Urinary Detection Times and Metabolite/Parent Compound Ratios After a Single Dose of Buprenorphine

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1 Urinary Detection Times and Metabolite/Parent Compound Ratios After a Single Dose of Buprenorphine Robert Kronstrand 1,*, Ingrid Nyström 1, Malin Andersson 1, Lina Gunnarsson 1, Staffan Hägg 2,3, Martin Josefsson 1, and Johan Ahlner 1 1 National Board of Forensic Medicine, Department of Forensic Genetics and Forensic Toxicology, Linkoping, Sweden; 2 Department of Clinical Pharmacology, Sahlgrenska University Hospital, Göteborg, Sweden; 3 Division of Clinical Pharmacology, Linköping University, Linköping, Sweden 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 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. Introduction Buprenorphine is an opioid acting as partial agonist and antagonist on the µ- and κ-opioid receptors. It has been used for the prevention or treatment of moderate to severe chronic pain with therapeutic doses of mg in the form of sublingual tablets (Temgesic ). In most European countries including Sweden, buprenorphine is also used in treatment of heroin addiction as an alternative to methadone (1,2). The doses used on this indication range from 1 to 32 mg/day. Until 2005, sublingual buprenorphine for treatment of opioid * Author to whom correspondence should be addressed. Robert Kronstrand, National Board of Forensic Medicine, Department of Forensic Genetics and Forensic Toxicology, Artillerigatan 12, SE , Linkoping, Sweden; robert.kronstrand@rmv.se. dependence (Subutex ) was increasingly prescribed in Sweden and a rapid spread to the black market was noted. Therefore, restrictions on the prescription of Subutex came in Despite these restrictions, there has been a steady increase in the request for forensic buprenorphine analysis. During 2007, the National Board of Forensic Medicine, which serves all of Sweden, screened approximately 20,000 and confirmed 400 urine samples (2%) as positive for buprenorphine. A majority of these samples came from either the prison boards or the police (petty drug offences) but an increase in requests from forensic autopsy cases has also occurred. Recently, a new formulation of buprenorphine for treatment of opioid dependence, Suboxone, was introduced on the Swedish market. This formulation contains not only buprenorphine but also the µ-receptor antagonist naloxone. In the ratio 4:1, naloxone has no pharmacological effect after oral or sublingual administration (3,4). It will only exert its opioid receptor antagonist effect if the tablet is injected. In this way, the new formulation should reduce the potential for abuse. Buprenorphine is usually administered sublingually because it has a very low bioavailability after oral administration owing to an extensive first-pass metabolism. After sublingual administration the maximum plasma concentration is reached after 40 min to 3.5 h. In humans, buprenorphine is metabolized primarily in the liver by N-dealkylation to norbuprenorphine (norbuprenorphine) through CYP3A4 (5). Both the metabolite and the parent drug undergo extensive conjugation to glucuronides that are excreted in the urine (5,6). Norbuprenorphine is an active metabolite but the conjugated metabolites seem to be inactive (5). Urine concentrations of free buprenorphine and norbuprenorphine have been reported to be low, with mean percentages of 6% and 35% (7) or 10% and 22%, as reported by Blom et al. (8), and thus a hydrolysis step is recommended before analysis, unless the conjugates are analyzed directly (9,10). Even though glucuronide reference materials are available, many laboratories tend to use a hydrolysis before analysis and thus measure the total concentration of the compounds (7,11 17). Feng et al. (12) reported the quantitative hydrolysis of buprenorphine-glucuronide using β-glucuronidase 586 Reproduction (photocopying) of editorial content of this journal is prohibited without publisher s permission.

