Urinary buprenorphine concentrations in patients treated with suboxone as determined by liquid chromatography-mass spectrometry and CEDIA immunoassay

We report on the utility of urine total buprenorphine, total norbuprenorphine, and creatinine concentrations in patients treated with Suboxone (a formulation containing buprenorphine and naloxone), used increasingly for the maintenance or detoxification of patients dependent on opiates such as heroin or oxycodone. Patients received 8-24 mg/day buprenorphine. Two-hundred sixteen urine samples from 70 patients were analyzed for both total buprenorphine and total norbuprenorphine by liquid chromatography-mass spectrometry (LC-MS-MS). Buprenorphine concentrations in all 176 samples judged to be unadulterated averaged 164 ng/mL, with a standard deviation (SD) of 198 ng/mL. Nine samples (4.2%) had metabolite-parent drug ratios < 0.02, and 33 (15.3%) had no detectable buprenorphine. The metabolite/parent drug ratio in 166 samples had a range of 0.07-23.0 (mean = 4.52; SD = 3.97). Fifteen of 96 available urine samples (16.7%) had creatinine less than 20 mg/dL. We also found sample adulteration in 7 (7.3%) available samples. Using a 5 ng/mL urine buprenorphine cutoff, the sensitivity and specificity of the Microgenics homogeneous enzyme immunoassay versus LC-MS-MS were 100% and 87.5%, respectively. The 5 ng/mL cutoff Microgenics CEDIA buprenorphine assay results agreed analytically with LC-MS-MS in 97.9% of samples.     

Validation of the Immunalysis microplate ELISA for the detection of buprenorphine and its metabolite norbuprenorphine in urine

The purpose of this study was to validate the Immunalysis Buprenorphine Microplate enzyme-linked immunosorbent assay (ELISA) for the detection of buprenorphine in urine samples. Sixty-nine urine samples were obtained from volunteers on the Subutex treatment program and from routine samples submitted to the laboratory for buprenorphine testing. For ELISA analysis, samples were diluted 1:10 with K(2)HPO(4) (0.1M, pH 7.0). The limit of detection was calculated as 0.5 ng/mL buprenorphine. The intra-assay and interday precision was 3.8% (n = 10) and 8.6% (n = 50) respectively at 1 ng/mL buprenorphine. At a low concentration of norbuprenorphine (1 ng/mL), the immunoassay demonstrated a cross-reactivity of 78%. A higher cross-reactivity of 116% was observed at a higher concentration of norbuprenorphine (10 ng/mL). Dihydrocodeine, codeine, tramadol, morphine, propoxyphene, methadone, and EDDP were tested at concentrations of 10 ng/mL and 10,000 ng/mL and demonstrated no cross-reactivity with the assay. For liquid chromatography-tandem mass spectrometry (LC-MS-MS), deuterated internal standard mixture, 1M acetate buffer (pH 5.0), and b-glucuronidase were added to the standards and samples, which were then incubated for 3 h at 60 degrees C. After incubation, 3 mL K(2)HPO(4) (0.1M, pH 6.0) was added and the pH altered to pH 6.0 using 1M KOH. Buprenorphine and norbuprenorphine were subsequently extracted by solid-phase. Twenty-one samples were confirmed positive and 48 samples were confirmed negative by LC-MS-MS. Using a cut-off value of 0.5 ng/mL buprenorphine, the immunoassay demonstrated a sensitivity and specificity of 100%.     

Urine naloxone concentration at different phases of buprenorphine maintenance treatment

