Simutaneous Analysis Of GHB And Its Precursors In Urine Using LC-MS/MS

The method presented here is the first demonstration of the use of LC-MS/MS for the simultaneous analysis of GHB and its precursors in urine samples. The method is simple and rapid (total analysis time of <12 min). It offers sufficient sensitivity to enable the measurement of endogenous levels of GHB and to identify exogenous ingestion.

Benefits

• It enables the simultaneous quantification of the GHB and the precursors in a single analysis
• It involves fewer manipulations and is less time-consuming

Gamma-hydroxybutyrate ( GHB) is a metabolite of gamma-aminobutyric acid (GABA) and plays the role of a central neurotransmitter and neuromodulator. Since GHB is a normal component of mammalian metabolism, it is present in all tissues of the body. Typical urinary GHB concentrations are < 10 mg/L^1,2. In some countries GHB is used clinically as an intravenous anaesthetic and as a treatment for narcolepsy, alcoholism and opiate withdrawal. Over the last few years, GHB has been gaining popularity amongst club-goers as the recreational drug (Figure 1) where it is taken for its ability to produce feelings of euphoria and to enhance sexuality^3-5. As a result of its potent prosexual effects, GHB has also been increasingly implicated in drug-facilitated sexual assault ^6,7. Ingestion of the chemical precursors of GHB i.e. gamma-butyrolactone (GBL) and 1,4-butanediol (1,4-BD) also results in similar physiological effects since they are rapidly converted to GHB in the body⁸. Raised awareness of the effects of these drugs and their potential for misuse, in addition to their ease of availability, has resulted in a dramatic increase in the demand for their analytical determination in both biological specimens and putative drug preparations. The purpose of this study was to develop and validate a sensitive LC-MS/MS procedure that would enable the simultaneous quantification of GHB, GBL and 1,4-BD in urine.

Samples

Calibrators and quality control (QC) samples.

Control urine was spiked with GHB, GBL and 1,4-BD to yield a series of calibrators at the following concentrations; 0, 1, 2, 5, 10, 20, 50 and 80 mg/L. Low and high QC samples were prepared by spiking control urine with the drugs to yield concentrations of 4 and 40 mg/L, respectively.

Authentic Samples

One hundred and eighty two authentic human urine samples were collected from club-goers attending a post dance-club ‘chill-out’ venue and were the result of 2 separate raids by the Belgian Police Department. The samples were analysed for GHB and the precursors using the newly-developed LC-MS/MS procedure. For comparative purposes, the samples were also analysed for GHB using a routinely used GC-MS procedure.

Sample Preparation

Urine samples were diluted (1:20) with an internal standard solution ( GHB-d6 and GBL-d6, at a concentration of 2 mg/L in deionised water).

Multiple reaction monitoring (MRM) transitions were determined for GHB, the precursors and the internal standards i.e. GHB-d6 and GBLd6 (Table 1). Figure 2 shows some examples of product ion spectra.

A series of urine calibrators was prepared. Following preparation i.e. simple dilution, the samples were analysed using LC-MS/MS. Figure 3 shows the MRM chromatograms obtained following the analysis of a control urine sample and the same sample enriched with GHB, GBL and 1,4-BD.

Quantification was achieved by integration of the area under the specific MRM chromatogram. For GHB and GBL, responses were calculated in reference to the integrated area of their respective deuterated internal standards. For 1,4-BD the response was calculated by reference to that of GHB-d6. Linear responses were obtained for GHB and 1,4-BD over the range investigated (1-80 mg/L). GBL produced a linear response over the range 1-50 mg/L.

Precision, intra-assay and interassay variation (as % CV) were all found to be highly satisfactory at < 7%. Analytical recoveries ranged from 90-107% (Table 2).

The limit of quantification was defined as the concentration of the lowest calibrator which was calculated to be with in ± 20% of the nominal value and with a % CV less than 20%. For all of the analytes of interest, this criteria was met by the 1 mg/L calibrator and was sufficient to determine endogenous levels of GHB in the urine.

To investigate any potential interference in GHB quantification by its naturally occuring isomers i.e. alpha and beta-hydroxybutyric acid, standards were analysed using the developed LC-MS/MS method. Both compounds were shown to be chromatographically resolved from GHB and thus would not interfere in the quantification of the latter (Figure 4).

The utility of the LC-MS/MS method was demonstrated by the analysis of one hundred and eighty-two authentic urine samples. Seven samples contained GHB at concentrations > 2 mg/L. The same seven samples were independently identified by the more time-consuming, labour-intensive GC-MS method.

Only two, of these seven samples, were above the recommended interpretive cut-off concentration of 10 mg/L and were 956 mg/L and 1411 mg/L, respectively. These two samples were also positive for GBL. None of the authentic urine samples contained 1,4-BD.

The method presented here is the first demonstration of the use of LC-MS/MS for the simultaneous analysis of GHB and its precursors in urine samples. The method is simple and rapid (total analysis time of <12 min). It offers sufficient sensitivity to enable the measurement of endogenous levels of GHB and to identify exogenous ingestion.

The LC-MS/MS results obtained following the analysis of authentic samples, correlated with the more labour-intensive, time-consuming (~1 hour) GC-MS method.

The procedure offers several advantages over other published techniques;

1. It enables the simultaneous quantification of the GHB and the precursors in a single analysis; this can facilitate the identification of the chemical basis of any seized putative drug preparations or if present in the biological specimen, can provide information of the chemical nature of the ingested drug.

2. It involves fewer manipulations and is less time-consuming.

Although the data presented here indicate that the actual prevalence of GHB-positives might be quite low, the hype and publicity surrounding these drugs has led to a dramatic increase in the number of requests for their analysis in biological samples (and particularly in urine). The simplicity and speed of the described LC-MS/MS technique, should prove a useful means to meet this current increased demand on laboratories.

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