GHB - Forensic Science Journal

Forensic Science Journal 2006;5:41-54 Available online at:fsjournal.cpu.edu.tw

FORENSIC SCIENCE JOURNAL SINCE 2002

Determination of gamma-hydroxybutyrate (GHB) in biological specimens by simultaneous extraction and chemical derivatization followed by GC-MS 1,

Sheng-Meng Wang, * Ph.D.; Yun-Seng Giang,1 Ph.D.; Min-Jen Lu,1 B.S.; Tsung-Li Kuo,2 Ph.D. 1Department

of Forensic Science, Central Police University, Taoyuan33304, Taiwan, ROC 2, Graduate School of Forensic Medicine, National Taiwan University, Taiwan, ROC

Received: September 26, 2006 /Received in revised form: October 19, 2006 /Accepted: October 23, 2006

Abstract Urine and chicken liver fortified with gamma-hydroxybutyrate (GHB) were pretreated with in-situ liquid-liquid extraction/chemical derivatization (LLE-ChD) or in-situ solid-phase extraction/chemical derivatization (SPE-ChD) followed by gas chromatography-mass spectrometry (GC-MS). GHB as its N,O-bis(trimethylsilyl)-trifluoroacetamide (BSTFA) derivative was recovered from urine in 23.7 % through the LLE-ChD procedure, in contrast to 60.7 % via the SPE-ChD counterpart. In the selective ion monitoring (SIM) mode, the qualifier ions selected for GHB-BSTFA were m/z 233, 234 and 235, with m/z 233 being the quantifier ion. Meanwhile, m/z 239, 240 and 241 were the qualifier ions for GHB-d6-BSTFA, and m/z 239 the quantifier ion. These ions have made all the cross contributions between the analyte and the corresponding internal standard (IS) smaller than 3.0 %. The calibration curves plotted for GHB in urine is linear within 500－10000 ng/mL, with correlation coefficients typically exceeding 0.999. The limits of detection and quantitation obtained via LLE approach are 300 and 500 ng/mL, respectively, and those via SPE approach, 100 and 200 ng/g, respectively. For urine samples fortified with 4000 and 8000 ng/mL of GHB, up to 32.6 % of the GHB decomposed after a period of fifteen days. In acidic media, 23.8 % of γ-butyrolactone (GBL) on average were hydrolyzed into GHB, whereas 11.8 % of GHB were converted to GBL. Keywords: Gamma-hydroxybutyrate (GHB), γ-Butyrolactone, 1,4-Butanediol, Club drug, Gas chromatography-mass spectrometry (GC-MS), Urine drug testing, Drug urinalysis

Introduction Following the footsteps of such “club drugs” as MDMA, FM2, ketamine, etc., the newly emerging gammahydroxybutyrate (IUPAC name: γ-hydroxybutyric acid; common name: GHB) has also been increasingly abused in a similar fashion among teenagers and young adults over the past couple years. It has often been used for criminal offenses, mostly as a date-rape drug. GHB is a substance naturally present in trace

* Corresponding author, e-mail:[email protected]

amount within mammal species. Properties of neurotransmitter or neuromodulator are generally given to this substance [1]. GHB was first synthesized in 1961 for anaesthetic use [2]. In Europe it has more often been used in the treatments of alcoholism [3-6], opiate withdrawal syndrome [7,8], and narcolepsy [9-11], while in the States it is just being evaluated for treating the latter. Oral administration of GHB by the abuser is followed by rapid gastric and/or intestinal absorption

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Forensic Science Journal 2006; Vol. 5, No. 1

of the drug, with its half-life being ca. 0.3－1.0 h. The climax of GHB level in urine for a 100-mg/kg oral dose is around 110 mg/dL and is usually reached no later than 4 h after the ingestion [12,13]. Regardless of the dosage, no more than 5 % of the orally ingested GHB will be detected intact in urine at any time, no urinary intact GHB at all will be detected at 12 h after the ingestion, and nearly all of the metabolites will be gone from the urine within the first 24 hours [1]. In contrast, the peak

of blood GHB for a 25-mg/kg oral dose comes as high as 8 mg/dL yet as early as 0.5 h after the ingestion [14]. With this dosage most GHB user feel dizzy and sleepy. Thus, although the climaxes of GHB in both blood and urine shift toward later periods as the dosage increases [15], the fact is that the window of detection of GHB is very short in these fluids and its presence is very difficult to prove after a rape case [1,16]. The metabolism of GHB is illustrated in Fig. 1.