In spite of the benefits of buprenorphine‐naloxone co‐formulation (BNX) in opioid maintenance treatment, the naloxone component has not prevented parenteral use of BNX. Current laboratory methods are not sufficient to differentiate between therapeutic and illicit use of buprenorphine, and little is known about urine naloxone concentrations. Measurement of urine naloxone, together with buprenorphine and norbuprenorphine, might help to determine the naloxone source and administration route. A liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) method was developed and validated for this purpose. Naloxone, buprenorphine, and norbuprenorphine total concentrations were measured in urine samples from opioid‐dependent patients before and during stable and unstable phases of maintenance treatment with BNX. The limit of quantification in urine was 1.0 µg/L for naloxone, buprenorphine and norbuprenorphine. Before treatment, all samples contained buprenorphine but the median naloxone concentration was 0 µg/L. During the maintenance treatment with BNX all urine samples were positive for naloxone, buprenorphine and norbuprenorphine. The naloxone concentration at a stable phase of treatment (median 60 µg/L, range 5–200 µg/L) was not different from the naloxone concentration at an unstable phase (70 µg/L, 10–1700 µg/L). Applying an upper limit of 200 µg/L to the sample, the median naloxone/buprenorphine ratio was higher in the high than in the low naloxone concentration group (0.9 vs 0.3, respectively). This study suggests that naloxone in urine can act as an indicator of compliance with BNX. Parenteral use of BNX was associated with a high naloxone/buprenorphine ratio. Negative naloxone with positive buprenorphine suggests the use/abuse of buprenorphine alone. Copyright © 2013 John Wiley & Sons, Ltd.     

Quantitative testing of buprenorphine and norbuprenorphine to identify urine sample spiking during office-based opioid treatment

Background: Patients may spike urine samples with buprenorphine during office-based opioid treatment to simulate adherence to prescribed buprenorphine, potentially to conceal diversion of medications. However, routine immunoassay screens do not detect instances of spiking, as these would simply result in a positive result. The aim of this study was to report on the experience of using quantitative urine testing for buprenorphine and norbuprenorphine to facilitate the identification of urine spiking. Methods: This is a retrospective chart review of 168 consecutive patients enrolled in outpatient buprenorphine treatment at an urban academic medical setting between May 2013 and August 2014. All urine samples submitted were subjected to quantitative urine toxicology testing for buprenorphine and norbuprenorphine. Norbuprenorphine-to-buprenorphine ratio of less than 0.02 were further examined for possible spiking. Demographic and clinical variables were also extracted from medical records. Clinical and demographic variables of those who did and did not spike their urines were compared. Statistically significant variables from the univariate testing were entered as predictors of spiking in a regression analysis. Results: A total of 168 patients were included, submitting a total of 2275 urine samples. Patients provided on average 13.6 (SD = 9.9) samples, and were in treatment for an average 153.1 days (SD = 142.2). In total, 8 samples (0.35%) from 8 patients (4.8%) were deemed to be spiked. All of the samples suspected of spiking contained buprenorphine levels greater than 2000 ng/mL, with a mean norbuprenorphine level of 11.9 ng/mL. Spiked samples were submitted by 6 patients (75.0%) during the intensive outpatient (IOP) phase of treatment, 2 patients (25.0%) during the weekly phase, and none from the monthly phase. Regression analysis indicated that history of intravenous drug use and submission of cocaine-positive urine samples at baseline were significant predictors of urine spiking. Conclusions: Even though only a small number of patients were identified to have spiked their urine samples, quantitative testing may help identify urine spiking during office-based opioid treatment with buprenorphine.     

Effect of tramadol use on three point-of-care and one instrument-based immunoassays for urine buprenorphine

We report that use of the popular analgesic tramadol can cause false-positive urine buprenorphine results. We examined the extent of tramadol cross-reactivity in three point-of-care urine buprenorphine immunoassays (ACON, QuikStrip, and ABMC) and an instrument-based one (Cedia). We tested 29 urine samples from patients known to be taking tramadol. Ten different samples tested positive for urine buprenorphine by at least one immunoassay. Samples with positive buprenorphine screens by immunoassay were tested for total buprenorphine and total norbuprenorphine content by liquid chromatography-tandem mass spectrometry (LC-MS-MS), which confirmed that seven of the 10 positive samples were false-positives. The remaining three positive immunoassay samples had insufficient quantity for LC-MS-MS testing. No false-positives were detected with the ACON (10 ng/mL calibration cutoff) or the Cedia assay (using a 20 ng/mL calibration cutoff). All four false-positive Cedia results (using a 5 ng/mL cutoff) in this study tested negative using the ACON device. Our data suggest that tramadol use can cause false-positive urine buprenorphine immunoassays, and this effect appears to be assay-dependent. Tramadol interference with the Cedia assay is clinically relevant, especially if the 5 ng/mL calibration cutoff is used.     

