA) due to its potential incapacitating effects. 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 [6–8]. Gamma-butyrolactone (GBL) and 1,4-butanediol (1,4-BD) are sold as solvents prevalently for industrial use; they represents precursors/analogous of GHB and they are converted very quickly and easily into GHB after ingestion, however, they are not present endogenously [9,10]. In forensic investigations it is usually required to determine if any GHB detected is due to endogenous production or exogenous ingestion and it can generate interpretative problems in particular in cadaveric samples due to the post mortem production [11,12]. The first aim of this paper is to measure the post mortem concentration of endogenous GHB in whole blood and urine samples of 30 GHB free-users, who have been divided according to the post-mortem interval (PMI) in three groups, trying to evaluate the role of PMI in affecting post mortem levels. The second aim of this research was to determine the new formation of GHB in vitro in blood and urine samples of the three groups, which have been stored at three different temperatures ( 20 8C, 4 8C and 20 8C) over a period of one month. 2. Materials and methods 2.1. Reagents 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); sulphuric acid (98%) and ethyl acetate were purchased from Merck (Milan, Italy); BSTFA +1% TMCS derivatising agent was purchased Supelco (Bellefonte, USA). 3. Extraction and analysis The analyses were performed following the published method suggested by Elliott [13], which has been adapted and revalidated to the conditions of the present study according to the guidelines of Peters et al. [14]. For the extraction, in a 4 mL capped vial 100 mL blood or urine were added, followed by 50 mL of GHB-D6 internal standard (10 mg/L in 0.005 M H2SO4). After vortex mixing, five hundred microliters of ethyl acetate extraction solvent was added and vortex mixed for 30 s followed by centrifugation at 5000 rpm for 5 min. Five hundred microliters of upper (solvent) layer was transferred to a glass vial and evaporated to dryness at 45 8C under nitrogen flow. Seventy-five microliters of BSTFA 1% TMCS derivatizing agent was added followed by brief vortex mixing and incubation at 90 8C for 5 min in a heating block. After cooling and further brief vortex mixing, the solution was transferred to a GC–MS vial insert ready for injection. The injection volume was 1 mL. The GC/MS analysis was carried out using an Agilent Technologies (AT) model 6890 N GC
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coupled with an AT mod. 5973 Inert mass selective detector and an AT 7683 Series automatic sampler at the following chromatography conditions: DB5-MS capillary column (30 m, 0.32 mm i.d.) with a temperature gradient starting at 60 8C (for 2 min) and ramping to 180 8C (20 8C/min for 6 min) then ramping to 250 8C (50 8C/min for 1 min) and finally 70 8C post-run. The mass spectrometer operated in the electron impact (EI) mode with ionization energy of 70 eV. The ion source temperature was 230 8C, quadrupole temperature 150 8C and solvent delay 2 min. The following ions were measured in selected ion monitoring (SIM) mode: derivatized GHB (GHB-diTMS): 233-target, 248, 147, 117, 73, IS GHB-D6 (GHB-D6-diTMS): 239-target, 120. Quantification was based on peak area ratios of the target ion relative to the respective internal standard (IS). 4. Sensitivity and linearity The lower limit of quantification (LLOQ) for GHB was defined as the lowest standard on the calibration curve and it was 0.5 mg/L, with a peak response at least 10 times (S/N 10) that of the blank response. Two calibration curves were used, each of them have included 7 points. The following GHB calibrators: 0.5, 1, 5, 10, 15, 30 and 40 mg/L were prepared in blank (pre-screened) equine plasma for the analysis of blood samples and in human urine (endogenous GHB concentration determined to be 0.20). A first analysis of GHB concentration was performed in blood (collected from femoral veins for all cases) and urine samples immediately after autopsy and then 1 mL aliquots were transferred in 2 mL Eppendorf capped tubes and all samples were stored at three different temperatures: 20 8C, 4 8C and 20 8C and extracted and analyzed at three days, 1 week, 2 weeks, 3 and 4 weeks in duplicate. No preservatives were added to the specimens. 6. Determination of creatinine concentrations and creatinine normalization procedure In urine samples, the creatinine (CR) concentration was performed by using a Beckman Synchron CX3 Clinical Analyzer. GHB concentrations in urine specimens were creatinine-normalized and the procedure used for CR normalization was based on a previously published method [15], using the following equation:
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Table 1 Main features of the three groups divided according to the post-mortem interval. Mean age (years) (S.D.)
Sex
Mean PMI (hours) S.D.(range)
Place of death/discovery
Mean ET (8C) and HR (%) (S.D.)
