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GHB Urine Concentrations After Single-Dose Administration in Humans

. Author manuscript; available in PMC: 2008 Feb 27.

Published in final edited form as: J Anal Toxicol. 2006;30(6):360–364. doi: 10.1093/jat/30.6.360

Abstract

Gamma-hydroxybutyric acid (GHB) is used as an illicit drug and is implicated in drug-facilitated sexual assault, but it also has some therapeutic uses. Detection of GHB in urine is important for forensic testing and could be of clinical benefit in overdose management. Urine GHB concentration-time profiles have not been well-characterized or correlated with doses used therapeutically. GHB levels were measured by gas chromatography–mass spectrometry in urine collected over 24 h from 16 adults administered single doses of 50 mg/kg GHB (Xyrem®) alone and combined with 0.6 g/kg ethanol. Peak GHB urine concentrations averaged 150–200 mg/L and occurred in the 0–3 h urine collection. Significant variability in GHB urine levels between individuals was observed. Caucasians had lower urine concentrations than other races/ethnicities (p = 0.03). Men had lower GHB levels than women in the first 3 h after dosing (p = 0.038). Coingestion of ethanol did not significantly affect renal clearance of GHB, but urine GHB concentrations were lower in the first 3 h when ethanol and GHB were coingested (p = 0.039). At a proposed cut-off of 10 mg/L to distinguish endogenous versus exogenous GHB levels, 12.5% of the samples collected from 3 to 6 h, 81.3% of samples collected from 6 to 12 h, and 100% of urine specimens collected from 12 to 24 h were below this level. We conclude that the detection time for GHB in urine may be shorter than the previously reported 12-h window in some people taking therapeutic doses of GHB.

Introduction

Gamma-hydroxybutyrate (GHB) has been used as an anesthetic drug, body-building/athletic enhancing supplement, and substance of abuse and implicated as an agent in drug-facilitated sexual assault. Because of its abuse potential and association with serious adverse effects, including deaths, GHB was classified as a Schedule 1 controlled substance in 2000. The FDA approved a pharmaceutical form of GHB, sodium oxybate (Xyrem), in 2002 for limited use in the treatment of narcolepsy (1). GHB is also used therapeutically in Italy for treatment of alcohol withdrawal and dependence.

GHB is rapidly absorbed after oral administration with maximal plasma concentrations reached in about 30–50 min (2,3). It is rapidly metabolized in part by beta-oxidation and in part to succinic acid and then via the tricarboxylic acid pathway. Less than 5% of ingested GHB is excreted unchanged in the urine (4). The plasma elimination half-life averages 40–60 min after single oral doses of 12.5 to 50 mg/kg (2–4).

GHB is also generated in vivo as a metabolic product of gamma-aminobutyric acid (GABA). Because endogeneous GHB concentrations in plasma and urine are readily detectable by highly sensitive analytical methods currently utilized for forensic testing purposes, interpretation of antemortem and postmortem GHB levels has been a topic of recent interest. Although there is no consensus on the optimal blood concentration to distinguish endogenous GHB levels from those indicative of recent GHB ingestion, a plasma level of 4–5 mg/L is proposed as an appropriate cutoff concentration (5,6). Interpretation of postmortem blood levels can be difficult, as studies have shown that factors, such as collection tube additive and storage temperature, dramatically affect GHB levels. In one study of 26 postmortem blood samples, sodium-fluoride-preserved specimens stored at 4°C had a 50% lower GHB concentration than non-fluoride-preserved samples (mean 19 mg/L vs. 32 mg/L) (7). At room temperature storage, unpreserved blood samples had a threefold higher GHB concentration (57 mg/L) than fluoride-preserved blood.

Postmortem urine does not exhibit the same variability in GHB levels as blood (7,8). Because of this apparent stability as well as the ease of collection, urine is generally considered the optimal specimen for testing for the presence of GHB. GHB levels reported in forensic cases include a urine level of 308 mg/L in a case of alleged drug-facilitated sexual assault (9), a level of 714 mg/L in a person arrested for driving under the influence of drugs, and overdose levels ranging from 432 to 2407 mg/L (10,11).