Development and GC-MS validation of a highly sensitive recombinant G6PDH-based homogeneous immunoassay for the detection of buprenorphine and norbuprenorphine in urine

Buprenorphine is now increasingly prescribed as an alternative to methadone for the treatment of heroin addiction. Because of its potency (dosage usages from 0.2 mg to 8 mg), the drug concentrations in body fluids are normally very low. Here, we report the first recombinant glucose-6-phosphate dehydrogenase (G6PDH)-based homogeneous immunoassay (EMIT-type assay) for free buprenorphine and free norbuprenorphine in urine. The antibody used in this assay cross-reacts nearly identically with buprenorphine and norbuprenorphine and, at the same time, has less than 1% cross-reactivity with a wide range of commonly prescribed opiates, particularly those structurally related compounds such as morphine, codeine, and dihydrocodeine. More importantly, this assay has a low detection limit of 1 ng/mL for buprenorphine or norbuprenorphine. Further evaluation of this technique using gas chromatography-mass spectrometry (GC-MS) of authentic urine samples demonstrated that the accuracy of the assay is greater than 95%. Because this assay is designed to measure the free drugs in urine, it resulted in simplification for GC-MS or liquid chromatography-MS confirmation methods that did not require urine hydrolysis before solid-phase or liquid-liquid extraction.     

Analysis of buprenorphine, norbuprenorphine, and their glucuronides in urine by liquid chromatography-mass spectrometry

Buprenorphine is used for the treatment of chronic pain and also in treatment of heroin addiction as an alternative to methadone. As the availability of buprenorphine increases, so does the risk for abuse and the pressure on forensic and clinical laboratories to analyze for it. Buprenorphine and its dealkylated metabolite are excreted in urine, almost exclusively as glucuronides. The aim of the present study was to evaluate electrospray liquid chromatography tandem mass spectrometry (LC-MS-MS) for the rapid screening and quantitation of buprenorphine and its metabolites in urine. Three approaches were evaluated: (1) direct injection of diluted urine for measurement of glucuronides, (2) direct injection of diluted urine after enzymatic hydrolysis for the quantitation of buprenorphine and norbuprenorphine, and (3) quantitation of buprenorphine and norbuprenorphine after enzymatic hydrolysis and solid-phase extraction (SPE). One hundred six samples were subjected to procedure 1 and, when positive, further quantitated using procedure 2. Only samples with low analyte concentrations (< 20 microg/L) were subject to SPE. Concentrations of buprenorphine and norbuprenorphine in patients (N = 16) ranged between 31 and 1080 microg/L and 48-2050 microg/L, respectively. In suspected abusers (N = 33), the ranges were 2.3-796 microg/L and 5.0-2580 microg/L. In four of the authentic samples, both the buprenorphine and norbuprenorphine concentrations were below the 20- micro g/L cutoff. We concluded that LC-MS-MS analysis of the glucuronides provided an adequate screening method, but that the direct method for quantitation sometimes had to be complemented with a concentration by SPE, providing increased sensitivity, thus lowering the cutoff from 20 to 1 microg/L urine.     

Determination of buprenorphine and norbuprenorphine in urine and hair by gas chromatography-mass spectrometry.

