Quantitative analysis of buprenorphine and norbuprenorphine in urine using liquid chromatography tandem mass spectrometry.

Department of Clinical Biochemistry, Leeds Teaching Hospitals NHS Trust, Britannia House, Britannia Road, Morley, Leeds, LS27 0DQ, U.K.
Journal of analytical toxicology (Impact Factor: 2.63). 06/2006; 30(4):238-44. DOI: 10.1093/jat/30.4.238
Source: PubMed

ABSTRACT Buprenorphine is an opioid analgesic drug that is used as an alternative to methadone to treat heroin addiction. Established methods for the analysis of buprenorphine and its metabolites in urine such as gas chromatography-mass spectrometry (GC-MS) involve complicated sample extraction procedures. The aim of the present study was to develop a sensitive yet straightforward method for the simultaneous analysis of buprenorphine and norbuprenorphine in urine using liquid chromatography-MS-MS. The method comprised an enzymatic hydrolysis using Patella vulgata b-glucuronidase, followed by centrifugation and direct analysis of the supernatant. The limits of detection and quantitation were < 1 microg/L for buprenorphine and < 1 and 4 microg/L, respectively, for norbuprenorphine. Assay coefficients of variation (CVs) were < 15%, with the exception of concentrations close to the limit of quantitation, where CVs were below 20%. In direct comparison with an established GC-MS protocol, the method showed minimal negative bias (8.7% for buprenorphine and 1.8% for norbuprenorphine) and was less susceptible to sample carryover. The extent of conjugation in unhydrolyzed urine was investigated and found to be highly variable, with proportions of unconjugated buprenorphine and norbuprenorphine of 6.4% [range 0% to 67%; standard deviation (SD) 9.7%] and 34% (range 0% to 100%; SD 23.8%), respectively.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: A liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of buprenorphine (BUP), norbuprenorphine (NBUP), buprenorphine glucuronide (BUP-Gluc), and norbuprenorphine glucuronide (NBUP-Gluc) in human urine was developed and fully validated. Extensive endogenous and exogenous interferences were evaluated and limits of quantification were identified empirically. Analytical ranges were 5-1,000 ng/mL for BUP and BUP-Gluc and 25-1,000 ng/mL for NBUP and NBUP-Gluc. Intra-assay and interassay imprecision were less than 17% and recovery was 93-116%. Analytes were stable at room temperature, at 4 degrees C, and for three freeze-thaw cycles. This accurate and precise assay has sufficient sensitivity and specificity for urine analysis of specimens collected from individuals treated with BUP for opioid dependence.
    Analytical and Bioanalytical Chemistry 09/2008; 392(5):903-11. DOI:10.1007/s00216-008-2326-z · 3.58 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: With the increasing use of buprenorphine in treatment of opiate addiction and pain management, it is important that laboratories be able to assess patient compliance. The presented procedure is simple, efficient, and employs gas chromatography-mass spectrometry (GC-MS) technology available to most laboratories. The specimen is hydrolyzed with beta-glucuronidase prior to liquid-liquid extraction at a basic pH. The evaporated extract is derivatized to form the tertiary-butyl-dimethyl-silyl derivatives of buprenorphine and norbuprenorphine prior to analysis by GC-MS in the electron impact mode. Confirmation of the analytes is based on comparing the ion abundance ratios of the analytes to those of a contemporaneously analyzed standard. The qualitative ion abundance ratios are required to be within 20% of those of the standard for acceptance. Quantification is based on the ion ratios of the analytes to those of their corresponding deuterated analogues. Linearity was obtained for buprenorphine in the range of 1 to 2000 microg/L with a correlation coefficient (R) exceeding 0.999 and for norbuprenorphine from 1 to 1000 microg/L with R exceeding 0.997. Percent recoveries for the buprenorphine and norbuprenorphine were 71% and 75%, respectively. It was found that the recovery of norbuprenorphine could be enhanced to 100% by a simple "salting-out" modification to the procedure.
    Journal of analytical toxicology 11/2008; 32(8):626-30. DOI:10.1093/jat/32.8.626 · 2.63 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The objective was to estimate the detection times and metabolite/parent compound ratios in urine after a single dose of buprenorphine. Eighteen healthy volunteers received a single dose of 0.4 mg buprenorphine sublingually. Urine samples were collected prior to dosing and at 2, 4, 6, 8 12, 24, 48, 72, and 96 h post-dose. The samples were screened using cloned enzyme donor immunoassay (CEDIA) reagent and quantitation was performed with liquid chromatography-tandem mass spectrometry (LC-MS-MS) with a cut-off of 0.5 ng/mL for buprenorphine and norbuprenorphine. The mean time of continuous positive results was 9 h (range 4 to 24 h) with CEDIA, whereas for an LC-MS-MS method it was 76 h (range 23-96 h) for buprenorphine, and for norbuprenorphine all samples were positive at 96 h. Some subjects had positive CEDIA results after a negative sample, owing to differences in creatinine concentration. The time when the ratio norbuprenorphine/buprenorphine exceeded 1 was estimated at 7 h. The metabolite/parent ratio may be used to estimate the time of intake even though the individual ratios showed an increased variation the more distant the collection time. We believe that using this ratio, rather than the actual concentrations, it is possible to compensate for urine dilution and different doses, and to improve interpretation.
    Journal of analytical toxicology 11/2008; 32(8):586-93. DOI:10.1093/jat/32.8.586 · 2.63 Impact Factor
Show more