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Window of Detection of -Hydroxybutyrate in Blood and Saliva

¹ Institut de Médecine Légale, 11 rue Humann, F-67000 Strasbourg, France

² Centre Hospitalier du Havre, 76083 Le Havre, France

^aauthor for correspondence: fax 33-3-90-24-33-62, e-mail pascal.kintz{at}wanadoo.fr

-Hydroxybutyrate (GHB) is an endogenous constituent of the brain, produced by the metabolism of -aminobutyric acid and believed to play a role in neurotransmission (1). GHB is a suppressant of the central nervous system that is used in some countries as an anesthetic adjunct and is also sometimes used in treating sleep disorders and alcoholism (2). The primary effects of GHB are those of a depressant and can include reducing inhibition, increasing libido, and causing amnesia, seizure, unconsciousness, coma, and death.

In recent years, GHB has gained popularity as an illicit drug. It is abused by young people at nightclubs or raves, mainly for its euphorigenic and hallucinogenic effects. The drug has been promoted among bodybuilders because it is alleged to possess anabolic properties. Moreover, criminals can use GHB to narcotize potential victims, and therefore, the drug is used in drug-facilitated sexual assaults resulting from sedation and reduced inhibitions (3).

GHB has a half-life of 20–53 min, which appears to increase with higher doses (4). Because the drug is rapidly eliminated from the body, GHB exposure is difficult to document.

To compare the window of detection of GHB, blood and saliva were collected after a single exposure. A GHB dose of 60 mg/kg (total, 4680 mg) was administered orally to a 54-year-old male (78 kg) on an empty stomach, under close medical supervision in an intensive care unit. Approval by local authorities was obtained, along with the written consent of the volunteer. The drug was dissolved in 200 mL of mineral water. The volunteer did not take any prescribed or over-the-counter medications.

Blood (in EDTA tubes) and saliva (in plastic tubes, without stimulation) were collected simultaneously for 5 h.

We analyzed GHB in whole blood by gas chromatography–mass spectrometry (GC-MS) after acetonitrile precipitation in the presence of GHB-d₆ followed by silylation using a published procedure (5). A modification of the method was used for saliva. Briefly, saliva samples (20 µL) were supplemented with 500 ng of GHB-d₆ and precipitated with 50 µL of acetonitrile. After vortex-mixing and centrifugation, the supernatant was evaporated to dryness. The residue was derivatized by adding 30 µL of N,O-bis(trimethylsilyl)trifluoroacetamide plus 10 g/L trimethylchlorosilane, and then incubating for 20 min at 60 °C.

An aliquot of the derivatized extract (1 µL) was injected into the column of a Hewlett Packard gas chromatograph (6890 Series) via a Hewlett Packard autosampler (7673). The flow of carrier gas (helium; purity grade, N 55) through the column (HP5-MS capillary column; 50 g/L phenylsiloxane–950 g/L methylsiloxane; film thickness, 0.25 mm; 30 m x 0.25 mm i.d.) was 1.0 mL/min. The injector temperature was 250 °C, and splitless injection was used with a split-valve off-time of 1.0 min in the pulsed mode. The column oven temperature was programmed to increase from an initial temperature of 36 °C, maintained for 1 min, to 100 °C at 10 °C/min, and then to 295 °C at 30 °C/min.

The detector was a Hewlett Packard 5973 operated in the electron-impact mode. The electron multiplier voltage was set to 600 V above the electron-impact-tune voltage.

Under the chromatographic conditions used, there was no interference with GHB and the internal standard by any extractable endogenous materials present in saliva. GHB–ditrimethylsilyl derivative was stable for 24 h after derivatization. GHB was identified and quantified on the basis of its retention times (± 1% from that of the reference materials) and the abundance of three diagnostic ions (with ion ratios within ± 20% of the reference materials). GHB was eluted at 8.30 min and monitored using m/z ions 159, 204, and 233. Quantification was performed by comparing the areas of ions m/z 233–239 (GHB-d₆).

Response for GHB was linear in the range of 2–300 mg/L (r = 0.994). The within-run imprecision was 13%, as determined by analysis of 8 replicates of 20 µL of drug-free saliva with a final GHB concentration of 50 mg/L. The extraction recovery (n = 3 at 50 mg/L) was 69.3%. The limit of detection (signal-to-noise ratio >3) of GHB was 1 mg/L.

Concentrations of GHB in blood and saliva are reported in Table 1 . The peak blood concentration was observed 20 min after administration. The increase of GHB in blood at 60 min can be explained by the fact that both the oral absorption and the elimination of GHB are capacity-limited processes. This was also suggested by Palatini et al. (4). Because only one participant was involved, no attempt to calculate any pharmacokinetic values was undertaken.

The analysis of the saliva specimens demonstrated that oral contamination was rapidly washed out after 20 min. A second oral contamination was possible as an explanation for the increase of the saliva/blood ratio to >1 at 90 min because the participant vomited 5 min before sampling. Overall, we believe that the saliva/blood ratio for GHB is <1. The increased salivary GHB at 90 min may be a consequence of the increase in blood at 60 min. To date, we have found no other report that documents GHB excretion in saliva. Because of a saliva/blood ratio <1, oral fluid may not be a suitable specimen to extend the window of detection of GHB.

References

1. Vayer P, Mandel P, Maitre M. -Hydroxybutyrate, a possible neurotransmitter. Life Sci 1987;41:1547-1557.[Medline] [Order article via Infotrieve]
2. Gallimberti L, Ferri M, Ferrara SD, Fadda F, Gentile N, Gessa GL, et al. -Hydroxybutyric acid for treatment of alcohol withdrawal syndrome. Lancet 1989;30:787-789.
3. ElSohly MA, Salamone SJ. Prevalence of drugs used in cases of alleged sexual assaults. J Anal Toxicol 1999;23:141-146.[Web of Science][Medline] [Order article via Infotrieve]
4. Palatini P, Tedeschi L, Frison G, Padrini R, Zordan R, Orlando R, et al. Dose-dependent absorption and elimination of -hydroxybutyric acid in healthy volunteers. Eur J Clin Pharmacol 1993;45:353-356.[Web of Science][Medline] [Order article via Infotrieve]
5. Louaagie HK, Verstraete AG, De Stoete CJ, Baetens DG, Calle PA. Sudden awakening from a near coma after combined intake of -hydroxybutyric acid (GHB) and ethanol. Clin Toxicol 1997;35:591-594.

This Article Extract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Web of Science (10) Citing Articles via Google Scholar Google Scholar Articles by Kintz, P. Articles by Ludes, B. Search for Related Content PubMed PubMed Citation Articles by Kintz, P. Articles by Ludes, B. Related Collections General Clinical Chemistry Drug Monitoring and Toxicology