Buprenorphine, which is used in France as a substitution drug for opioid addiction, is widely abused, and several fatal cases have been reported. In order to confirm a recent intoxication or to establish retrospectively chronic abuse, a simple and reliable gas chromatographic-mass spectrometric method was developed and validated for quantitation of buprenorphine and its active metabolite norbuprenorphine in urine and hair. Two milliliters of urine or 50 mg of pulverized hair was submitted to a pretreatment (enzymatic hydrolysis for urine and decontamination with dichloromethane followed by incubation in 0.1 M HCI for hair). Buprenorphine-d4 was chosen as the internal standard. Selective solid-phase extraction with Bond Elut Certify columns provided recoveries higher than 85% for urine and 43% for hair. By using a mixture of MSTFA/TMSIM/TMCS (100:2:5), buprenorphine and norbuprenorphine produced stable silylated derivatives. The detection was carried out with a quadrupole mass detector working in El selected ion monitoring mode. Ions at m/z 450 and 468 were chosen for the quantitation of buprenorphine and norbuprenorphine, respectively (m/z 454 was used for the internal standard). Limits of quantitation were 0.25 and 0.20 ng/mL, respectively, for buprenorphine and norbuprenorphine in urine and 0.005 ng/mg for the two compounds in hair. Calibration curves were linear from 0 to 50 ng/mL in urine and from 0 to 0.4 ng/mg in hair. Between-day and within-day precisions were less than 8.4% in hair and 6.1% in urine for both molecules in all cases. This method was applied to urine and hair samples collected from patients in a withdrawal treatment program and demonstrated its good applicability in routine analysis and its benefit for clinicians. This technique, which requires instruments already available to many toxicology laboratories, offers an attractive alternative to more sophisticated techniques.     

The in vivo glucuronidation of buprenorphine and norbuprenorphine determined by liquid chromatography-electrospray ionization-tandem mass spectrometry

The opioid partial agonist medication, buprenorphine (BUP), and its primary metabolite, norbuprenorphine (NBUP), are extensively glucuronidated. Sensitive analytical methods that include determination of buprenorphine-3-glucuronide (BUPG) and norbuprenorphine-3-glucuronide (NBUPG) are needed to more fully understand the metabolism and pharmacokinetics of buprenorphine. A method has now been developed that uses solid-phase extraction followed by liquid chromatography-electrospray ionization-tandem mass spectrometry. BUP-d4, NBUP-d3, and morphine-3-glucuronide-d3 were used as internal standards. The lower limit of quantitation was 0.1 and 0.5 ng/mL for each of the analytes in 1-mL of human plasma and urine, respectively, except for NBUP in urine in which it was 2.5 ng/mL. The analytes were stable under the following conditions: plasma and urine at room temperature, up to 20 hours; plasma and urine at -20 degrees C for 119 and 85 days, respectively; plasma freeze-thaw, up to 3 cycles; processed sample, up to 96 hours at -20 degrees C and up to 48 hours on the autosampler; stock solutions at room temperature and at -20 degrees C, up to 6 hours and 128 days, respectively. In plasma collected from 5 subjects on maintenance daily sublingual doses of 16 mg BUP and 4 mg naloxone, respective 0- to 24-hour areas under the curve were 32, 88, 26, and 316 ng/mL x h for BUP, NBUP, BUPG, and NBUPG. In urine samples respective percent of daily dose excreted in the 24-hour urine were 0.014%, 1.89%, 1.01%, and 7.76%. This method allowed us to determine that NBUPG is a major metabolite present in plasma and urine of BUP. Because urinary elimination is limited ( approximately 11% of daily dose), the role of NBUPG in total clearance of buprenorphine is not yet known.     

Simultaneous determination of buprenorphine and norbuprenorphine in serum by high-performance liquid chromatography-electrospray ionization-mass spectrometry

Buprenorphine is a strong narcotic analgesic. It is also used in the substitution therapy for opium alkaloid addicts. The aim of this paper was to develop and validate a highly sensitive high-performance liquid chromatography-electrospray ionization-mass spectrometry method for simultaneous determination of buprenorphine and norbuprenorphine in human serum. The developed methodology was then applied to real clinical cases in a clinical toxicology setting. Extraction of analytes has been done using solid-phase extraction. Chromatographic separation was achieved on a LiChroCART column with a Purospher RP-18e cartridge, and for detection an LCQ mass spectrometer with an ion trap analyzer was used. Quantitation of buprenorphine and norbuprenorphine was performed in a single ion monitoring mode (m/z 468 buprenorphine, m/z 414 for norbuprenorphine) in order to increase the sensitivity of the method. The standard curves for both compounds were linear over the range of 0.2-10 ng/mL (r2 > 0.995). The quantitation limit was 0.2 ng/mL for both analytes. The method was used for quantitation of both buprenorphine and norbuprenorphine in the serum of 15 patients undergoing the buprenorphine substitution therapy. Serum concentrations ranged between 0.36 and 4.60 ng/mL for buprenorphine and 0.21 and 2.50 ng/mL for norbuprenorphine, with buprenorphine single dosages from 0.8 to 6.0 mg.     

