Forensic Science International 195 (2010) 7377

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Forensic Science International

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Buprenorphine and major metabolites in blood specimens collected for drug analysis in law enforcement purposes
Stephanie Oechsler, Gisela Skopp *
Institute of Legal Medicine and Trafc Medicine, University Hospital, Voss-Str. 2, 69115 Heidelberg, Germany

A R T I C L E I N F O

A B S T R A C T

Article history: Received 19 August 2009 Received in revised form 4 November 2009 Accepted 17 November 2009 Available online 16 December 2009 Keywords: Buprenorphine Norbuprenorphine Glucuronides LCESIMS/MS Authentic samples

A liquid chromatographic/electrospray ionization tandem mass spectrometric method for the quantication of buprenorphine (BUP), norbuprenorphine (NBUP), buprenorphine-3-b-D-glucuronide (BUPG) and norbuprenorphine-3-b-D-glucuronide (NBUPG) in serum samples was developed and validated. Pre-treatment of BUP and NBUP was by liquidliquid extraction, while glucuronides were favourably isolated by solid phase extraction. Separation in 2 separate runs (2 5 min) was achieved using isocratic elution. The method was applied to 20 authentic serum specimens collected for law enforcement purposes where BUP intake had been indicated. The parent drug was not detectable in half of the specimens at a lower limit of detection of 0.2 ng/mL, whereas NBUP could be determined from any sample but one. NBUPG is the major metabolite present, which could be identied along with BUPG in all samples under investigation. In authentic specimens it could be advisable to monitor BUP metabolites along with the parent drug. 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Buprenorphine (BUP), a thebaine derivative, is used in low doses of 0.30.6 mg for pain management, and has been approved for the treatment of opioid dependency with an acceptable daily intake of up to 32 mg [1,2]. A preliminary communication of agonist and antagonistic effects was already given in 1972, but even to date the mechanisms of action of BUP are not fully understood. The drug has been described as a partial agonist at the mu receptor, and can also bind to kappa and delta opioid receptors. It blocks epsilon receptors at low doses, and has recently been shown to interact with the ORL-1 receptor [3]. BUP is metabolised through N-dealkylation mainly by CYP3A4 and CYP2C8 to form norbuprenorphine (NBUP) [4]. NBUP is likely to contribute to the pharmacology of BUP acting as a full, but less active agonist at the mu opioid receptor [3]. Both, BUP and NBUP are further metabolized by phase II glucuronidation via UGT1A1, 1A3 and 2B7 or UGT1A1 and 1A3 to produce buprenorphine-3-b-D-glucuronide (BUPG) and norbuprenorphine-3-b-D-glucuronide (NBUPG), respectively (Fig. 1) [5,6; S. Oechsler, unpublished results]. Gas chromatography and liquid chromatography preferably coupled to mass spectrometric detection (GC/MS, LC/MS or LC/MS/ MS) have been applied for BUP analyses in a variety of matrices [7]. Only 2 articles present results on the direct determination of BUP,

NBUP and respective glucuronides in blood; one comprises 2 clinical samples, the other 13 samples collected from 5 subjects, respectively, undergoing a control session concerning the interaction of BUP with anti-HIV-medications [2,8]. In the present study, a LCESIMS/MS method for direct quantication of BUP, NBUP and their glucuronides was applied to 20 blood samples collected for law enforcement purposes. Specimens were also checked for blood alcohol, other drugs or comedication. The present data may complement the few published ones. The objective was to determine whether in random samples (1) NBUP blood concentration is comparable with or even exceeds the concentration of BUP and (2) glucuronide concentrations exceed those of the corresponding unconjugated compounds.
2. Materials and methods 2.1. Chemicals BUP-hydrochloride and 1-chlorobutane were purchased from Sigma/Aldrich (Steinheim, Germany). NBUP, NBUPG, BUP-d4, NBUP-d3 as standard solutions were provided by LGC Standards (Wesel, Germany), BUPG was supplied by ElSohly Laboratories (Oxford, MS, USA), acetonitrile, methanol, sodium hydroxide and acetic acid were obtained from Roth (Karlsruhe, Germany). Sodium carbonate, sodium hydrogen carbonate and ammonium hydroxide solution (27%) were from Merck (Darmstadt, Germany). Bond Elut C8 SPE columns (1.0 mL) were obtained from Varian (Darmstadt, Germany). 2.2. Preparation of standard solutions Different working solutions of the analytes were prepared by appropriate dilution from 100 mg/mL stock solutions in methanol. The internal standard solution (ISTD) at 1 mg/mL BUP-d4 and NBUP-d3, respectively, was prepared by