2 from E. coli after 2 h of incubation at 37 C and at ph 6.8 (12). Their study did not include evaluation of the hydrolysis of norbuprenorphine-glucuronide. However, Lisi et al. (13) optimized the hydrolysis of norbuprenorphine-glucuronide in a urine sample from a volunteer who was administered 0.2 mg buprenorphine. The hydrolysis required very high concentrations of the enzyme β-glucuronidase from H. pomatia, even at a norbuprenorphine-glucuronide concentration as low as 6 µg/l. Kronstrand et al. (11) studied the hydrolysis using β-glucuronidase from E. coli and found that the cleavage of the buprenorphine glucuronides was much easier to break than that of norbuprenorphine. Differences in glucuronide hydrolysis have also been reported for codeine, morphine, and other opioids (18,19). To be able to use a ratio between parent compound and metabolite quantitative hydrolysis is of great importance. Urine concentrations of buprenorphine from both drug addicts and patients have been reported (7,11,14,16,20). George et al. found concentrations between 28 and 1458 µg/l of buprenorphine and 28 to 1843 µg/l of norbuprenorphine in patients receiving decreasing doses of buprenorphine (16). They also concluded that the ratio between norbuprenorphine and buprenorphine was unrelated to the dose regimen. Kronstrand et al. reported buprenorphine concentrations between 31 and 1080 µg/l in patients and concentrations in abusers from 2.9 to 796 µg/l (11). Cirimele et al. (20) found buprenorphine concentrations ranging from only 1 µg/l up to 1052 µg/l in patients in treatment for heroin addiction, whereas Vincent et al. (14) reported concentrations from 1.0 to 3.3 µg buprenorphine/mg creatinine and µg norbuprenorphine/mg creatinine in five buprenorphine abusers. Thus, as expected concentrations in living humans seem to vary substantially due to the dose intake and the time of sampling. Also, postmortem concentrations Table I. Demographics of the 18 Subjects Sex Age Height Weight Subject (Male/Female) (years) (cm) (kg) BMI* 1 M M F F M F M M M F M F F M F F F F * Body mass index. range extensively (21 25). Tracqui et al. reported postmortem urine concentrations of µg/l in 20 fatalities involving buprenorphine (21). We have recently investigated buprenorphine-related overdose deaths and found that in deaths where buprenorphine was the major (or only) cause of death, the urine concentrations were low and suggested a recent abstinence before the last dose, whereas other cases had much higher metabolite concentrations than parent compound (25). Thus, analysis of urine in buprenorphine-related deaths could help to rule out or confirm recent abstinence. One of the most frequently asked inquiries from prison boards regarding urine sample results is when the drug was ingested. The information regarding buprenorphine excretion is very limited and the aim of this study was to describe the excretion of buprenorphine into urine and to investigate the norbuprenorphine/buprenorphine ratio changes over time. In this way, we aimed to establish a method to estimate the time for buprenorphine use from urine data. Materials and Methods Chemicals and reagents The reference materials buprenorphine, norbuprenorphine, buprenorphine-d 4, and norbuprenorphine-d 3 were purchased from Cerilliant (Round Rock, TX). All organic chemicals were of gradient or analytical grade and all inorganic solvents were of analytical grade. Enzymatic hydrolysis was performed using β- glucuronidase (E. coli, 200U/mL) from Roche (Mannheim, Germany). Drug-free urine was obtained from laboratory personal and was tested negative for buprenorphine and norbuprenorphine before use as matrix for controls and calibrators. Study design The study was approved by the Regional Research Ethics Committee in Linköping (#M5-06). Eighteen healthy volunteers were recruited by advertising. There were no specific inclusion criteria. Participation in any other study during the time for this study was considered an exclusion criterion, as Figure 1. Mean excretion curves for buprenorphine and norbuprenorphine corrected for creatinine concentration. N = 18. Error bars represent standard error of the mean (s.e.m.). The two curves cross at approximately 7 h after dose. 587

