Laboratory Diagnosis of 1,4-BD and GHB Overdose by Routine Urine Organic Acid Analysis

To the Editor:

Gamma-hydroxybutyric acid (GHB), or 4-hydroxybutyric acid, is an endogenous neurochemical that is formed in the human brain from gamma-aminobutyric acid (GABA) metabolism 1. GHB's natural presence in the body, coupled with a human study that demonstrated its influence on the release of growth hormone 2, resulted in its early popularity as a dietary health supplement among bodybuilders seeking its purported muscle-building effects through “natural” growth hormone release 3-5. More recently, GHB has gained popularity as a “club drug” for its purported euphoriant and natural psychedelic effects, as a sleep aid, and as a sexual enhancement aid 3-5. Moreover, GHB has been used in the commission of drug-facilitated “date rape” 3-5. Although the manufacture, distribution, and possession of illicit GHB were recently illegalized 6, 1,4-butanediol (1,4-BD) has recently emerged as GHB prodrug. Undergoing rapid in vivo enzymatic biotransformation to GHB by alcohol dehydrogenase and aldehyde dehydrogenase 1, 1,4-BD has been reported to produce similar toxicity as GHB 3-5. Coma, severe respiratory depression, apnea, and deaths have occurred from overdoses with 1,4-BD and GHB 3-5. Despite attempts to restrict GHB as a federal Schedule I drug and 1,4-BD as a Class I Health Hazard, emergency department encounters with these overdoses have steadily increased during the past decade 7. Moreover, GHB precursors such as 1,4-BD have been recently reported to account for up to 71% of GHB-related overdoses 7.

However, laboratory detection of GHB and 1,4-BD for accurate diagnosis remains a significant challenge. Routine hospital toxicology screens do not detect GHB or 1,4-BD 3-5. Several techniques employing gas chromatography (GC)/mass spectrometry (MS) have been developed to measure specifically blood and urine concentrations of GHB and 1,4-BD but are not routinely available at hospital laboratories 3-5. Presently, confirmation of acute GHB or 1,4-BD overdoses generally requires that a hospital laboratory send blood and urine specimens to one of a few national reference laboratories that have adapted these specialized, specific GC/MS methods for detecting GHB and 1,4-BD 3-5. Hence, the diagnosis of acute overdoses with GHB or 1,4-BD is often made presumptively by a patient's clinical presentation. However, the urine organic acid analysis may provide an alternative laboratory method for confirming clinically suspected acute overdose with GHB or 1,4-BD. As an in vivo catabolite of GABA, 4-hydroxybutyric acid (GHB) is among approximately 250 endogenous organic acids the urine organic acid analysis can detect.

We conducted a controlled, longitudinal murine study to evaluate the diagnostic accuracy of routine urine organic acid analysis for detecting GHB after acute 1,4-BD overdose. Twenty-five male CD-1 mice (Charles River Laboratories, Cambridge, MA) were divided into five groups (N = 5 in each group). On the experiment day, each group of mice was placed into separate metabolic cages for urine collection in two phases. In the control phase of the study, baseline urine collection was obtained from each group for a period of 4 h. Immediately after this phase, all mice were given 1,4-BD 600 mg/kg intraperitoneal. Urine was then collected for a period of 4 h during this overdose phase. All urine samples were frozen at − 80°C until the day of analysis. On the day of urine organic acid analysis, all frozen samples were thawed and underwent solid phase extraction (SPE) and derivatization with bis(trimethyl-silyl) trifluoroacetamide (BSTFA) in the following procedure. One milliliter of each sample was spiked with 50 μL of 1 g/L 2-ketocaproic acid as internal standard, followed by oximation with 0.2 mL of 75 g/L hydroxylamine at 60°C for 30 min and 50 uL of 2.4 g/L pentadecanoic acid. Then it went through double organic extraction with ethyl acetate and ether. The subsequent organic layers were spiked with 50 uL of 1 g/L tetracosane as external standard, dried down, and derivitized with 100 uL of BFSTA at 60°C for 10 min. The product was diluted with 900 uL of hexane, yielding a final volume of 1.0 mL. They were then analyzed on GC 5890/MS 5792 (Hewlett-Packard Co., Wilmington, DE), using MSD Productivity ChemStation Software. Operators of the urine organic acid analysis were blinded to the urine samples. The total extraction time was about 2 h, and total instrument run time was 33 min.