Several studies have been conducted to determine typical endogenous urine GHB levels. In one study of eight non-GHB users, urine levels ranged from 0 to 6.63 mg/L (12). In a study of 50 female volunteers, the highest urine GHB concentration measured was 1.46 mg/L (13). Another study found a maximal level of 3 mg/L in 119 GHB-free subjects (5). From these studies, cut-off concentrations of 5 mg/L (2,13,14) and 10 mg/L (5,15) have been proposed to distinguish endogeneous urine GHB concentrations from levels indicative of GHB ingestion.

There are currently no commercially available assays for the rapid detection of GHB in urine that could be used in a hospital setting to diagnose GHB overdose. In addition, negative emergency toxicology screening results can hamper investigations of alleged drug-facilitated sexual assault. Even broad-spectrum toxicology testing is frequently negative for drugs that are rapidly cleared such as GHB. This is particularly true when there is a delay from the incident until medical evaluation. Of 1179 specimens collected from victims of alleged assault, 4% were positive for GHB (16). However, 32% of samples were collected after 24 h, and therefore, this would underestimate the actual incidence of GHB use in these incidents.

Urine GHB concentration-time profiles have not been well characterized or correlated with doses used therapeutically or in controlled human studies. In one study involving eight GHB-naïve volunteers administered a single 25-mg/kg dose of GHB, a peak mean urine GHB level of 230 ± 86.3 mg/L was reported at 60 min after dosing, with a detection window of 720 min (2). The influence of factors, such as race, gender, and coingested drugs, on GHB urine concentrations has not been investigated. In this paper, we present data on GHB concentrations in timed urine collections taken over 24 h in men and women administered 50 mg/kg of GHB alone and in combination with 0.6 g/kg ethanol.

Methods

Subjects

Study subjects were healthy adults (7 men and 9 women) aged 22 to 34 years. Seven participants (44%) were white, 6 (38%) were Asian/Pacific Islander, 1 (6%) was Latino, and 2 (12%) reported multiple ethnicities. Subjects completed a medical history, physical examination, and screening laboratory tests prior to enrollment. Exclusion criteria included any significant medical conditions, pregnancy, lactation, or obesity. Illicit or prescription drug use (other than oral contraceptives) and more than light use of alcohol (> 3 drinks/week) or GHB and its derivatives (defined as greater than 2 times in last 6 months) were cause for exclusion from the study. Volunteers provided written informed consent prior to enrollment. The Committee on Human Research at the University of California, San Francisco approved the study.

Procedures

The study was a randomized double-blind, four-arm, crossover design. Subjects were given placebo, ethanol, GHB, or ethanol-plus-GHB in random order. Subjects were admitted to the General Clinical Research Center at San Francisco General Hospital for four 24-h visits after an overnight fast from food, caffeine, and tobacco. Subjects were asked not to drink alcoholic beverages for three days prior to each study day. Predose samples of blood were tested for the presence of GHB and ethanol, and urine was tested for illicit drugs. The washout period between treatments was a minimum of two days. At 8:00 a.m. on study days, subjects received 0.3 g/kg ethanol (in the form of vodka) or placebo in orange or cranberry juice. Fifteen minutes later, the subjects received GHB 50 mg/kg (Xyrem, Orphan Medical Co., Minnetonka, MN) or placebo, and a second dose of 0.3 g/kg ethanol or placebo in juice. Subjects were asked to rest in supine position for 3 h after dosing. Subjects were closely monitored for 24 h after dosing and discharged the next day. Urine was collected in intervals of 0–3, 3–6, 6–12, 12–24, and post-24 h after dosing. Blood samples were collected through an intravenous catheter at 0, 15, 30, 45, 60, and 90 min and 2, 3, 4, 5, 6, 12, and 24 h after dosing. Blood levels and pharmacodynamic results of this study have been reported separately (4).