* Corresponding author. Tel.: +49 6221 568926; fax: +49 6221 565252. E-mail address: gisela.skopp@med.uni-heidelberg.de (G. Skopp). 0379-0738/$ see front matter 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2009.11.015

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S. Oechsler, G. Skopp / Forensic Science International 195 (2010) 7377 analysis of each concentration level on 8 different days. Imprecision and accuracy, expressed as % relative standard deviation of measured concentrations, were required to be less than 15%. Recovery was performed by comparing the analytical results for extracted samples at 5 and 20 ng/mL with neat standard solutions representing 100% recovery. Stability was tested separately for each compound by fortifying serum aliquots (20 ng/mL of BUP, NBUP, BUPG, NBUPG, respectively) which were subsequently stored at 22 8C, 4 8C and 40 8C for 7 days. Stability was considered acceptable if samples quantied within 20% of target. Carry over was checked by injecting a blank sample immediately after a sample spiked at 100 ng/mL concentrations. The matrix effect was assessed by comparing analyte peak area in serum specimens from 5 different sources fortied with the analytes and ISTD after extraction to peak areas of samples at the same nominal concentrations prepared in mobile phase. Matrix suppression or enhancement was estimated as previously suggested by Matuszewski et al. [10]. Fig. 1. Major metabolic pathways of buprenorphine according to [6,22]. 2.7. Authentic samples

dilution from 100 mg/mL stock solutions in methanol. Quality control (QC) solutions containing BUP, NBUP, BUPG and NBUPG were prepared at two different working concentrations (5, 20 ng/mL). BUP and NBUP were preferably isolated by liquidliquid-extraction yielding a high recovery of the unconjugated compounds, whereas glucuronide metabolites were extracted by solid-phase-extraction using NBUP-d3 as the ISTD, for deuterated analogues are not commercially available. 2.3. Liquidliquid-extraction of BUP and NBUP Twenty ve microliters of 1 M NaOH, 1 mL 1-chlorobutane/acetonitrile (4:1 by vol.) and 10 mL ISTD were added to 500 mL of serum separated from whole blood, calibrators or QC solutions. After having been vortexed (1 min) and shaken (10 min), samples were centrifuged at 19,700 g (10 min). Eight hundred microliters of the supernatant was evaporated to dryness under nitrogen at 40 8C, and dried residues were reconstituted in 50 mL of the mobile phase. A 10 mL aliquot was injected into the LCMS/MS system. 2.4. Solid-phase extraction of BUPG and NBUPG Two hundred fty microliters of serum, calibrator or QC solution, respectively, 10 mL NBUP-d3 and 0.5 mL sodium carbonate buffer pH 9 were combined, vortexmixed and centrifuged (19,700 g, 10 min). The supernatant was applied to a SPE cartridge preconditioned with 2 mL methanol, 1 mL water, and 1 mL buffer pH 9 followed by 100 mL ammonium hydroxide solution. Subsequently, the column was washed with 1 mL buffer pH 9 and dried for 30 min under vacuum. Analytes were eluted with 1 mL of methanol/acetic acid (98:2 by vol.) which was dried down under nitrogen, and reconstituted with 50 mL of the mobile phase. Ten microliters was injected into the LCMS/MS system. 2.5. LCESIMS/MS The concentration of BUP, NBUP and respective glucuronides was determined by LCESIMS/MS (API 4000, Applied Biosystems, Darmstadt, Germany) in 2 separate runs, 5 min each, with a TurboIon1 ionization source operated in the positive-ion mode. The mass analyzer was coupled to an Agilent model 1100 series LC system (binary pump and autosampler, Agilent, Waldbronn, Germany). Chromatographic separation was achieved using a Phenomenex Luna column C18(2) (150 mm 2.00 mm, Phenomenex, Torrance, CA, USA), and a mobile phase of 4 mM ammonium acetate buffer pH 3.2/acetonitrile/methanol (60:32:8 by vol.) at a ow rate of 0.25 mL/min for any analyte. Peak areas and peak area ratios were determined using Analyst 1.4.1 software (Applied Biosystems, Darmstadt, Germany). Quantication was achieved by calculating the peak area ratios for the ion transitions of the particular analyte and its internal standard, and by referring the values to the particular calibration curve. The following transitions were used: BUP (-d4), m/z 468 (472) ! 468 (472); NBUP (-d3), m/z 414 (417) ! 414 (417); BUPG, m/z 644 ! 468, NBUPG m/z 590 ! 414. 2.6. Method validation Five non-zero samples (BUP, NBUP, BUPG: 135 ng/mL, NBUPG: 250 ng/mL) were prepared for calibration, in addition to a blank sample (matrix sample processed without ISTD) and a zero sample (matrix sample processed with ISTD). The method was validated for linearity, sensitivity (limits of detection (LOD) and quantitation (LOQ) were estimated from the particular calibration lines according to DIN 32645 [9]), imprecision, recovery, stability and carry over. The matrix effect was assessed according to Matuszewski et al. [10]. Linearity of the calibration range was determined by least-squares linear regression, and acceptable linearity was achieved when the coefcient of correlation was >0.99. Imprecision and accuracy were determined by replicate