There was a library match for 4-hydoxybutyric acid, or GHB, in 5/5 mouse overdose urine samples, with a retention time of 9.4 min, molecular weight (MW) 248, methylene unit (MU) 1225, and ion pattern inclusive of 233 m/z (Fig. 1). Conversely, 0/5 mouse control urines had a library match for GHB. The urine organic acid analysis demonstrated 100% sensitivity, specificity, positive predictive value, and negative predictive value as a diagnostic test for detecting GHB following acute 1,4-BD overdose. This study also addresses the important possibility of interference with the assay by endogenous GHB, which could result in false positive results. In our study, the urine organic acid analysis did not falsely detect GHB in the mouse control urines collected before acute 1,4-BD overdose. Hence, it appears that the urine organic acid analysis has specificity for distinguishing between endogenous and exogenous GHB. It is possible that endogenous GHB, which comprises only 0.16% of in vivo GABA metabolism 1, results in such low urine GHB concentrations that it was below the detection limit of the urine organic acid screen.

FIGURE 1 Gas chromatograhic separation of organic acids from mouse urine prior to 1,4-BD administration (A.) and after 1,4-BD administration (B.). Note the prominent chromatogram peak that appears at 9.49 min after administration of 1,4-BD (B.). Amplification of chromatogram peak at 9.49 min (C.) and its mass spectrum analysis (D.), which had a library match for 4-hydroxybutyric acid/GHB in the organic acid library that was searched.

While the data of the present study support the use of the urine organic acid analysis for laboratory diagnosis of 1,4-BD and GHB overdoses, several aspects of this diagnostic application are potentially limiting and warrant additional studies. First, GHB and 1,4-BD standards or calibrators were not used in this instrumental analysis. Good laboratory practice and laboratory certifying bodies would require the use of such calibrators for the purpose of identifying these agents in a patient's urine. Second, the identification of GHB by this analysis relied on a library matching program for mass spectra, which is not a reportable independent finding. Third, the diagnostic application of this urine organic acid analysis for 1,4-BD and GHB would require additional validation in several respects: demonstration of a calibration-response relationship (definable and repeatable), demonstration of accuracy (vs. another accepted method, vs. primary reference material, vs. recovery studies), demonstration of selectivity over background and interferences (limits of detection), and demonstration of precision (instrumental, inter-, and intra-assay) at appropriate levels. Fourth, while this instrumental approach (with added validation) could potentially be useful in the laboratory diagnosis of 1,4-BD and GHB overdoses, most hospitals do not perform the urinary organic acid analysis in-house, and if available in-house, the instrument is usually operated only during limited time periods, which may well surpass the diagnostic and interventional utility of this test in the emergency department.

In summary, our preliminary data indicate that the urine organic acid analysis is a sensitive and specific diagnostic tool for confirming clinically suspected cases of acute overdoses with GHB and 1,4-BD. When available in a hospital, it may serve as an accurate, more accessible diagnostic test for laboratory confirmation of acute overdoses with GHB and its precursors than the GHB-specific GC/MS assay available at only a few commercial laboratories nationwide. Accurate, accessible, timely laboratory diagnosis of patients with these overdoses will be important for appropriate medical management, particularly as potential antidotes are being identified 8-10.

Lawrence S. Quang, M.D.^*

Medical Director, Greater Cleveland Poison Control Center, Rainbow Babies and Children's Hospital, University Hospitals Health System, Department of Pediatrics, Case Western Reserve University School of Medicine

^*Corresponding author:

11100 Euclid Ave., Cleveland, OH, 44106, USA

Telephone: [phone], Fax: [phone], E-mail: [email protected]

Harvey L. Levy, M.D.

Division of Genetics/Metabolic Diseases, Children's Hospital Boston/Harvard Medical School, Boston, Massachusetts, USA

Terence Law BS, MT(ASCP)SC

Clinical Chemistry Laboratory, Department of Laboratory Medicine, Children's Hospital Boston/Harvard Medical School, Boston, Massachusetts, USA

Malhar C. Desai, M.S.

Timothy J. Maher, Ph.D.

Department of Pharmaceutical Sciences, Massachusetts College of Pharmacy and Health Sciences, Boston, Massachusetts, USA

Edward W. Boyer, Ph.D., M.D.

Michael W. Shannon, M.D., M.P.H.

Alan D. Woolf, M.D., M.P.H.

Massachusetts/Rhode Island Regional Poison Control System, Program in Clinical Pharmacology/Toxicology, Children's Hospital Boston/Harvard Medical School, Boston, Massachusetts, USA

Acknowledgments

This research was funded by National Institute on Drug Abuse (NIDA)/National Institutes of Health (NIH) grant # 1 RO3 DA15951-03 (Dr. Quang) and National Institute of Child Health and Human Development (NICHHD)/NIH grant # 1 T32 HD40128-03 (Dr. Quang).