A potential new metabolite of gamma-hydroxybutyrate: sulfonated gamma-hydroxybutyric acid.

Detection of gamma-hydroxybutyric acid (GHB) became crucial in many clinical and forensic settings due to its increasing use for recreational purposes and drug-facilitated sexual assault. Its narrow window of detection of about 3-12 h in urine represents a major problem. Analogous to ethyl glucuronide, the recently identified GHB-glucuronide exhibits a longer window of detection than the parent drug. It appeared reasonable that a sulfonated metabolite of GHB (GHB-SUL) will also be formed. Due to the lack of an appropriate standard, GHB was incubated with a human liver cytosolic fraction to produce GHB-SUL. Following development of a liquid chromatography/tandem mass spectrometry (LC-MS/MS) assay to measure GHB and GHB-SUL, authentic urine samples (n = 5) were tested for GHB-SUL. These investigations revealed detectable signals of both GHB and GHB-SUL, strongly indicating that GHB is not only glucuronidated but also sulfonated. Given that sulfonated metabolites generally have longer half-life times than the corresponding free drugs, GHB-SUL may serve as a biomarker of GHB misuse along with its glucuronide.

Simultaneous quantification of triazolam and its metabolites in human urine by liquid chromatography-tandem mass spectrometry.

A simple, simultaneous, sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for the determination of triazolam and its metabolites, α-hydroxytriazolam (α-OHTRZ) and 4-hydroxytriazolam (4-OHTRZ), in human urine was developed and validated. Triazolam-d4 was used as the internal standard (IS). This analysis was carried out on a Thermo(®) C(18) column, and the mobile phase was composed of acetonitrile/H(2)O/formic acid (35:65:0.2, v/v/v). Detection was performed on a triple-quadrupole tandem MS using positive ion mode electrospray ionization, and quantification was performed by multiple reaction monitoring mode. The MS-MS ion transitions monitored were m/z 343.1 → 308.3, 359.0 → 331.0, 359.0 → 111.2, and 347.0 → 312.0 for triazolam, α-OHTRZ, 4-OHTRZ, and triazolam-d(4), respectively. The lower limits of quantification of the analytical method were 0.5 ng/mL for triazolam, 5 ng/mL for α-OHTRZ, and 0.5 ng/mL for 4-OHTRZ. The within- and between-run precisions were less than 15%, and accuracy was -12.33% to 9.76%. The method was proved to be accurate and specific, and it was applied to a urinary excretion study of triazolam in healthy Chinese volunteers.

Development of an enzyme-linked immunosorbent assay for fentanyl and applications of fentanyl antibody-coated nanoparticles for sample preparation.

A sensitive enzyme-linked immunosorbent assay (ELISA) was developed for the detection of fentanyl in serum and urine. The ELISA used an indirect competitive method produced by coating the plate with thyroglobulin conjugated with fentanyl hapten. Antibodies against fentanyl-hemocyanin were detected by a goat-anti-rabbit antibody conjugated with alkaline phosphatase. Calibration standard curves ranged from 0.5ng/ml to 50mug/ml (IC(50)=10ng/ml), and the limits of detection were 0.5 and 1.0ng/ml for serum and urine, respectively. The intra- and inter-assay variations were less than 8% and 10%, respectively. The antibody produced against fentanyl completely cross-reacted with p-fluorofentanyl, thienylfentanyl and 3-methylthienylfentanyl, cross-reacted highly with carfentanil (85%), but was considered non-cross-reactive with alpha-methylfentanyl (5%), sufentanil (<1%), alfentanil (<1%) and lofentanil (<1%). Nano-sized iron oxide magnetic particles coated with the developed fentanyl antibody were capable of specific binding and releasing of fentanyl from urine samples. This enabled the drug to be effectively pre-concentrated and decreased the limit of detection by approximately one order of magnitude. The analytical background noise was significantly reduced to enable fentanyl detection at concentrations originally below chromatographic limit of detection. The change of platform for antibody binding with nanoparticles demonstrated a novel use of antibodies for sample preparation and should facilitate drug screening by traditional ELISA.

Gamma-hydroxybutyrate serum levels and clinical syndrome after severe overdose.

STUDY OBJECTIVE: We discuss a prospective case series of patients who present with a severe gamma-hydroxybutyrate intoxication with confirmatory serum and urine gamma-hydroxybutyrate levels. METHODS: Patients with a clinical suspicion of gamma-hydroxybutyrate-like drug overdoses and a Glasgow Coma Scale score of 8 or lower were identified from July 1998 through January 1999. Serial serum specimens and a single urine specimen were collected. The levels of gamma-hydroxybutyrate were performed by gas chromatography-mass spectrometry. RESULTS: All 16 suspected severe gamma-hydroxybutyrate overdose patients had significant serum or urine levels of gamma-hydroxybutyrate. Serum levels ranged from 45 to 295 mg/L, with a median of 180 mg/L (interquartile range [IQR] 235 to 118 mg/L). Patients who developed a Glasgow Coma Scale score of 3 had serum levels that ranged from 72 to 300 mg/L, with a median of 193 mg/L (IQR 242 to 124 mg/L). The time of awakening ranged from 30 minutes to 190 minutes, with a median of 120 minutes (IQR 150 to 83 minutes). Quantitative serum gamma-hydroxybutyrate levels did not correlate with the degree of coma or the time to awakening. Urine levels ranged from 432 to 2,407 mg/L, with a median of 1,263 mg/L (IQR 1,550 to 796 mg/L). Mild transitory hypoventilation occurred in 5 of the 16 patients. CONCLUSION: All of our patients with clinically suspected severe gamma-hydroxybutyrate overdose were confirmed to have significant serum and urine levels of exogenous gamma-hydroxybutyrate. They presented with severe coma that lasted 1 to 2 hours. Transient hypoventilation occurred in one third of these patients.

A study of urinary endogenous gamma-hydroxybutyrate (GHB) levels.

In recent years, the use of gamma-hydroxybutyrate (GHB), as a recreational drug has prompted forensic toxicology laboratories to incorporate the analysis for GHB into their routine screening procedures. GHB, being a natural occurring constituent of the human body, presents a challenge for forensic toxicologists in that endogenous levels and exogenous levels of GHB need to be differentiated in case samples. This study was designed to determine typical urinary endogenous levels of GHB in humans based on the analysis of urine samples voluntarily provided by 55 male and female subjects ranging in age from 6 to 59 years. All samples were initially screened for the presence of GHB utilizing a hydrolysis method designed to quantitatively convert the GHB in urine samples to gamma-butyrolactone (GBL) followed by the liquid-liquid extraction and analysis of any GBL present by gas chromatography-mass spectrometry (GC-MS). As a confirmation test, samples were then extracted by a solid-phase extraction technique, derivatized to GHB di-TMS, and analyzed by GC-MS. The median concentration determined for the 55 subjects was 1.3 mg/mL (mean = 1.65 microg/mL, range 0.9 microg/mL to 3.5 microg/mL, standard deviation 0.68 microg/mL). The results of this study confirm the previously suggested cutoff of 10 microg/mL for routine forensic analyses.