Analysis

Plasma and urine samples were stored frozen at −20°C until the time of analysis. A novel gas chromatography–mass spectrometry method for the analysis of GHB in plasma and urine was developed and validated, which is described in detail in a separate paper (17). Limits of detection for GHB were found to be 0.5 mg/L in plasma and 0.25 mg/L in urine. For this analysis, we established a limit of quantitation of 5 mg/L because blood and urine concentrations below this threshold are in the range of endogenous GHB levels (2,12,14). Urine samples were also tested for creatinine concentration to adjust GHB levels for urine flow. Renal clearance of GHB (CLR) was estimated by dividing the total amount of GHB recovered in the urine in 24 h by the area under the plasma concentration-versus-time curve (AUC) for the same time period.

Results

The mean and range of GHB concentrations in timed urine collections for the 16 subjects who completed the study are shown in Table I. Results are expressed as measured concentrations of GHB and as adjusted ratios of micrograms GHB per milligrams creatinine measured in the same urine sample. Mean values for CLR categorized by treatment, gender, and race are shown in Table II. There was no significant effect of these factors on calculated GHB CLR. Urine GHB concentrations for all subjects and mean values are shown graphically in Figure 1.

Table I.

Mean Urine GHB Levels with Standard Deviations and Ranges in 16 Adults Given Single Oral Dose of 50 mg/kg Sodium Oxybate

0–3 h
3–6 h
6–12 h
12–24 h
Mean (SD*) Range Mean (SD) Range Mean (SD) Range Mean (SD) Range
GHB (mg/L) 168.1 (138.9) 14.9–598 157.3 (208.4) 0–787 3.8 (8.0) 0–35.9 < 5 < 5
GHB/creatinine (μg/mg) 939.0 (168.1) 80–4176 250.9 (157.3) 0–1082 5.6 (3.8) 0–70.9 0 0

Table II.

Average GHB Renal Clearance Rates Categorized by Gender, Race/Ethnicity, and Ethanol Coingestion*

All (n = 16) Men (n = 7) Women (n = 9) Asians (n = 6) Caucasians (n = 7) Other Races (n = 3)
CLR (mL/min) GHB alone 17.8 ± 13.1 18.3 ± 15.8 17.4 ± 10.9 20.2 ± 11.7 17.9 ± 15.7 12.8 ± 12.5
CLR (mL/min) GHB and ethanol 19.2 ± 17.4 23.9 ± 24.7 15.2 ± 6.7 30.6 ± 26.3 12.1 ± 5.9 16.9 ± 11.5

Figure 1.

Figure 1

GHB concentrations in serial timed urine collections in 16 healthy volunteers administered 50 mg/kg GHB alone (solid diamonds); or with 0.6 g/kg ethanol (open triangles). Mean values represented by horizontal lines. p = 0.039 for GHB versus GHB and ethanol in 0–3 h period.

GHB levels were higher in the first (0–3 h) urine collection with GHB alone (mean 203.6 mg/L) versus GHB and ethanol (mean 132.6 mg/L) (p = 0.039). With the exclusion of one outlier, however, these differences were not significantly different in the 0–3 h or in subsequent collections. When adjusted for urine creatinine concentration, ethanol coingestion had no effect on urine GHB, with a mean concentration of 898 versus 980 μg GHB/mg creatinine for GHB alone and combined with ethanol, respectively, in the 0–3 h period (p = 0.79).