Cause of death
First group (n = 10)
42.7(14.2)
7 M/3 F
27 3.6(26–36)
Hospital (6)Street (3) Private flat (1)
18.3(5.6)46(7.8)
Second group(n = 10)
49.6(12.5)
6 M/4 F
59 11.7(37–72)
Private flat (5) Basement (4) Car parking (1)
15.9(6.4)51(11.2)
Third group(n = 10)
47.7(15.1)
7 M/3 F
142.4 45.6(73–192)
Private flat (7) Basement (2) Stable (1)
15.2(4.4)49(12.8)
RTA (3) Cardiac diseases (3) PT (2) Hanging (1) Firearm injuries (1) Cardiac diseases (3) PT (2) Stabbing injuries (2) Hanging (2) Strangulation (1) Cardiac diseases (4) Firearm injuries (2) Stroke (2) Stabbing injuries (1) Hanging (1)
ET: environmental temperature; HR: humidity rate; RTA: road traffic accident; PT: pulmonary thromboembolism.
Concentration CR normalized = GHB concentration (CR reference)/(CR specimen). The CR reference utilized in all corrections was 100 mg/dL [15]. 7. Statistical analysis The statistical analysis of data was performed by using the analysis of variance in order to compare the three groups, and ttest (for the comparison of groups two by two). The STATA software 11.0 version was used. 8. Results In the first group the GHB mean concentration measured after autopsy was: 2.14 mg/L (range 0.54–3.21 mg/L) in blood and [(Fig._1)TD$IG]
3.90 mg/g (range 0.60–4.81 mg/g) in urine; in the second group it was: 5.13 mg/L (range 1.11–9.60 mg/L) in blood and 3.93 mg/g (range 0.91–7.25 mg/g) in urine; in the third group it was: 11.8 mg/ L (range 3.95–24.12 mg/L) in blood and 9.83 mg/g (range 3.67– 21.90 mg/g) in urine (see Figs. 1 and 2). For urine samples, GHB concentrations were creatininenormalized, although there was no statistically significant difference (p > 0.20) between concentrations of endogenous GHB before and after creatinine-normalization. The results obtained in blood and urine samples showed a statistically significant difference among groups (p < 0.001) in the first analysis performed immediately after autopsy. In particular, in blood samples the mean difference was +2.99 mg/L (p < 0.01) for the second group and +9.66 mg/L (p < 0.0001) for the third group, both in comparison to the first group. In urine samples, the
Fig. 1. (A) Blood samples (First group, PMI range 24–36 h). (B) Blood samples (Second group, PMI range 37–72 h). (C) Blood samples (Third group, PMI range 73–192 h). (A–C) GHB mean concentration (mg/L) measured after autopsy and mean values obtained at three different storage temperatures: 20 8C, 4 8C and 20 8C and extracted and analyzed at three days, 1 week, 2 weeks, 3 and 4 weeks. (D) Blood samples. In blood samples the mean increase Is 2.99 mg/L (p < 0.01) for the second group and 9.66 mg/L (p < 0.0001) for the third group, both in comparison to the first group.
[(Fig._2)TD$IG]
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Fig. 2. (A) Urine samples (First group, PMI range 24–36 h). (B) Urine samples (Second group, PMI range 37–72 h). (C) Urine samples (Third group, PMI range 73–192 h). (A–C) GHB mean concentrations after creatinine normalization (mg/g) measured after autopsy and mean values obtained at three different storage temperatures: 20 8C, 4 8C and 20 8C and extracted and analyzed at three days, 1 week, 2 weeks, 3 and 4 weeks. (D) Urine samples. The mean increase is not significant for the second group (0.03 mg/g; p = 0.98), whereas it is significant for the third group (5.93 mg/L; p < 0.005), both in comparison to the first group.