Fig. 1 Metabolism of GHB GHB and its metabolites are currently not one of the NIDA-5 standardly tested for in the basic drug test, nor are they included in the extended drug tests [17]. This along with the short detection period and the inherent cross-interferences with the analysis has made GHB test much more expensive to give than the basic test. Nevertheless, in recent years, the tremendous increase in the abuse of GHB has prompted worldwide forensic toxicology laboratories to incorporate its analysis into their routine screening procedures. So far as the analysis of GHB is concerned, although GHB is a naturally occurring constituent of human body, the average level of endogenous GHB is reportedly so low (ranging 0.9－3.5 µg/mL with a mean of 1.65

µg/mL) as to be negligible compared to the proposed 1000-µg/mL cutoff concentration [18]. However, two organic solvents structurally similar or related to GHB, i.e., γ-butyrolactone (GBL) and 1,4-butanediol (1,4-BD; simply denoted BD in this paper), if involved in the sample, may complicate or interfere with the analysis. On the one hand, once absorbed into human body these two solvents are readily converted into GHB under the catalysis of alcohol dehydrogenase and lactonase as is shown in Fig. 2. On the other hand, the acidic conditions employed in some LLE, SPE, solidphase micro-extraction (SPME), and/or derivatization procedures or even during the course of instrumental analysis may well transform in vitro the GHB into

GHB urinalysis　43

GBL through an intramolecular esterification reaction [19-26]. It is therefore paramount that the quantification of GHB can be differentiated from those of GBL and BD, or kept free from the interferences of the latter two. Beside the improvements that have been made on the above stated sample preparation methods [22-26], isotope dilution GC-MS protocols employing GHB-d6 as IS [21,24] as well as capillary electrophoresis (CE) and high performance liquid chromatography (HPLC)

free from degradation of GHB to GBL commonly occurring on the GC injection port or column have also been reported [27,28]. Since GC-MS as a confirmatory test has proved most evidential and worldwide adopted for drug testing [22,29,30], the present study is devoted to the development of an analytical scheme comprising a compatible sample preparation procedure, optimal GC-MS parameters/conditions, and quality data interpretation.

Fig. 2 In-vivo transformation of 1,4-BD into GHB, and in-vivo interconversion between GHB and GBL. Experimental Materials Authentic GHB (1 mg/mL in methanol), GBL (1 mg/mL in methanol), and GHB-d6 (100 µg/mL in methanol) were purchased from Cerilliant Co., USA. They were each diluted tenfold with methanol to serve as the respective stock solutions, i.e., 100 µg/mL for GHB and GBL, and 10 µg/mL for GHB-d6. The derivatizing agents, N,O-bis(trimethylsilyl)-trifluoroacetamide (BSTFA) fortified with 1% trimethylchlorosilane, was

ordered from Aldrich Chemical Co., USA. The SPE cartridge, CLEAN SCREENTM ZSGHB020, containing 200 mg of CSGHB sorbent in a 10-mL column was obtained from United Chemical Technologies, Inc. All the other chemicals/solvents used in this study were in analytical or reagent grade, were from local chemical suppliers, and were directly used without further purification. Sample collection and spiking