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

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.     

Liquid chromatography-mass spectrometric analysis of buprenorphine and its N-dealkylated metabolite norbuprenorphine in rat brain tissue and plasma

INTRODUCTION: A specific, accurate, and reproducible liquid chromatography-mass spectrometric (LC/MS) method was developed and validated that allows simultaneous measurement of the centrally acting analgesic buprenorphine and its major metabolite, norbuprenorphine, in rat brain and plasma samples. METHODS: A 96-well plate solid phase extraction (SPE) procedure was developed for buprenorphine and norbuprenorphine using mixed-mode cation-exchange reversed-phase sorbent. An LC method using a C8 column with isocratic mobile phase (80:20 water/acetonitrile with 20 mM ammonium acetate and 0.1% acetic acid) was developed for reproducible and selective separation. A quadrupole mass spectrometer with atmospheric electrospray ionization source under positive ion mode was used for detection. d4-Buprenorphine and d3-norbuprenorphine were used as internal standards. RESULTS: The calibration curves for buprenorphine and norbuprenorphine in plasma and brain tissue were linear within the range of 7 to 8333 ng/ml (plasma) and 5 to 5000 ng/g (brain). The lower limit of quantification for both buprenorphine and norbuprenorphine from brain tissue was 5 ng/g, and from plasma was 7 ng/ml. Assay accuracy and precision of back-calculated standards were within +/-15%. DISCUSSION: This method will be useful for investigation of buprenorphine's mechanism of action and clinical profile.     

Evaluation of buprenorphine CEDIA assay versus GC-MS and ELISA using urine samples from patients in substitution treatment

As buprenorphine becomes more clinically used in heroin substitution treatment, there is an increasing need for methods suitable for high-volume screening. In this study, a new immunochemical test based on CEDIA technology was evaluated for the use in clinical urine drug testing. The method was compared with an existing ELISA method and a gas chromatography-mass spectrometry (GC-MS) method on urine specimens from patients in heroin substitution treatment. The precision of the CEDIA assay was < 9% both within- and between-day at levels at and above the cutoff limit of 5 microg/L. The concordance in qualitative results with an existing ELISA method was 96.8%. The CEDIA measuring range was extended by diluting urine samples 100-fold with saline, and the results agreed well (slope of regression line was 1.09, r(2) = 0.968) with GC-MS. The sensitivity of CEDIA in detecting authentic specimen containing buprenorphine at levels >or= 5 microg/L was 99.5%. Cross-reactivity causing false-positive response was discovered in patients receiving prescribed dihydrocodeine. The urine concentration of total buprenorphine in urine from patients prescribed daily doses between 0.2 and 24 mg ranged from 0.5 to 2900 microg/L. The concentration of the metabolite norbuprenorphine was usually higher, and the median ratio of buprenorphine to norbuprenorphine was 0.23 (95% were below 1). We conclude that the CEDIA assay is suitable for application in high-volume screening of buprenorphine for urine drug testing.     

Urine drug testing of chronic pain patients. III. Normetabolites as biomarkers of synthetic opioid use