Twenty blood specimens collected from subjects suspected to be under the inuence of drugs and requested for analysis of BUP were investigated. In each case, there was an indication on BUP administration by voluntary information during the police stop. Furthermore, a few subjects reported on the daily dose of BUP. Determination of blood alcohol (headspace gas chromatography), monitoring (immunoassay, screening with gas chromatographymass spectrometry mode or high pressure liquid chromatography-UV detection) or conrmation (gas chromatographymass spectrometry) of drugs other than BUP was also performed if requested. Upon submission of the samples, serum was separated from whole blood as soon as samples were submitted, and was kept frozen at 20 8C until analysis.

3. Results The separation of the 4 compounds was achieved in a total of 10 min (Fig. 2). As already noticed by Murphy and Huestis [8], a deuterium isotope effect could be noted resulting in BUP-d4 eluting 0.18 min earlier than BUP. Some of the validation data are summarized in Table 1. The linear dynamic range was 135 ng/mL for BUP, NBUP and BUPG, and 250 ng/mL for NBUPG. Imprecision, accuracy and analytical recovery were within the required ranges. There was no matrix effect, except for the glucuronides. Ion enhancement of about 29% could be observed for BUPG levels !20 ng/mL. However, 20 ng/mL are far in excess of the levels present in authentic samples (Table 2). Suppression could be noticed with NBUPG in a similar range as that reported for BUPG by Murphy and Huestis [8]. Thus, levels of NBUPG may be underestimated. No analytes of interest were detected in a blank sample injected immediately following analysis of a 100 ng/mL sample. All 4 analytes proved to be stable over 7 days at all storage conditions. Concentrations determined from authentic specimens are shown in Table 2 by BUP concentration in an ascending order, along with alcohol and further medication or drug ndings. The daily dose of BUP is given as far as it had been reported. Interestingly, BUP was not detectable in 50% out of all specimens, whereas NBUP could be identied in any but a single sample. BUPG and NBUPG were present in all samples under investigation. NBUPG levels considerably exceeded those of NBUP as well as of BUP. Some BUPG concentrations were higher than BUP concentrations, whereas some were not; however, mean and median values were consistently lower compared to those of BUP, NBUP and NBUPG. Concomitant use of alcohol and drugs other than BUP was observed in 14 out of the 20 samples. Morphine could be detected in 4 cases, and cannabis was most often seen in subjects with positive BUP ndings. Blood alcohol levels (n = 7) ranging from 0.03% to 0.26% showed an irregular pattern of distribution. 4. Discussion Although methadone is the substance most frequently prescribed in substitution treatment in Germany, its use has dropped

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Fig. 2. Chromatogram of an authentic sample (case 17) with 4.6 ng/mL buprenorphine (BUP), 1.8 ng/mL norbuprenorphine (NBUP), 1.9 ng/mL buprenorphine glucuronide (BUPG) and 17.0 ng/mL norbuprenorphine glucuronide (NBUPG).