Subgroup comparisons of men versus women and 3 racial/ethnic groups of Asians, Caucasians, and “other” showed some significant differences in GHB urine concentrations. A gender effect in GHB urine concentration adjusted for urine creatinine was observed in the first 0–3 h urine collection. The mean adjusted GHB concentration was 1442 ± 1330 μg/mg creatinine for females and 354 ± 168 μg/mg creatinine for males (p = 0.038). There was no significant gender effect observed in subsequent urine collections or in any of the unadjusted GHB concentrations. Significant racial/ethnic differences in GHB urine levels were also observed. Caucasians had lower GHB urine levels than Asians and people of other races, as shown in Figure 2 (p = 0.03). This effect was observed with both adjusted and unadjusted GHB measurements, indicating that it is not related to differences in urine flow. None of the seven Caucasians had measurable GHB in urine collected after 6 h.

Figure 2.

Figure 2

GHB urine levels adjusted for urine creatinine concentration in Asians (solid diamonds, n = 6); Caucasians (solid squares, n = 7,); and other ethnicities (solid circles, n = 3). Data are means ± standard errors. p < 0.05 for Caucasians versus Asians at 0–3 h and 6–12 h and for Caucasians compared with other ethnicities at all time intervals.

Discussion

This study provides new data on the urine concentration-time profile of GHB after controlled oral dosing in healthy volunteers. At the moderate GHB dose of 50 mg/kg used in this study, we found that the duration of detection of GHB in urine is less than 12 h. We discovered that coingestion of ethanol reduces absolute GHB urine levels in the first 3 h after dosing but not creatinine-corrected GHB concentrations. This observation is consistent with the effect of ethanol to increase urine flow rate. Urine concentrations of GHB showed substantial inter-individual variability, with race and gender differences observed.

Peak GHB urine concentrations averaged 150–200 mg/L and generally occurred in the 0–3 h urine collection. We are aware of only two other human studies that have examined the urinary excretion of GHB (2,18). In one study, a single healthy volunteer took 1- and 2-g doses of GHB, and the peak urine concentration was 29 mg/L, and the elimination time was less than 10 h (18). In the other study, eight GHB-naïve volunteers (4 men) were administered a single 25-mg/kg dose of GHB, and the peak urine level was 230 ± 86.3 mg/L in the 60 min urine collection, and the detection duration was 12 h (2). Taking into consideration the 50% lower GHB dose, this prior study shows significantly higher peak GHB levels than found in our study. The reason for this discrepancy in findings may be because peak concentrations would be expected to be higher at 60 min than over a 3-h collection period.

The mean GHB urine level was lower in the first 3 h after dosing with GHB and ethanol versus GHB taken alone; however, this effect was not apparent when GHB levels were adjusted for urine creatinine. Ethanol dosing also did not affect GHB renal clearance rates. These findings could be explained by a short-term diuresis induced by ethanol (19), resulting in urine dilution and lower GHB concentrations in the first collection interval. Because ethanol is commonly coingested with GHB, and may be consumed repeatedly in some social settings, this urinary dilution effect could potentially result in false-negative toxicology test results for GHB.

The range of urine GHB concentrations varied widely in the first 6 h after dosing, and some individuals had maximal GHB levels that were several-fold higher than mean values. Adjusting GHB levels for urine creatinine measurements did not reduce this variability, indicating that factors other than renal clearance, such as variations in metabolism, may determine individual differences in urinary concentration profiles of GHB. Differences in urine concentrations between Caucasians and other races, and between men and women may be related to differences in bioavailability, rate of absorption, or alterations in drug metabolizing enzyme activity. Although significant gender and racial differences in pharmacokinetic parameters were not observed (4), this study was not specifically designed to detect such differences.

The detection time of GHB in urine at a cut-off concentration of 10 mg/L has been reported to be 12 h (2,20). From our study findings, 12.5% of urine samples collected from 3 to 6 h, 81.3% of samples collected from 6 to 12 h, and 100% of urine specimens collected from 12 to 24 h after dosing would be below this proposed 10 mg/L threshold level (Figure 3). At a higher cut-off concentration of 50 mg/L, proposed for one enzyme-based GHB assay currently under development (21), 34% of samples collected from 3 to 6 h and 100% of samples collected from 6 to 12 h would be interpreted as negative. We conclude that an assay with better test sensitivity is needed for clinical diagnostic utility, particularly in cases of suspected drug-facilitated sexual assault with delay to presentation.