3 were pregnancy or nursing. Ongoing medication with opiates, opioids, or benzodiazepines, or a documented abuse of any of these substances were considered an exclusion criterion, as were allergies. Ten females aged 21 to 27 (mean 23) and eight males aged 22 to 33 (mean 27) were recruited. Their mean body mass indexes were 22.1 and 23.2, respectively. The subject demographics are shown in Table I. In the morning of the first day, subjects administered 0.4 mg of buprenorphine sublingually (Temgesic ). The subjects provided urine samples prior to dosing and approximately at 2, 4, 6, 8, 12, 24, 48, 72, and 96 h post dose. During the first 10 h of the study, the subjects remained at the clinic and were monitored for adverse effects. Samples The urine samples were collected in plastic bottles and were transferred into 10-mL tubes within 24 h. One aliquot was subjected to immunoassay and two 1-mL aliquots and one 2-mL aliquot were frozen in 10-mL glass tubes pending LC MS MS analysis. The urine samples were first screened for buprenorphine using CEDIA reagent with a cut-off of 5 µg/l, and the urine creatinine concentration was determined with the Jaffé method. These two analyses were performed on an Advia 1650 from Bayer AB (Gothenburg, Sweden). Instrumentation The LC MS MS analysis was performed on a PerkinElmer series 200 chromatography system consisting of two 200 micro pumps, a hot-pocket column oven, a 200 auto sampler, and a SCIEX API 2000 MS MS instrument (Applied Biosystems, Toronto, ON) equipped with an electrospray interface. Ion spray voltage was set to 5000 V. Nitrogen was used as nebulizer gas (25 psi), auxiliary gas (50 psi heated to 300 C), curtain gas (30 psi), and as collision-activated dissociation gas (set on 5). A mm Zorbax phenyl analytical column with 3.5-µm particle size (Agilent Technologies, Kista, Sweden) was used. Mobile phase A was a 10:10:80 mixture of acetonitrile/methanol/20mm ammonium formate buffer (ph 3.0), and mobile phase B was a 35:35:30 mixture. The system was run in a linear gradient from Table II. Individual Analytical Results From Each Subject Given 0.4 mg Buprenorphine Time BUP* NorBUP* Ratio Creatinine Subject (h) (µg/l) (µg/l) NorBUP/BUP CEDIA (g/l) FP NEG NEG n.a. n.a. n.a. n.a. 4.0 n.a. n.a. n.a. n.a FP NEG NEG FP NEG NEG FP NEG NEG FP NEG NEG NEG * BUP = Buprenorphine and NorBUP = norbuprenorphine. n.a. = Not available because no sample was obtained. Table continues on next page 588

4 75% A-phase to 10% A-phase during 5 min, followed by a 1-min equilibration with 90% A-phase. The total flow rate was 0.25 ml/min. The column oven was set at 30 C. A 10-µL aliquot of the samples was injected. Extraction and quantification of buprenorphine and norbuprenorphine Samples were analyzed in batches of 17 samples and three controls at 2 µg/l, 50 µg/l, and a hydrolysis control made of pooled samples. Quantitation of buprenorphine and norbuprenorphine was performed using LC MS MS as described by Kronstrand et al. (11). In brief, to 1.0 ml of urine were added 25 µl of internal standard (buprenorphined 4 and norbuprenorphine-d 3 2 mg/l), 300 µl 0.5 M BIS-TRIS propane buffer (ph 6.8), and 40 µl β-glucuronidase. The tube was capped and incubated in a water bath (with orbital shaking) at 37 C for 20 h and the ph was then adjusted to 6.1 with 4 ml of 0.2 M phosphate buffer. Thereafter, solid-phase extraction was performed. BondElute Certify columns were activated and conditioned with 2 ml of methanol followed by 2 ml of 0.2 M phosphate buffer (ph 6.1). The sample was drawn through the column, which was then rinsed with 2 ml of deionized water, 2 ml of 0.1M HCl, and 2 ml of methanol. Then the column was dried for 5 min (10 in. Hg), and the analytes were eluted with 2 ml of a mixture of dichloromethane/2-propanol (80:20) containing 2% ammonia (25%). The eluate was evaporated in a TurboVap at 40ºC and 5 psi nitrogen. The residue was reconstituted in 100 µl of mobile phase. The calibration range of the original method was µg/l. In this study, the concentrations were expected to be low, and the calibrator concentrations were set at 0.5, 2, 10, 25, 50, and 100 µg/l. Standard solutions were added to 5 ml batches of negative urine. One milliliter from each calibration batch was pipetted and treated as authentic samples. Calibrations were performed in duplicates to minimize random errors as one calibration curve could be used for several series of samples. To estimate the imprecision at the lowered calibration level, five controls at 0.5 µg/l were analyzed within one batch. The coefficients of variation were 7.1% (buprenor- Table II. Individual Analytical Results From Each Subject Given 0.4 mg Buprenorphine (Continued) Time BUP* NorBUP* Ratio Creatinine Subject (h) (µg/l) (µg/l) NorBUP/BUP CEDIA (g/l) FP NEG NEG FP NEG NEG FP NEG NEG n.a. n.a. n.a. n.a. 6.0 n.a. n.a. n.a. n.a FP NEG NEG FP NEG NEG Table continues on next page * BUP = buprenorphine and NorBUP = norbuprenorphine. n.a. = Not available because no sample was obtained. 589

