MSACL 

24. Monitoring Buprenorphine Treatment and Compliance using LC-MS/MS
Mon 4:36 PM - PosterSplash Track 3
Kara Lynch
UCSF
Kara L. Lynch, Weiming Ruan, Jean S. Branch and Alan H. B. Wu

Department of Laboratory Medicine, University of California San Francisco, CA
Introduction:
Buprenorphine (BUP) is a semi-synthetic opiate analgesic used in the treatment of opiate addiction. BUP is metabolized by CYP3A4 to norbuprenorphine (NBUP), followed by glucuronidation of BUP and NBUP. A sublingual co-formulation of BUP with naloxone (Suboxone®) was produced in attempts to deter diversion and intravenous misuse. However, multiple cases of diversion and abuse have been reported. The two FDA approved BUP immunoassays have limitations which make monitoring compliance difficult, including cross-reactivity with other abused opiates. Also, due to the long half-life of BUP and its metabolites, immunoassay results can stay positive for up to two weeks after last use. The purpose of this study was to determine the feasibility of monitoring treatment compliance through the use of an LC-MS/MS assay.

Methods:
An LC-MS/MS method was developed and validated for the determination of BUP, NBUP, buprenorphine-glucuronide (BUPG) and norbuprenorphine-glucuronide (NBUPG) in urine. After the addition of deuterated internal standards, the urine samples were extracted by SPE using Oasis MCX Cartridges (Waters). Chromatographic separation was performed on a Synergi Polar reverse-phase 2.5-µm column (100 x 2.0 mm) using a mobile phase consisting of 1mM ammonium formate adjusted to pH3 and acetonitrile. BUP and metabolites were monitored in MRM-IDA-EPI mode (Applied Biosystems 3200QTRAP®MS/MS) using the following transitions, 468.3/396.2 (BUP), 414.3/83.1 (NBUP), 644.3/468.3 (BUPG) and 590.3/414.3 (NBUPG). Intraday and interday precision, for all analytes, was between 4.0% and 10.6%, recovery was between 84% and 95% and ion suppression was <33%. Calibration curves were linear for all analytes over the concentration range 2-1000 ng/mL. Limits of detection and quantitation for all analytes were 1-2 ng/mL and 1-10 ng/mL, respectively.

Results:
Over 100 urines from patients prescribed BUP were tested. These included samples from noncompliant patients who reported missed doses and patients who were taking CYP3A4 inhibitors concurrently. The average values for compliant patients were 4 (BUP), 162 (NBUP), 562 (BUPG) and 1177 ng/mL (NBUPG). These values were significantly decreased for patients who reported not taking BUP for two or more days (1, 71, 130 and 339 ng/mL). The levels of NBUP and NBUPG were significantly decreased for patients taking CYP3A4 inhibitors (1, 9, 447 and 103 ng/mL). There was a positive correlation between BUP metabolite levels and BUP dose. Repeat patient samples showed low within-subject variability.

Conclusion:
The BUP and BUP metabolite levels determined by the LC-MS/MS assay are useful for monitoring BUP treatment compliance and circumvent problems posed by the established immunoassays.
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