44


Urine previously certified negative for all drugs was contributed by a voluntary student from the Central Police University. Drugs-free chicken liver was bought at a local supermarket in Taipei and was homogenized with physiological saline prior to spiking. To simulate the actual situation in Taiwan, all spiking steps were performed while the blank urine and chicken liver had not yet been refrigerated, but all specimens to be analyzed were preserved at 4℃ prior to the analysis. The serial calibrators used for plotting the method calibration curve were each prepared by adding an appropriate amount (10, 20, 50, 100, 200 µL for urine; 5, 10, 25, 50, 100 µL for chicken liver) of 100-µg/mL GHB solution to a screw-cap topped test tube containing 2 mL of blank urine or saline-homogenized chicken liver (containing 1 g of liver tissue), and 100 µL of 10-µg/mL GHB-d6 solution. (This would make the corresponding urinary spikes contain 500, 1000, 2500, 5000, and 10000 ng/mL, respectively, of GHB and 500 ng/mL of GHB-d6 and make the corresponding chicken liver spikes represent 500, 1000, 2500, 5000, and 10000 ng/g, respectively, of GHB and 500 ng/g of GHB-d6.) This study did not involve case work. If a real-case specimen is to be analyzed for GHB, simply 100 µL of 10-µg/mL GHB-d6 needs to be added at this starting step.

a screw-cap topped test tube was added 100 µL of pH 6 phosphate buffer. After 3 min of vortex, the mixture was passed (under reduced pressure, 20 mmHg) through an SPE cartridge that had been preconditioned in succession with 3 mL of methanol, 3 mL of D.I. water, and 0.5 mL of pH 6 phosphate buffer. The foregoing sample test tube was thoroughly rinsed with 1 mL of freshly prepared CH3OH/NH4OH (99:1,v/v) and the filtration under reduced pressure was repeated. The two portions of filtrate were combined in another screwcap topped test tube, purged at 60℃ with nitrogen gas to dryness, and then cooled to ambient temperature. The residues were re-dissolved with 200 µL of N,Ndimethylformamide (DMF) and 2 mL of n-hexane. After 10 min of vortex and 5 min of centrifugation (3000 rpm), the lower layer was transferred to a screw-cap topped concentration tube and purged at 60℃ with nitrogen gas to dryness. A 100-µL portion of BSTFA was added. The mixture was incubated with vortex at 70℃ for 20 min, cooled to ambient temperature, and then purged with nitrogen gas to dryness. Bring the volume to 100 µL with EA. A 1-µL aliquot of the reconstituted solution was injected for the GC-MS analysis.

General procedure of simultaneous LLE and BSTFAChD

The GC-MS analyses were carried out using a Hewlett-Packard HP-5890 Series II gas chromatograph coupled to an HP-5971 Series mass selective detector (MSD). The GC column used was an HP-5 MS capillary column (25 m * 0.2 mm I.D., 0.33μm film thickness). The GC was operated in the splitless mode (i.e., purge off) when performing injection with the aid of an HP-7673 autosampler, but 1 min later the purge valve was turned on. The injector temperature was 250℃. For the analysis of BSTFA-derivatized GHB, the column temperature was programmed from 60 to 100℃ at 15℃/ min, then from 100 to 250℃ at 25℃/min, with the initial temperature held for 2 min and final temperature held for 5 min. Helium of 99.999 % purity was used as the carrier gas at a flow-rate of 1 mL/min. Effluents from the GC column was transferred via a transfer line held at 280℃ to a 70-eV electron impact (EI) ionization source held at 180℃. The instrument was first operated in full-scan mode where relevant total ion current (GC-EIMS TIC) chromatograms and mass spectra were acquired to look into the fragmentation nature (with the aid of a software named “High ChemTM Mass Frontier Version 1.0”) of the previously unexplored BSTFA-derivatized analytes,

To 2 mL of urine or saline-homogenized chicken liver tissue fortified with GHB and GHB-d6 (see “sample collection and spiking” subsection) in a screw-cap topped test tube were added 0.2 mL of 0.5 N HCl and 6 mL of ethyl acetate (EA). After 10 min of vortex (LLE), the mixture was subjected to centrifugation at 3000 rpm for 5 min. The supernatant was transferred to a screw-cap topped concentration tube and purged with nitrogen gas to dryness. A 100-µL portion of BSTFA was added. The mixture was incubated with vortex at 70℃ for 20 min, cooled to ambient temperature, and then purged with nitrogen gas (Model N-EVAPTM112 equipped with twenty four Teflon tubes, Organomation Associates Inc., USA) to dryness. Bring the volume to 100 µL with EA. A 1-µL aliquot of the reconstituted solution was injected for the GC-MS analysis. Simultaneous SPE and BSTFA-ChD To 2 mL of urine fortified with GHB and GHB-d6 (see “sample collection and spiking” subsection) in