5 Table II. Individual Analytical Results From Each Subject Given 0.4 mg Buprenorphine (Continued) Time BUP* NorBUP* Ratio Creatinine Subject (h) (µg/l) (µg/l) NorBUP/BUP CEDIA (g/l) FP NEG NEG FP NEG NEG n.a. n.a. n.a. n.a. 4.0 n.a. n.a. n.a. n.a. 6.0 n.a. n.a. n.a. n.a. 8.0 n.a. n.a. n.a. n.a n.a. n.a. n.a. n.a NEG FP NEG NEG NEG FP NEG NEG FP NEG NEG * BUP = buprenorphine and NorBUP = norbuprenorphine. n.a. = Not available because no sample was obtained. Table continues on next page phine) and 6.8% (norbuprenorphine). Samples with concentrations lower than 0.5 µg/l were re-analyzed using 2 ml urine. Results In total, the 18 subjects provided 170 urine samples. Due to failures to mictate, 10 urine samples were not possible to obtain. The concentrations of the analytes varied substantially between the subjects even though the same dose was administered. Peak concentrations of buprenorphine ranged from only 4.4 to 60 µg/l and norbuprenorphine peak concentrations ranged from 5.7 to 26 µg/l. The excretion profiles were similar between the study subjects with initial higher concentrations of buprenorphine than of norbuprenorphine that later changed. The time when the ratio between norbuprenorphine and buprenorphine turned higher than 1 was approximately 7 h as derived from Figure 1 where the mean concentration profiles over 96 h of all subjects are shown. Individual data are shown in Table II. The mean time for the last consecutive positive using CEDIA (> 5 ng/ml) was calculated to 9 h (range 4 to 24 h) for 17 subjects. Subject 12 never provided a positive sample and no samples were obtained during the first day. Owing to differences in urine dilution a negative sample could sometimes be followed by a positive. The corresponding detection times using the LC MS MS method was 76 h for buprenorphine and 96 h for norbuprenorphine using the 0.5 µg/l cut-off. However, when using 2 ml urine for the extraction, 14 of the subjects were positive also for buprenorphine in the last sample at 96 h. In Figure 2, the mean ratios between norbuprenorphine and buprenorphine are depicted. Figure 3 shows the relationship between the mean ratio and sampling time during the first 24 h. The relationship was linear during the first 24 h after intake and then a significant curvature was observed. After administration of buprenorphine, all subjects suffered from sedation and vertigo, but at different degrees. In addition, other common adverse events reported during 590

6 the first study day were nausea (12 subjects), vomiting (7 subjects), and tremor (7 subjects); however, two or more subjects also reported sweating, impaired hearing, dry mouth, and headache. Discussion We have shown that buprenorphine and its metabolite, norbuprenorphine, can be detected in the urine up to 96 h after a single dose and that the concentration of norbuprenorphine became higher than that of the parent compound within a few hours after intake. The urine concentrations of a drug and its metabolites show a considerable scatter owing to differences in dose, uptake, distribution, metabolism, and excretion, but also depend on urine dilution. Thus, solely the concentration of a drug in urine is not suitable for estimations of time of intake. Metabolite/parent compound ratios, on the other hand, may prove useful as the ratio is unaffected by urine dilution, and as Table II. Individual Analytical Results From Each Subject Given 0.4 mg Buprenorphine (Continued) Time BUP* NorBUP* Ratio Creatinine Subject (h) (µg/l) (µg/l) NorBUP/BUP CEDIA (g/l) FP NEG NEG FP NEG NEG FP NEG NEG n.a. n.a. n.a. n.a NEG * BUP = buprenorphine and NorBUP = norbuprenorphine. n.a. = Not available because no sample was obtained. shown by George et al. (16), independent of buprenorphine dose. Still, interindividual differences in metabolism will affect the ratio. Buprenorphine is metabolized through CYP3A4, which have shown some polymorphism even though it is not bimodal. As seen in Table II, the individual ratios differed significantly between the subjects. One