Figure 3.

Figure 3

Percentage of subjects whose urine GHB concentration is less than the proposed cut-off concentrations of 10 mg/L (solid) and 50 mg/L (cross-hatched) at each urine collection time interval.

In summary, we found that urine concentrations vary widely after oral administration of a 50-mg/kg dose of GHB to healthy young adults. Coingestion of ethanol did not significantly affect renal clearance of GHB, but it did lower urine GHB concentrations in the first 3 h. Caucasians had lower urine concentrations than other races/ethnicities, and urine GHB levels for all Caucasian subjects were below the cutoff concentration of 5 mg/L after 6 h. Men had lower GHB levels than women in the first 3 h after ingestion, but no gender differences were observed in later urine collections. We conclude that the detection time for GHB in urine may be shorter than the previously reported 12-h window in some people taking therapeutic doses of GHB.

Acknowledgments

This work was supported by Public Health Service grants DA 14935 and DA 12393 from the National Institute on Drug Abuse (NIDA), a clinical pharmacology training grant award (T32 GM007546-28), and a General Clinical Research Center Award (M01RR00083-41) from the National Institutes of Health. The Xyrem used in this study was generously provided by Orphan Medical Co. (Minnetonka, MN). We gratefully thank Dr. Neal Benowitz for his helpful guidance on this study; Gina Lowry for subject recruitment and assistance with the clinical study; Faith Allen for her expertise in protocol and data management; and the nurses and staff of the GCRC for the care of the research subjects.