GC-MS analysis

GHB urinalysis　45

and then in selected ion monitoring mode (GC-EIMS SIM) to further evaluate the qualifier and quantifier ions and run the formal analysis. The calibration curves were produced by plotting the quantifier-ion-abundance ratio (analyte : IS) obtained from the SIM measurement against the concentration of the analyte in the fortified samples. Each peak-area ratio used was the mean of triplicate analyses. Results and discussion Mass chromatography Possessing two protic moieties on each of their molecules, GHB, GHB-d6, and BD were readily diderivatized with BSTFA (denoted di-TMS-GHB, diTMS-GHB-d6, and di-TMS-BD, respectively, where “TMS” stands for a “trimethylsilyl group”). In

Fig. 3

the GC-EIMS TIC chromatogram shown in Fig. 3, while di-TMS-GHB and di-TMS-BD are well separated, diTMS-GHB and di-TMS-GHB-d6 are also sufficiently resolved in the sense of analyte-IS quantifier ion pair cross contribution which is crucial to the accuracy of quantitation (see the subsection that follows). In contrast, the intramolecularly dehydrated GBL was neither capable of BSTFA-ChD nor co-detectable with the other three compounds in the GHB-oriented analysis although GBL and GHB were more or less inter-convertible under the acidic conditions adopted for sample preparation. Thus, the GC-EIMS TIC chromatogram obtained (with the same instrumental parameters as those for Fig. 3) upon the direct injection of 1 µL of underivatized GBL in ethyl acetate is shown separately in Fig. 4.

The isotope dilution GC-EIMS TIC chromatogram obtained for a urinary specimen containing 1,4-BD, GHB-d6, GHB, and GBL after the LLE/BSTFA-ChD pretreatment. GBL was not co-detected in this GHBoriented analysis.

46


Fig. 4

The GC-EIMS TIC chromatogram obtained upon the direct injection of 1 µL of GBL in ethyl acetate. The instrumental conditions/parameters are the same as those for Fig. 3.

Selection of qualifier and quantifier ions As the analyses of the chromatographically noninterfering GBL and 1,4-BD are not our primary concern and their deuterium-labeled ISs are not commercially available, we shall focus the discussion on the GCEIMS analysis of GHB and GHB-d6. Judging from our previous experience in analyzing amphetamines [31] and ketamine [32] as well as the relative mass difference between di-TMS-GHB and its deuteriumlabeled IS, satisfactory GC resolution between the latter two compounds might need the use of an IS labeled with more than eight or nine deuterium atoms. Being more available, however, the d6-labeled IS was used throughout the present study. This along with the demand of rapid analysis in forensic practice did lead to superficially inadequate GC separation between di-TMSGHB and di-TMS-GHB-d6 (Fig. 3). The appreciable overlap between the analyte and IS peaks caused their respective fragment ions to be mutually mixed up. Fortunately, through the more appropriate yet easier selection of qualifier/quantifier ions described below, the essential or effective resolution and hence the accuracy and precision for the to-be-routine GC-EIMS SIM analyses can still be secured.