mean difference was not significant for the second group (+0.03 mg/g; p = 0.98), whereas it was significant for the third group (+5.93 mg/g; p < 0.005), both in comparison to the first group. Throughout the period of investigation up to 4 weeks, the comparison of storage temperatures within each group showed in blood samples a mean difference at 20 8C compared to 20 8C of + 0.29 mg/L (first group at 4 weeks), +0.71 mg/L (second group at 4 weeks) and +1.62 mg/L (third group at 4 weeks); these differences in concentration were not statistically significant at the 10% level. In urine samples a mean difference at 20 8C compared to 20 8C was of +0.62 mg/g (first group at 4 weeks), +0.64 (second group at 4 weeks) and +1.67 (third group at 4 weeks); these increases were not statistically significant at the 10% level. During the period of investigation, the highest mean increase of concentration in blood, was found at 3 weeks in the third group at the storage temperature of 20 8C (2.7 mg/L), which is not significant (p = 0.29). Differently from blood, the highest mean increase of concentration in urine, was found at 4 weeks in the third group at the storage temperature of 20 8C (2.41 mg/L), which is not significant (p = 0.31). 9. Discussion and conclusions Endogenous GHB concentrations in cadaveric blood and urine are often markedly higher than living subjects, showing variable ranges depending on different studies [16,17]; Kintz et al. [17] tested GHB endogenous concentrations in 71 cardiac blood specimens and the results obtained were in the range 0.4– 409 mg/L, with a major distribution in the range 10–40 mg/L and a concentration >50 mg/L was observed only in 14 cases. In five cases Kintz et al. [17] compared the corresponding GHB concentrations in cardiac and femoral blood and the results
obtained showed a mean concentration in femoral blood (30.4 mg/ L) more than four times lower than cardiac blood (133.5 mg/L). In the study here presented, blood specimens (30 cases) were collected always from femoral veins and taking into account the three postmortem intervals the following range of values was obtained: 0.54–24.12 mg/L (blood samples analyzed immediately after autopsy). These values fall within the ranges above reported. Regarding postmortem urine samples different concentrations have been reported in literature; Elliott [18] presented the following range 0–18 mg/L, whereas Moriya et al. [19] reported a mean concentration of 0.6 1.2 mg/L. In our study, taking into account the three postmortem intervals the following range of values was obtained: 0.58–22.13 mg/L (urine samples analyzed immediately after autopsy); the upper value of this range is higher (4.13 mg/L) than the upper value reported by Elliott [18], who measured GHB concentrations in urine samples from 40 fatalities unrelated to GHB, but in that case the PMI ranged from 2 to 62 days. Moreover, GHB concentrations in urine samples after creatinine-normalization did not show a significant difference (p > 0.20). These results are consistent with data obtained by LeBeau et al. [20], who evaluated intra and interindividual variations in urinary concentrations of endogenous GHB in living subjects. However, this does not suggest that creatinine-normalization is not beneficial. Such an endeavor removes the effect that urine dilution has on the interpretation of endogenous concentrations [20]. Because numerous factors can affect post mortem GHB production, in particular the length of time after death and the temperature at which cadavers are placed [21,22], different mechanisms are thought to be involved, e.g. residual enzyme activities and putrefactive bacteria, especially during the early postmortem period [11,23,24]; for this reason the Authors in order
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to evaluate the role of PMI for GHB production both in blood and urine specimens, have selected three groups with no statistical significant differences (p > 0.20) in terms of age, sex, exposure to environmental temperature and humidity. The results obtained, allow us to affirm that the PMI strongly affects the post mortem production of GHB in blood and urine samples, taking into consideration the statistically significant difference among groups (p < 0.001) in the first analysis performed after autopsy. It is interesting to note that in blood samples the mean difference of concentrations in the second and third group, both in comparison to the first group was statistically significant (+2.99 mg/L; p < 0.01 and 9.66 mg/L; p < 0.0001, respectively) whereas, in urine samples only the mean difference of the third group in comparison to the first group was significant (+5.93 mg/g; p < 0.005). Taking into consideration that the estimation of PMI is a crucial step in forensic investigation and sometimes this estimation can be very difficult to achieve [25], the three ranges of PMI here reported, according to which the 30 GHB-free users have been divided, may effectively help in the interpretation of post-mortem GHB levels in order to distinguish between endogenous production and exogenous administration. Regarding the new formation of GHB in vitro both in blood and urine samples of the three groups, which have been stored at three different temperatures ( 20 8C, 4 8C and 20 8C) over a period of one month, it showed no statistically significant increases at the 10% level. During the period of investigation, the highest mean increase of concentration in blood, was found at 3 weeks in the third group at the storage temperature of 20 8C (2.7 mg/L), which is not significant (p = 0.29), whereas the highest mean increase of concentration in urine, was found at 4 weeks in the third group at the storage temperature of 20 8C (2.41 mg/L), which is not significant (p = 0.31). Although there was no significant increases of GHB in vitro both in blood and urine samples throughout the period of investigation (4 weeks) at three different temperatures ( 20 8C, 4 8C and 20 8C) the lowest increases were found both in blood and urine at 20 8C (see Figs. 1–2), therefore we recommend the latter ( 20 8C) as optimal storage temperature. This study, although preliminary, for the small number of cases analyzed (n = 30), may provide a valid support in addressing open issues related to the behavior of gamma-hydroxybutyrate in postmortem blood and urine and certainly further investigations will be performed in order to increase current evidences. Conflict of interest statement The authors confirm that this article content has no conflicts of interest. Acknowledgement This work was funded by the Deutsche Forschungsgemeinschaft, grant no. 1208/6.
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