reason was the different intervals between urine samples in the beginning of the first day. Several subjects failed to leave urine samples and buprenorphine and metabolites accumulated in the urine until later times, increasing the range of ratios for each sampling time. However, this reflects the authentic samples from casework. After 48 h, the ratio showed considerable scatter and the mean values at 48, 72, and 96 h were in the same order. This indicated that buprenorphine and norbuprenorphine have similar excretion rates and Vincent et al. (14) came to a similar conclusion when following the decline in urinary concentrations in some buprenorphine addicts in a detoxification center. Using the ratio, an estimation of the time from intake to sampling can be made. A ratio less than 0.5 indicated very recent use, and a ratio of 1 indicated use within 7 to 10 h. A ratio of about 3 indicated use 1 day before sampling and this correlated well with the study of George et al. (16) where daily urine samples from patients treated with buprenorphine had ratios of 3, independently of the dose given and with that of Böttcher et al. (15), who found a mean ratio of 4.3. In the present study, ratios higher than 4 indicated use farther back in time, but the relationship showed a very distinct curvature after 48 h and an estimation of the time of intake became difficult. The mean detection time with the CEDIA reagent was 9 h, and considering the cut-off of 5 ng buprenorphine per ml urine, this was in accordance with the method specifications and the actual buprenorphine concentrations in the samples as measured by LC MS MS. An earlier study has also shown that the CEDIA reagent performed well at the proposed cut-off (15). However, the metabolite was present in significant concentrations even after 96 h. The specificity of the reagent towards buprenorphine resulted in negative outcome even short times after ingestion of buprenorphine, and thus failed to identify even a recent intake. A limitation of the data from our study is the single intake as compared to multiple doses that is likely to occur among abusers. In chronic users, the norbuprenorphine/buprenorphine ratio might be overestimated because of residual norbuprenorphine from former intakes. However, the similar 24-h ratios 591

7 Board of Forensic Medicine, Sweden. Ingela Jacobsson is acknowledged for excellent clinical assistance. References Figure 2. Mean norbuprenorphine/buprenorphine ratios for the 18 subjects during the 96 h urine was collected. Error bars represent s.e.m. The curve fit was performed only to depict the curvature. Figure 3. Mean norbuprenorphine/buprenorphine ratios during the first 24 h. The change in ratio over time could be described by a simple linear relationship, y = x. Error bars represent s.e.m. obtained in daily dosed patients do not support this (15,16). In Sweden, most requests for estimating the time of administration has so far been by the prison boards when a detained person became positive after weekend leave from the prison. In addition, the approach can be used in suspected buprenorphine overdose deaths to estimate the time since the last dose. In an earlier study by Seldén et al. (25), for example, the authors reported low norbuprenorphine/buprenorphine ratios in acute overdose cases, indicating a short period of survival after the last dose as well as a period of abstinence supported by low norbuprenorphine concentrations. Conclusions We have shown that a single low dosage of buprenorphine can be detected for four days using a confirmatory method, whereas using a screening method the detection time may shorten to less than one day. We have also shown that the ratio of norbuprenorphine and buprenorphine may be used to estimate the time of intake. Further studies using different doses of buprenorphine are warranted to investigate this issue. Acknowledgments This study was partly financed by a grant from the National 1. J. Kakko, L. Gronbladh, K.D. Svanborg, J. von Wachenfeldt, C. Ruck, B. Rawlings, L.H. Nilsson, and M. Heilig. A stepped care strategy using buprenorphine and methadone versus conventional methadone maintenance in heroin dependence: a randomized controlled trial. Am. J. Psychiatry 164(5): (2007). 2. J. Kakko, K.D. Svanborg, M.J. Kreek, and M. Heilig. One-year retention and social function after buprenorphine-assisted relapse prevention treatment for heroin dependence in Sweden: a randomised, placebo-controlled trial. Lancet 361(9358): (2003). 