Shown in Fig. 5 are the mass spectra of GBL, diTMS-1,4-BD, di-TMS-GHB, and di-TMS-GHB-d6 acquired in the full-scan mode. Those fragment ions which looked more intense and characteristic made good candidates of the respective qualifier and/or quantifier ions, and were further subjected to a detailed “analyteIS ion-pair cross-contribution evaluation” based on the total analysis (incorporating LLE-ChD and GC-EIMS SIM) of a working solution containing 5000 ng/mL each of GHB and GHB-d6 in methanol. Displayed in Table 1 are the relevant cross contributions calculated for the considered analyte-IS ion pairs. In the light of “minimized cross contribution,” the qualifier ions decided for the GC-EIMS SIM analysis of di-TMS-GHB were m/z 233, 234, and 235, and those for di-TMSGHB-d6 were m/z 239, 240, and 241, with their cross contributions typically below 3 %. Of these ions, the most abundant m/z 233 and 239 were decided to be the quantifier ions for di-TMS-GHB and di-TMS-GHB-d6, respectively. Since the base peak ion (m/z 147; assigned to be [TMS－O－DMS]＋, where “DMS” stands for a “dimethylsilyl group”) was common to di-TMSGHB and di-TMS-GHB-d6, it should be considered as neither a qualifier nor the quantifier ion.

GHB urinalysis　47

Fig. 5

Mass spectra of (a) GBL, (b) di-TMS-1,4-BD, (c)di-TMS-GHB, and (d) di-TMS-GHB-d6

48


Table 1 Relative intensities and cross contributionsa of ions with potential for designating the analyte and the IS

Analyte (di-TMS-GHB) (Full-scan) (SIM) % intensity Ion (m/z) Relative contributed by diintensity (%) TMS-GHB-d6 117 15.0 8.34

Ion (m/z) 120

IS (di-TMS-GHB-d6) (Full-scan) (SIM) % intensity Relative contributed by diintensity (%) TMS-GHB 5.46 2.20

b

147

100

NC

147

100

NCb

204

6.57

1.84

206

4.70

11.1

29.9

0.65

233c,d

27.9

1.72

239e,f

234d

5.55

1.85

240f

6.12

0.68

3.14

241f

2.84

0.73

235d

2.52

a

Relative intensities are based on full-scan data and expressed in percentage, while analogs’contributions (crosscontributions) are derived from SIM data and expressed also in percentage. b Not calculated. c Decided quantifier ion for the analyte. d Selected qualifier ion for the analyte. e Decided quantifier ion for the IS. f Selected qualifier ion for the IS.

Quantitation As the accuracy and precision for the measurement of drug level is highly crucial to the following legal interpretation of the results, every quantifier-ionabundance ratio used for plotting the calibration curves was the mean of triplicate analyses. Summarized in Table 2 are the relevant five-point calibration equations, linearity ranges, linear correlation coefficients (r2), and method limits of detection and quantitation (LODs/LOQs; previously defined [32,33]) for the

determination of GHB in urine and chicken liver by the two protocols under comparison. The SPE protocol afforded significantly lower LODs/LOQs than did the LLE protocol. Although there have been no officially adopted cutoff concentrations for GHBs, the LODs/ LOQs achieved hereby sufficiently meet the previously proposed 10-µg/mL cutoff for routine forensic GHB urinalysis, while that cutoff far overpasses the foregoing endogenous GHB level [18].

Table 2 Calibration equations, linearity ranges, linear correlation coefficients (r2), and method limits of detection and quantitation (LODs/LOQs) for the determination of GHB in urine and chicken liver by simultaneous extraction and BSTFA-ChD followed by GC-EIMS SIM Linearity range (ng/mL for urine; Calibration equation ng/g for chicken liver) (a) GC-EIMS SIM instrumental calibration a y=0.0023x-0.081

500－10000

Linear correlation coefficient (r2)

LOD (ng/mL for urine; ng/g for chicken liver)

LOQ (ng/mL for urine; ng/g for chicken liver)

0.999

(b) Method calibration for simultaneous LLE and BSTFA-ChD followed by GC-EIMS SIM Urine: y=0.0021x+0.7059

500－10000

0.999

400

500

Chicken liver: y=0.0023x+0.5331

500－10000

0.999

400

500

(c) Method calibration for simultaneous SPE and BSTFA-ChD followed by GC-EIMS SIM Chicken liver: y=0.0021x-0.0224 a

500－10000

0.999

Standard GHB unextracted and quantitative BSTFA-ChD presumed.