from 72.1% to 59.7% during 2002 to 2008 while that of BUP concurrently increased from 9.7% to 18.9% [11]. It has been suggested that BUP might be especially useful with pregnant women and low-dosed methadone patients as well as in detoxication and maintenance treatment [12]. The availability of appropriate assays to quantify BUP has been a major challenge to study concentrations in biological matrices. At present, only 2 articles describe the direct determination of BUP, NBUP and corresponding glucuronide metabolites in plasma by liquid chromatography/tandem mass spectrometry [2,8]. Analyses covered specimens collected from a total of 7 subjects enrolled in care centres. Although BUP medication assisted treatment has proven benecial in reducing harms associated with illegal drug use, violation against existing laws cannot be totally ruled out. Specimens taken for law enforcement purposes will differ from those taken in a controlled environment. The present results may therefore complement those observed in the maintenance treatment population. Analysis of BUP and major metabolites requires assays with low detection limits. Liquid chromatography coupled to tandem mass spectrometry enables quantitative analysis without the need of hydrolyzing glucuronide conjugates and derivatizing the extracted residue. Derivatization was reported to be difcult to reproduce

reliably for NBUP [13]. As already discussed by Polettini and Huestis [14] and Concheiro-Guisan et al. [7], it is necessary for BUP and NBUP to use the surviving parent ions in order to achieve the required sensitivity. BUP showed a fragment ion of little intensity (13% with reference to the base peak) at m/z 414, likely as a result of N-dealkylation. A still less intense fragment at m/z 396 was present, which could also be observed for NBUP [15]. BUPG and NBUPG produced very intense and almost unique fragments cleaving the glucuronide moiety, while the surviving ions proved difcult to fragment as already experienced for the free compounds. The use of an ion trap mass spectrometer allows more effective collisional experiments producing abundant product ions for identication purposes, which is a major limitation of this study. Nevertheless, quantication of BUP and NBUP was performed with the parent ion for reasons of sensitivity and precision even if such instrumentation had been available [7]. Although separate runs were performed for parent drugs and corresponding glucuronides, separation was achieved in a total of 10 min. Currently, the minimum time reported to simultaneously separate BUP, NBUP and their respective glucuronides was 13 min [16]. Also, employing separate runs and isolation steps enables isocratic elution. Isocratic methods are preferred over gradient elution which may be a source of imprecision in LCMS/MS. BUP

Table 1 Evaluation data including linearity, LOD and LOQ (ng/mL), accuracy (%, n = 8), imprecision (%, n = 8), recovery (%, mean SD, n = 6), matrix effect (%, mean SD, n = 5) BUP: buprenorphine, NBUP: norbuprenorphine; BUPG: buprenorphine glucuronide, NBUPG: norbuprenorphine glucuronide. BUP Range of linearity, >0.99 LOD LOQ Accuracy Imprecision Recovery 5 ng/mL 20 ng/mL Matrix effect 5 ng/mL 20 ng/mL 1-35 ng/mL 0.2 0.5 <5 <6 NBUP 1-35 ng/mL 0.2 0.4 <5 <5 BUPG 1-35 ng/mL 0.2 0.5 <4 <10 NBUPG 2-50 ng/mL 0.6 2.0 <5 <12

99 3.6 101 2.5 114 1.2 116 3.4

88 6.4 96 3.3 84 2.9 95 1.1

112 2.0 129 9.0 113 0.6 129 5.1

49 2.1 45 3.0 47 1.6 66 3.8

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Table 2 Concentrations (ng/mL) of buprenorphine (BUP), norbuprenorphine (NBUP) and corresponding glucuronide metabolites (BUPG, NBUPG) determined from 20 serum specimens, daily dose of buprenorphine, blood alcohol concentration (%) and further drug ndings (ng/mL); DD: daily dose of buprenorphine (mg), BAC: blood alcohol concentration (%), nd: non-detectable, positive: positive nding < LOQ, nr: not reported, THC: tetrahydrocannabinol, 11-OH-THC: 11-hydroxy-THC, THC-COOH: THC carboxylic acid, BZE: benzoylecgonine, and EME: ecgonine methyl ester. Case 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 BUP nd nd nd nd nd nd nd nd nd nd Positive Positive Positive 1.30 2.20 2.75 NBUP 1.58 1.20 0.67 nd Positive 3.82 2.19 6.89 1.78 2.66 4.69 1.35 1.40 0.50 4.40 Positive BUPG Positive Positive Positive 0.90 0.75 0.53 Positive Positive 0.57 Positive Positive 0.66 1.00 400 1.00 1.40 NBUPG Positive Positive Positive 2.40 2.60 2.90 3.66 15.2 32.5 40.2 Positive 7.47 10.7 6.70 10.4 7.20 DD nr nr nr nr 6 3 nr nr nr 2 12 nr 14-16 8 nr 16 BAC 0.26 nd nd 0.11 nd nd nd 0.03 nd 0.03 0.22 nd nd nd nd nd Drugs other than alcohol or BUP Methadone, not quantied nd nd nd nd 19 morphine nd nd 335 morphine, 9 codeine 25 morphine, 5 codeine nd 1.0 THC, 16 THC-COOH 1.5 THC, 6 THC-COOH, 243 morphine, 43 codeine 9.2 THC, 5.8 11-OH-THC, 60 THC-COOH, 114 clozapine 0.9 THC, 18 THC-COOH 160 cocaine, 424 BZE, 35 EME, 180 diazepam, 570 nordiazepam, 22 unitrazepam nd nd nd 59 cocaine, 100 BZE, 19 EME