Opioids are important therapeutic agents available to patients with moderate to severe pain. The synthetic opioids, buprenorphine, fentanyl, meperidine, methadone, and propoxyphene have been utilized for decades as analgesics. One of the major biotransformation pathways of these drugs occurs through N-demethylation leading to the formation and excretion of normetabolites. Normetabolites generally exhibit longer half-lives than the parent drug leading to accumulation with prolonged use. As part of continuing research efforts to improve monitoring programs of chronic pain patients undergoing opioid treatment, we evaluated the prevalence and relative abundance of normetabolites of buprenorphine, fentanyl, meperidine, methadone, and propoxyphene in patients? urine specimens. Selected sets of specimens were analyzed without prior immunoassay screening by liquid chromatography-tandem mass spectrometry for buprenorphine, fentanyl, meperidine, methadone, propoxyphene, and their respective normetabolites. Limits of quantitation (LOQ) were as follows: buprenorphine, 1 ng/mL; fentanyl, 0.5 ng/mL; meperidine, 50 ng/mL; methadone, 50 ng/mL; and propoxyphene, 50 ng/mL. LOQs for normetabolites were equal to the parent drug with the exception of norbuprenorphine (2.5 ng/mL). The percentage of positive specimens that contained normetabolite (only) ranged from 8.0% for EDDP (2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine) to 53.1% for norpropoxyphene. Inclusion of the five normetabolites in the test panel produced an increase in detection rates for parent drug use as follows: buprenorphine, 10.0%; fentanyl, 42.1%; meperidine, 98.7%; methadone, 8.7%; and propoxyphene, 113.2%. The authors conclude that testing for synthetic opioid normetabolites enhances the effectiveness of monitoring programs for pain patients.     

Urine concentrations of fentanyl and norfentanyl during application of Duragesic transdermal patches

A study of the urinary concentration of fentanyl (F) and its major metabolite norfentanyl (NF) in chronic pain patients treated with the Duragesic continuous release transdermal patches is presented. These patches are available in 10, 20, 30, and 40 cm(2) sizes releasing 25, 50, 75, and 100 microg/h F, respectively. F is rapidly and extensively metabolized, with NF as the major metabolite. Five hundred-forty six random urine specimens were collected from chronic pain patients wearing 25, 50, 75, or 100 ug F transdermal patches. Urine specimens were collected from hours after application to several days later after continuous F release. Each specimen was analyzed for F, NF, creatinine, and pH. Additionally, each was screened by enzyme immunoassay for the following: amphetamines, barbiturates, benzodiazepines, cocaine metabolite, methadone, phencyclidine, d-propoxyphene, opiates, and marijuana metabolites. All positive screening results were confirmed by gas chromatography-mass spectrometry (GC-MS). F and NF were isolated from urine by solid-phase extraction then identified and quantified by GC-MS in SIM mode. The LODs and LOQs for F and NF were 3 ng/mL, respectively. The results of F and NF analysis of urine form those wearing 25-microg patches (N = 142) was mean F, 47 ng/mL with a range of 0 to 983 ng/mL, and 97% of the specimens contained < 200 ng/mL and mean NF, 175 ng/mL with a range of 0-980 ng/mL, while 95% of the specimens contained < 400 ng/mL. The results of F and NF analysis of urine form those wearing 50 microg patches (N = 184) was: mean F, 74 ng/mL with a range of 0 to 589 ng/mL, and 92% of the specimens contained < 200 ng/mL and mean NF, 257 ng/mL with a range of 0-2200 ng/mL, and 98% of the specimens contained < 1000 ng/mL. The results of F and NF analysis of urine form those wearing 75 microg patches (N = 85) was mean F, 107 ng/mL with a range of 0 to 1280 ng/mL, and 98% of the specimens contained < 400 ng/mL and mean NF, 328 ng/mL with a range of 0-5630 ng/mL, and 99% of the specimens contained < 1000 ng/mL. The results of F and NF analysis of urine form those wearing 100 ug patches (N = 135) was mean F, 100 ng/mL with a range of 0 to 1080 ng/mL, while 96% of the specimens contained < 400 ng/mL and mean NF, 373 ng/mL with a range of 0-5730 ng/mL, and 95% of the specimens contained < 1000 ng/mL. The incidence of other drugs detected as a percentage the specimens was opiates, 48%, benzodiazepines, 43%; barbiturates, 3%; methadone, 4%; marijuana metabolite, 3%; and cocaine metabolite, 1%. With the exception of F and/or NF, no other drugs were detected in 25% of the specimens. These data demonstrate the wide variation in concentrations of F and NF in random urine specimens following application of Duragesic patches. However, these values obtained during therapeutic use far exceed concentrations previously reported in fatal poisoning. In general, one may expect to find urine NF concentrations 3-4 times higher than those of F.     