100

200

GHB urinalysis　49

Analyte recoveries indicative of method performances A five-pointed instrumental calibration curve (i.e., standard GHB unextracted and quantitative BSTFA-ChD presumed) was established in advance with the equation being y = 0.0023x-0.081 (Table 2). Five levels (within the working range, 500－10000 ng/mL) of urinary GHB spikes were then subjected respectively to the above described simultaneous extraction and BSTFA-ChD followed by GC-EIMS SIM analysis. The recoveries of GHB were obtained by dividing the hereby regressed concentration of GHB (actually recovered in the form

of di-TMS-GHB) by the corresponding concentration originally spiked. Thus, two series of recoveries of GHB from urine achieved via LLE and SPE, respectively, are displayed in Table 3. The SPE process with recoveries ranging 44.0－84.5 % and the mean being 60.7 % has proved much more effective than the LLE process with recoveries ranging 20.8－26.7 % and the mean being 23.7 %. The foregoing higher LODs/LOQs for the LLE protocol may be largely attributed to its poorer recoveries.

Table 3 Recoveries of GHB from urine achieved via LLE and SPE a

Level of GHB (ng/mL) b

Recovery (％） LLE

SPE

500

22.37

84.47

1000

20.77

66.07

2500

23.36

43.96

5000

25.32

46.42

10000

26.69

62.69

Mean = 23.70

Mean = 60.72

a b

Quantitative ChD presumed. All tested levels are within the LLE and SPE’ s linearity range, 500－10000 ng/mL.

Influences of temperature and time length on the efficiency of BSTFA-derivatization of GHB.

Peak area ratio (Analyte:IS)

The temperature (70℃) and time length (20 min) for the BSTFA-derivatization of GHB described in the experimental section was not adopted until they turned out to be the optimal. Fixed amounts of GHB and GHB-d6 in urine (2 mL × 5000 ng/mL) were subjected to the foregoing LLE-ChD procedure with the ChD being

carried out at 60, 70, 80, and 90℃, and for 5, 10, and 20 min as variants. The resulting di-TMS-derivatives were submitted to the GC-EIMS SIM analysis, and the relative ChD efficiencies in terms of analyte-to-IS ratio are portrayed in Fig. 6. Apparently, 70℃ and 20 min were the optimal conditions and were used throughout the study.

Fig. 6 Influences of temperature and time length on the efficiency of BSTFA-derivatization of GHB

50


Comparison among five solvents with respect to their efficiencies in extracting GHB from urine.

the relative LLE efficiencies in terms of analyte-to-IS ratio are contrasted in Table 4. Although chloroform/ isopropanol (80:20, v/v) is superior to the other four in doing the job, the second most efficient solvent, EA, was chosen for use throughout the study due to environmental protection consideration and its better compatibility with GC-MS analysis.

A fixed amount (6 mL) of EA, chloroform/ isopropanol (80:20, v/v), n-haxane, acetonitrile, and methylene chloride as the extraction solvent was respectively applied to the foregoing LLE-ChD of 2 mL of 5000-ng/mL GHB. The resulting di-TMS-derivatives were submitted to the GC-EIMS SIM analysis, and

Table 4 Efficiencies achieved for the LLE of GHBa from urine using five solventsb

Solvent Acetonitrile

Efficiency (in terms of analyte-to-IS peak-area ratio) 0

Ethyl acetate

2.55

n-Hexane

0

Chloroform/isopropanol (80:20, v/v)

4.92

Methylene chloride

0

a b

GHB used was 5000 ng/mL × 2 mL. The amount of solvent used for each extraction was 6 mL.

Optimization of LLE efficiency by pH adjustment The optimization process was performed in two steps. First, various normalities (12, 6, 3, 1, 0.5, and 0.1 N) of hydrochloric acid were respectively applied to the foregoing LLE-ChD procedure while keeping the other parameters unchanged. The resulting di-TMS-derivatives were submitted to the GC-EIMS SIM analysis, and the relative LLE efficiencies in terms of analyte-to-IS ratio are profiled in Fig. 7. The efficiency increases with decreasing HCl normality until a maximum is reached at 0.5 N, where the efficiency begins to turn down

drastically to that resulting from 0.1 N HCl. Afterward various amounts (100, 200, 300, 400, 500, and 600 µL) of the optimal (0.5 N) HCl were respectively applied to the same LLE-ChD procedure while keeping the other parameters invariant. The resulting di-TMS-derivatives were submitted to the GC-EIMS SIM analysis again, and the relative LLE efficiencies in terms of analyte-toIS ratio are profiled in Fig. 8. The optimal amount of 0.5 N HCl for the LLE turned out to be 200 µL, which is equivalent to pH 1-2.