References

  • 1.Fuller DE, Hornfeldt CS. From club drug to orphan drug: sodium oxybate (Xyrem) for the treatment of cataplexy. Pharmacother. 2003;23(9):1205–1209. doi: 10.1592/phco.23.10.1205.32756. [DOI] [PubMed] [Google Scholar]
  • 2.Brenneisen R, Elsohly MA, Murphy TP, Passarelli J, Russmann S, Salamone SJ, Watsone DE. Pharmacokinetics and excretion of gamma-hydroxybutyrate (GHB) in healthy subjects. J Anal Toxicol. 2004;28(8):625–630. doi: 10.1093/jat/28.8.625. [DOI] [PubMed] [Google Scholar]
  • 3.Scharf M, Lai A, Branigan B, Stover R, Berkowitz D. Pharmacokinetics of gammahydroxybutyrate (GHB) in narcoleptic patients. Sleep. 1998;21:507–514. doi: 10.1093/sleep/21.5.507. [DOI] [PubMed] [Google Scholar]
  • 4.Thai D, Dyer J, Benowitz N, Haller C. Pharmacokinetic interactions of GHB and ethanol: effects and interactions in humans. J Clin Psychopharmacol. doi: 10.1097/01.jcp.0000237944.57893.28. in press. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Elliott SP. Gamma hydroxybutyric acid (GHB) concentrations in humans and factors affecting endogenous production. Forensic Sci Int. 2003;133:9–16. doi: 10.1016/s0379-0738(03)00043-4. [DOI] [PubMed] [Google Scholar]
  • 6.Kintz P, Goulle JP, Cirimele V, Ludes B. Window of detection of gamma-hydroxybutyrate in blood and saliva. Clin Chem. 2001;47:2033–2034. [PubMed] [Google Scholar]
  • 7.Stephens BG, Coleman DE. In vitro stability of endogenous gamma-hydroxybutyrate in postmortem blood. J Forensic Sci. 1999;44(1):231. [PubMed] [Google Scholar]
  • 8.Fieler EL, Coleman DE, Baselt RC. γ-Hydroxybutyrate concentrations in pre- and postmortem blood and urine. Clin Chem. 1998;44:692. [PubMed] [Google Scholar]
  • 9.LeBeau MA, Montgomery MA, Miller ML, Burmeister SG. Analysis of biofluids for gamma-hydroxybutyrate (GHB) and gamma-butyrolactone (GBL) by headspace GC–FID and GC–MS. J Anal Toxicol. 2000;24:421–428. doi: 10.1093/jat/24.6.421. [DOI] [PubMed] [Google Scholar]
  • 10.Couper FJ, Logan BK. Determination of γ-hydroxybutyrate (GHB) in biological samples by gas chromatography–mass spectrometry. J Anal Toxicol. 2000;24:1–7. doi: 10.1093/jat/24.1.1. [DOI] [PubMed] [Google Scholar]
  • 11.LeBeau MA, Christenson RH, Levine B, Darwin WD, Huestis MA. Intra- and interindividual variations in urinary concentrations of endogenous gamma-hydroxbutyrate. J Anal Toxicol. 2002;26:340–346. doi: 10.1093/jat/26.6.340. [DOI] [PubMed] [Google Scholar]
  • 12.Sporer KA, Chin RL, Dyer JE, Lamb R. Gamma-hydroxybutyrate serum levels and clinical syndrome after severe overdose. Ann Emerg Med. 2003;42(1):3–8. doi: 10.1067/mem.2003.253. [DOI] [PubMed] [Google Scholar]
  • 13.Crookes CE, Faulds MC, Forrest ARW, Galloway JH. A Reference range for endogenous gamma-hydroxybutyrate in urine by gas chromatography–mass spectrometry. J Anal Toxicol. 2004;28(8):644–649. doi: 10.1093/jat/28.8.644. [DOI] [PubMed] [Google Scholar]
  • 14.McCusker RR, Paget-Wilkes H, Chronister CW, Goldberger BA. Analysis of gamma-hydroxybutyrate (GHB) in urine by gas chromatography–mass spectrometry. J Anal Toxicol. 1999;23:301–305. doi: 10.1093/jat/23.5.301. [DOI] [PubMed] [Google Scholar]
  • 15.Yeatman DT, Reid K. A study of urinary endogenous gamma-hydroxybutyrate (GHB) levels. J Anal Toxicol. 2003;27:40–42. doi: 10.1093/jat/27.1.40. [DOI] [PubMed] [Google Scholar]
  • 16.ElSohly MA, Salamone SJ. Prevalence of drugs used in cases of alleged sexual assault. J Anal Toxicol. 1999;23:141–146. doi: 10.1093/jat/23.3.141. [DOI] [PubMed] [Google Scholar]
  • 17.Thai DL, Haller CA, Benowitz NL, Jacob P., III Rapid and efficient method for the simultaneous measurement of gamma-hydroxybutyrate and 1,4-butanediol in human plasma and urine. Submitted to J Anal Toxicol [Google Scholar]
  • 18.Kavanagh PV, Kenny P, Feely J. The urinary excretion of gamma-hydroxybutyric acid in man. J Pharm Pharmacol. 2001;53(3):399–402. doi: 10.1211/0022357011775460. [DOI] [PubMed] [Google Scholar]
  • 19.Jones AW. Excretion of alcohol in urine and diuresis in healthy men in relation to their age, the dose administered, and the time after drinking. Forensic Sci Int. 1990;45:217–224. doi: 10.1016/0379-0738(90)90177-z. [DOI] [PubMed] [Google Scholar]
  • 20.Verstraete AG. Detection times of drugs of abuse in blood, urine, and oral fluid. Ther Drug Monit. 2004;26:200–205. doi: 10.1097/00007691-200404000-00020. [DOI] [PubMed] [Google Scholar]
  • 21.Bravo DT, Harris DO, Parson SM. Reliable, sensitive, rapid and quantitative enzyme-based assay for gamma-hydroxybutyric acid (GHB) J Forensic Sci. 2004;49(2):379–387. [PubMed] [Google Scholar]