3. M.Z. Mintzer, C.J. Correia, and E.C. Strain. A dose-effect study of repeated administration of buprenorphine/naloxone on performance in opioid-dependent volunteers. Drug Alcohol Depend. 74(2): (2004). 4. J. Mendelson and R.T. Jones. Clinical and pharmacological evaluation of buprenorphine and naloxone combinations: why the 4:1 ratio for treatment? Drug Alcohol Depend. 70(2S): S29 S37 (2003). 5. A. Elkader and B. Sproule. Buprenorphine: clinical pharmacokinetics in the treatment of opioid dependence. Clin. Pharmacokinet. 44(7): (2005). 6. E.J. Cone, C.W. Gorodetzky, D. Yousefnejad, W.F. Buchwald, and R.E. Johnson. The metabolism and excretion of buprenorphine in humans. Drug Metab. Dispos. 12(5): (1984). 7. E.J. Fox, V.A. Tetlow, and K.R. Allen. Quantitative analysis of buprenorphine and norbuprenorphine in urine using liquid chromatography tandem mass spectrometry. J. Anal. Toxicol. 30(4): (2006). 8. Y. Blom, U. Bondesson, and E. Anggard. Analysis of buprenorphine and its n-dealkylated metabolite in plasma and urine by selected ion monitoring. J. Chromatogr. B 338: (1985). 9. S. Hegstad, H.Z. Khiabani, E.L. Oiestad, T. Berg, and A.S. Christophersen. Rapid quantification of buprenorphine-glucuronide and norbuprenorphine-glucuronide in human urine by LC MS MS. J. Anal. Toxicol. 31(4): (2007). 10. W. Huang, D.E. Moody, and E.F. McCance-Katz. The in vivo glucuronidation of buprenorphine and norbuprenorphine determined by liquid chromatography electrospray ionization-tandem mass spectrometry. Ther. Drug Monit. 28(2): (2006). 11. R. Kronstrand, T.G. Selden, and M. Josefsson. Analysis of buprenorphine, norbuprenorphine, and their glucuronides in urine by liquid chromatography mass spectrometry. J. Anal. Toxicol. 27(7): (2003). 12. S. Feng, M.A. ElSohly, and D.T. Duckworth. Hydrolysis of conjugated metabolites of buprenorphine. I. The quantitative enzymatic hydrolysis of buprenorphine-3-beta-d-glucuronide in human urine. J. Anal. Toxicol. 25(7): (2001). 13. A.M. Lisi, R. Kazlauskas, and G.J. Trout. Gas chromatographic mass spectrometric quantitation of urinary buprenorphine and norbuprenorphine after derivatization by direct extractive alkylation. J. Chromatogr. B Biomed. Sci. Appl. 692(1): (1997). 14. F. Vincent, J. Bessard, J. Vacheron, M. Mallaret, and G. Bessard. Determination of buprenorphine and norbuprenorphine in urine and hair by gas chromatography mass spectrometry. J. Anal. Toxicol. 23(4): (1999). 15. M. Bottcher and O. Beck. Evaluation of buprenorphine CEDIA assay versus GC MS and ELISA using urine samples from patients in substitution treatment. J. Anal. Toxicol. 29(8): (2005). 592

8 16. S. George, C. George, and M. Chauhan. The development and application of a rapid gas chromatography mass spectrometry method to monitor buprenorphine withdrawal protocols. Forensic Sci. Int. 143(2 3): (2004). 17. M.A. ElSohly, W. Gul, S. Feng, and T.P. Murphy. Hydrolysis of conjugated metabolites of buprenorphine II. The quantitative enzymatic hydrolysis of norbuprenorphine-3-beta-d-glucuronide in human urine. J. Anal. Toxicol. 29(6): (2005). 18. Z. Lin, P. Lafolie, and O. Beck. Evaluation of analytical procedures for urinary codeine and morphine measurements. J. Anal. Toxicol. 18(3): (1994). 19. P. Wang, J.A. Stone, K.H. Chen, S.F. Gross, C.A. Haller, and A.H. Wu. Incomplete recovery of prescription opioids in urine using enzymatic hydrolysis of glucuronide metabolites. J. Anal. Toxicol. 30(8): (2006). 20. V. Cirimele, P. Kintz, S. Lohner, and B. Ludes. Enzyme immunoassay validation for the detection of buprenorphine in urine. J. Anal. Toxicol. 27(2): (2003). 21. A. Tracqui, P. Kintz, and B. Ludes. Buprenorphine-related deaths among drug addicts in France: a report on 20 fatalities. J. Anal. Toxicol. 22(6): (1998). 22. P. Kintz. Deaths involving buprenorphine: a compendium of French cases. Forensic Sci. Int. 121(1 2): (2001). 23. M. Reynaud, G. Petit, D. Potard, and P. Courty. Six deaths linked to concomitant use of buprenorphine and benzodiazepines. Addiction 93(9): (1998). 24. P. Kintz. A new series of 13 buprenorphine-related deaths. Clin. Biochem. 35(7): (2002). 25. T. Seldén, M. Roman, G. Thelander, H. Druid, and R. Kronstrand. Buprenorphine concentrations in post mortem whole blood and urine from suspected overdose cases. Presented at the Society of Forensic Toxicologists Annual Meeting, Raleigh, NC,