Fig. 7 Variation of efficiency with pH in the LLE of GHB from urine.

Peak area ratio (Analyte;IS)

GHB urinalysis　51

Fig. 8 Variation of efficiency with the amount of 0.5 N HCl used in the LLE of GHB from urine. In vitro interconversion between GHB and GBL Putting aside the previously mentioned negligible phenomenon of endogenous GHB, this subsection deals with the potential problem caused by the also foregoing interconversion between GHB and GBL, as 0.5 N HCl was routinely used for our LLE-ChD procedure. Shown in Table 5 are the estimated percentages of GBL in urine (5000 ng/mL×2 mL, initially) hydrolyzed into GHB after going through the LLE-ChD procedure employing various normalities (0.1, 0.5, 1, 3, 6, and 12 N) of 200-µL HCl. The percentage of GBL hydrolysis is directly dependent upon the amount of GHB recovered after the LLE-ChD process. Although 0.1 N HCl led to the smallest percentage of GBL hydrolysis (3 %),

that was attributed mainly to the above shown (Fig. 7) lowest LLE-ChD efficiency for GHB in urine under 0.1 N HCl. The truly smallest and most desirable GBL hydrolysis percentage should be the one estimated under 0.5 N HCl, i.e., an average of 23.8 % based on triplicate analyses, with the RSD being 2.9 %. The higher the HCl normality, the larger the GBL hydrolysis percentage. The largest GBL hydrolysis percentage was 47.5 % estimated for 12 N HCl. Since normally an individual should not have ingested any GBL and his endogenous GBL should also be negligibly low, we would usually ignore the error caused by GBL hydrolysis for the quantification of GHB.

Table 5 Percentages of GBL in urine (5000 ng/mL×2 mL, initially) hydrolyzed into GHB after going through the LLE-ChD procedure employing various normalities of 200-µL HCl

HCl normality

Final GHB level (ng/mL)

% GBL hydrolyzed into GHB

0.1

164 Trial 1 1222 Trial 2 1188 Trial 3 1152

3.3 24.5 23.8 23.1

Mean = 1187 RSD = 2.9 ﹪

Mean = 23.8

1256 1447 1468 2374

25.1 28.9 29.4 47.5

0.5

1 3 6 12

52


Table 6 presents the estimated percentages of GHB in urine (5000 ng/mL×2 mL, initially) that converted into GBL after going through the LLE-ChD procedure employing various normalities (0.1, 0.5, 1, 3, 6, and 12 N) of 200-µL HCl. The percentage of GHB esterification is proportional to the difference between the two GHB levels before and after the LLE-ChD process. Once again the percentage of GHB esterification estimated for 0.1 N HCl is unreliable due to the too poor GHB recovery after the LLE-ChD process. The percentage estimated for 0.5 HCl (average of triplicate analyses, 11.8 %; RSD, 3.5 %) is smaller than those for 6 and 12 N HCl (25.3 %), comparable with that for 3 N HCl,

but larger than that for 1 N HCl (2.2 %). Probably due to non-quantitative LLE recovery and ChD yield, the percentages estimated for the corresponding GHB esterification and GBL hydrolysis, apparently, will not correlate well with the theoretical equilibrium constant for GHB-GBL interconversion (unavailable, though). However, since the extent of GHB esterification is already significant under 0.5 N HCl, if it were not for the reason stated in the following subsection, it would be suggested that for routine GHB-case samples a relative percentage of 10 be added to the GHB outcome to compensate for the possible negative error.