Comparison of an automated and point-of-care immunoassay to GC-MS for urine oxycodone testing in the clinical laboratory

OxyContin, a controlled-release formulation of oxycodone, is increasingly abused. Monitoring patient compliance by urine drug testing may deter illegal diversion of OxyContin. Two urine immunoassays were evaluated with a 100 ng/mL cutoff for oxycodone. The Microgenics Corporation Oxycodone DRI on the Bayer ADVIA 1650 and a point-of-care (POC) immunoassay, Monitect Oxycodone POC from Branan Medical Corporation, were compared to gas chromatography-mass spectrometry (GC-MS) with a detection limit of 50 ng/mL free oxycodone. Between-day precision for DRI yielded coefficients of variation from 3.9% to 7.0% at 75 and 125 ng/mL. Fifty-two positive and 52 negative urines were tested. The DRI had a 100% agreement with GC-MS. Two positive specimens had free oxycodone < 50 ng/mL, but oxycodone metabolites, oxymorphone and oxycodone glucuronide > 100 ng/mL, were identified by GC-MS analysis. The POC assay had two false positives and 15 indeterminate (+/-) results. Codeine or hydrocodone was present in all but one of these samples. There was no interference with DRI from morphine, codeine, hydrocodone, hydromorphone, dihydrocodeine, or 6-monoacetyl morphine. Four-hundred and ninety urine samples were subsequently tested with DRI to estimate the oxycodone-positive rate at our hospital, and 47 (9.4%) were positive. The confirmation rate with GC-MS for free oxycodone, not including metabolites, was 93%. The Microgenics DRI offers good performance for oxycodone urine testing and is a better choice for the clinical laboratory than the POC assay. Confirmation of screened positive samples requires a method that can detect total oxycodone and oxymorphone.     

Liquid chromatographic-electrospray ionization mass spectrometric quantitative analysis of buprenorphine, norbuprenorphine, nordiazepam and oxazepam in rat plasma

A liquid chromatographic-mass spectrometric method with electrospray ionization is presented for the simultaneous determination of buprenorphine, nordiazepam and their pharmacologically active metabolites, norbuprenorphine and oxazepam, in rat plasma. The drugs were extracted from plasma by liquid-liquid extraction and chromatographically separated using a gradient elution of aqueous ammonium formate and acetonitrile. Following electrospray ionization, the analytes were quantified in the single ion storage mode. The assay was validated according to current acceptance criteria for bioanalytical method validation. It was proved to be linear from 0.7 to 200 ng/ml plasma for buprenorphine, 1.0 to 200 ng/ml for norbuprenorphine, 2.0 to 200 ng/ml for nordiazepam, and from 5.0 to 200 ng/ml for oxazepam. The average recoveries of buprenorphine, norbuprenorphine, nordiazepam and oxazepam were 89, 39, 88 and 82%, respectively, with average coefficients of variation ranging from 1.8 to 14.3%. The limits of quantitation for these drugs were 0.7, 1.0, 2.0 and 5.0 ng/ml, respectively, with associated precisions within 17% and accuracies within +/-18% of the nominal values. Both the intra- and inter-assay precision values did not exceed 11.3% for the four analytes. Intra- and inter-assay accuracies lay within +/-15% of the nominal values. The validated method was applied to the determination of buprenorphine, norbuprenorphine, nordiazepam and oxazepam in plasma samples collected from rats at various times after intravenous administration of buprenorphine and nordiazepam.     

Hydrolysis of conjugated metabolites of buprenorphine. I. The quantitative enzymatic hydrolysis of buprenorphine-3-beta-D-glucuronide in human urine

