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

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 mL 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