Table 6 Percentages of GHB in urine (5000 ng/mL×2 mL, initially) converted into GBL after going through the LLE-ChD procedure employing various normalities of 200-µL HCl

HCl normality

Final GHB level (ng/mL)

% GHB converted into GBLa

0.1

42 Trial 1 4407 Trial 2 4570 Trial 3 4258 Mean = 4412

99.2 11.9 8.6 14.8 Mean = 11.8

0.5 1 3 6 12 a

RSD = 3.5 ﹪

4890 4456 4189 3734

2.2 10.9 16.2 25.3

Estimated from the difference between the two GHB levels before and after the LLE-ChD process.

Stability of GHB in urine Keeping the above stated in vivo and in vitro instability of GHB in mind, we conducted an experiment to further understand the durableness of GHB urine specimens. Displayed in Table 7 are the percentages of GHB in urine (4000 and 8000 ng/mL×2 mL, initially) that converted automatically into GBL after 2, 3, and 15 days of preservation at 4℃. For the 4000-ng/mL specimens, no GHB esterification was discerned after 2 or 3 days of storage, but 32.6 % of GHB was converted into GBL after 15 days of storage. For the 8000-ng/mL specimens, 5.9, 7.4, and 29.5 % of the GHB was transformed into GBL after 2, 3, and 15

days, respectively, of preservation. That is, the higher the tested GHB level and the longer the sample stored, the more appreciable the GHB loss. Thus, depending on the firstly detected GHB level, an appropriate compensation needs to be made for the storage-loss of GHB. Theoretically, this addition to GHB level should have incorporated the contribution from the foregoing “relative 10 %”. Unfortunately, relevant data obtained hereby seem to correlate poorly. More detailed and precise evaluation and calibration are in need. At this time, it is still safe to suggest that for routine GHB-case samples a relative percentage of 10 be added to the GHB outcome to compensate for the possible negative error.

GHB urinalysis　53

Conclusions The results presented in this report demonstrated that simultaneous extraction and BSTFA-ChD followed by GC-EIMS SIM is a promising protocol for the determination of GHB in urine and other biological specimens. While the GC-EIMS SIM counterpart leaves no problem with the identification and quantification of GHB, the SPE protocol provides higher extraction efficiency and, hence, lower LOD/LOQ than the LLE protocol. However, the LLE approach is better suited for the exploration of many basic yet unestablished analytical properties of the newly targeted GHB, such as solvent and pH effects on the extraction efficiency, possible source of analytical errors, and sample durableness. For either of the proposed protocols to be more efficient, accurate, and precise, we shall undertake more detailed evaluation and calibration with respect to the above stated properties. Acknowledgment Financial support by the National Bureau of Controlled Drugs, Department of Health, Republic of China under Grant No. DOH91-NNB-1011 is gratefully acknowledged. References 1. Kintz P, Cirimele V, Jamey C, Ludes, B. Testing for GHB in hair by GC/MS/MS after a single exposure application to document sexual assault. J Forens Sci 2003; 8(1):195-200 . 2. L a b o r i t H, J o u a n y J M, G e r a l d J, F a b i a n i F. Generalities concernant l’etude experimentale de l’emploi clinique du gamma hydroxybutyrate de Na. Aggressologie 1960; :407. 3. Addolorato G, Castelli E, Stefanini GF, Casella G, Caputo F, Marsigli L, Bernardi M, Gasbarrini G. An open multicentric study evaluating 4-hydroxybutyric acid sodium salt in the medium-term treatment of 179 alcohol dependent subjects. Alcohol Alcoholism 1996; 1(4):341-5. 4. Agabio R, Colombo G, Loche A, Lobina C, Pani ML, Reali R, Gessa GL. Gamma-hydroxybutyric acid reducing effect on ethanol intake: evidence in favour of a substitution mechanism. Alcohol Alcoholism 1998; 3(5):465-74. 5. Galloway GP, Frederick SL, Staggers FE Jr, Gonzoles M, Stalcup SA, Smith DE. Gammahydroxybutyrate: an emerging drug of abuse that

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