Buprenorphine, which is a powerful analgesic, a substitution drug for opioids widely used in Europe, and a promising new drug currently undergoing clinical trials in the treatment of opioid dependence in the U.S., is excreted in human urine mainly as glucuronide conjugates. In gas chromatographic-mass spectrometric analysis, the urine specimens must be first hydrolyzed to release buprenorphine from its glucuronide conjugates. In order to evaluate the existing hydrolysis methods and to find the optimal hydrolysis conditions, buprenorphine-3-beta-D-glucuronide (B3G) was synthesized. Urine fortified with synthetic B3G was hydrolyzed using acid, base, and beta-glucuronidases from different source species, including Helix pomatia, Escherichia coli, and Patella vulgata. Glusulase, a preparation containing both beta-glucuronidase (H. pomatia) and sulfatase, was also tested. Whereas both acidic and basic hydrolysis were ineffective, quantitative hydrolysis could be achieved by using beta-glucuronidases under appropriate conditions. However, we found that there was a marked difference in the reactivity of these enzymes (E. coli > H. pomatia > P. vulgata). The optimal incubation conditions for enzymatic hydrolysis of B3G were 2 h at 37 degrees C for E coli and 4 h at 60 degrees C or 16 h at 37 degrees C for H. pomatia. Using 1000 Fishman units of either of these two enzymes, effective hydrolysis could be achieved even when the B3G concentration was as high as 2000 ng/mL. Glusulase was equally effective toward B3G if the fortified urine samples were incubated with 25 microL of this enzyme for 1 h at 60 degrees C.     

Validation of a liquid chromatography-tandem mass spectrometry method for the simultaneous determination of 26 benzodiazepines and metabolites, zolpidem and zopiclone, in blood, urine, and hair

A liquid chromatography-tandem mass spectrometry method was developed for the simultaneous quantification of 26 benzodiazepines and metabolites, zolpidem and zopiclone, in blood, urine, and hair. Drugs were extracted from all matrices by liquid-liquid extraction with 1-chlorobutane. Chromatography was achieved using a XTerra MS C18 column eluted with a mixture of methanol and formate buffer. Data were acquired using positive electrospray ionization and multiple reaction monitoring using one precursor ion/product ion transition per compound. Quantification was performed using 13 deuterated analogues. Further confirmation of the identity of the compounds was achieved through a second injection of positive samples, monitoring two transitions per compound. The limits of quantification for all benzodiazepines ranged from 1 to 2 ng/mL in blood, 10 to 25 ng/mL in urine, and 0.5 to 10 pg/mg in hair. Linearity was observed from the limit of quantification of each compound to 200 ng/mL, 1000 ng/mL, and 1000 pg/mg for blood, urine, and hair, respectively (r2 > 0.99). Precision for quality control samples, spiked at three concentrations, was calculated (CV < 20% in most cases). Extraction recoveries for the three matrices ranged from 25.1 to 103.8%, except for one compound (cloxazolam in urine). Ion suppression was studied for all matrices. The validated assay was applied to authentic blood, urine, and hair samples from forensic cases.     

Enzyme immunoassay validation for the detection of buprenorphine in urine

A solid-phase enzyme immunoassay involving microtiter plates was proposed by Microgenics to screen buprenorphine in urine. The intra-assay precision at 10 ng/mL was 7.7% (coefficient of variation). The immunoassay was determined to have no cross-reactivity with codeine, dihydrocodeine, morphine, ethylmorphine, 6-monoacetylmorphine, methadone, pholcodine, propoxyphene, dextromoramide, and dextromethorphan at 1 and 10 mg/L. A low cross-reactivity (3% at 1 ng/mL) was observed at low concentrations of norbuprenorphine. After comparing this new immunological test (Singlestep ELISA) for 76 urine specimens with our validated high-performance liquid chromatography-electrospray mass spectrometry (HPLC-ES-MS) procedure, an optimum cutoff concentration of 2 ng/mL was determined for the kit. At this cutoff, the screening assay was able to determine more than 90% of true results with 43.4% true positives and 48.7% true negatives. Four positive urines (5.3%) were not confirmed by HPLC-ES-MS. In only one case, the negative urine test was confirmed as positive by HPLC-ES-MS (buprenorphine: 62.5 ng/mL). Buprenorphine concentrations determined by HPLC-ES-MS ranged from 1.2 to 1052 ng/mL. Of the four potential adulterants (hypochloride 50 mL/L, sodium nitrite 50 g/L, liquid soap 50 mL/L, and sodium chloride 50 g/L) that might be added to a positive urine specimen, none were able to cause a false-negative response by the immunoassay. The results of this study support the concept that the Singlestep ELISA for buprenorphine determination in urine should be considered as a new, valided screening procedure.