17 18 19 20 Mean Median

4.60 5.20 5.70 7.20 4.14 4.60

1.80 17.0 2.50 0.90 3.25 1.80

1.90 5.80 1.20 1.40 1.62 1.00

17.0 56.9 12.3 6.50 14.66 8.94

4 5 4 2 6.2 4.5

0.14 nd nd 0.10

and NBUP as well as BUPG and NBUG were well separated, whereas BUPG co-eluted with NBUP-d3. However, there was no interference from in-source transformation or from the selected-reaction monitoring transition for the glucuronide compounds and the internal standard. Chromatographic conditions were adjusted in order to elute BUPG as close as possible to NBUP-d3. In addition, liquidliquid extraction of BUP and NBUP was superior to solid phase extraction. For example, Concheiro-Guisan et al. [7] reported extraction efciencies as low as 3947% for BUP and NBUP. Due to the very low concentrations of BUP and NBUP and their rapid isolation from serum a 2nd extraction step was considered to be acceptable. Furthermore, it is important to consider stability of glucuronides when interpreting data from biological matrices, for degradation may increase free drug concentration. The excellent stability of BUP, NBUP and their glucuronides in serum when stored under a variety of conditions for a time period of 7 days is comparable to that of morphine and respective glucuronides [17]. The present data may complement those reported from other working groups. For example, Moody et al. [18] found BUP to be stable in plasma stored at room temperature for up to 24 h. Drug stability of BUP, NBUP and corresponding glucuronides was also investigated in processed samples stored on the autosampler at 10 8C for up to 72 h, and in drug-fortied placenta stored at 22 8C or 4 8C for 16 and 72 h, respectively [7]. Also, no deterioration or loss was observed for any analyte in meconium under similar conditions [16]. Daily doses of BUP, as far as reported, were in the range usually administered when used for treatment of opioid dependency. Measured concentrations of BUP and NBUP were up to 7.2 and 17.0 ng/mL serum, with mean values of 4.14 and 3.25 ng/mL, respectively. The data available for BUP therapeutic or toxic ranges are as limited as conicting. Repetto and Repetto [19] dened therapeutic ranges of BUP to be less than 5 ng/mL plasma, while Kintz [20] reported therapeutic ranges of BUP evaluated from clinical studies to be in the range of 220 ng/mL. Concentrations ranging from 0.3 to 7.7 ng BUP/mL blood (mean: 3.5 ng/mL) and

from 0.3 to 16.2 ng NBUP/mL blood (mean: 2.9 ng/mL) have been reported from 13 death cases involving BUP. These gures are in the same order of magnitude as the present ones. However, no fatality involving BUP alone was observed. All death cases involved a concomitant intake of a psychotropic agent; in 9 out of these 13 cases, benzodiazepines were present; and a concomitant intake of narcotics or cocaine was observed in 3 cases, respectively. In the present study, psychotropic agents were detected in 14 out of the 20 specimens investigated. Interestingly, opioid ndings were abundant in specimens where BUP was not detectable, while cocaine and cannabinoids were present in 2 and 4 out of the specimens with positive BUP ndings. It is not known whether under-treatment or illegal use of BUP has caused or contributed to concomitant use of heroin and methadone. Also, the number of specimens is yet rather small to further discuss pattern of concomitant use of psychotropic drugs during BUP maintenance. The present data revealed no consistent pattern of parent drug and metabolites. Interestingly, the median NBUP concentration was lower than that of BUP. CYP3A4 which is the enzyme primarily involved in NBUP formation is inhibited by a host of prescription drugs such as HIV protease inhibitors, or azol antifungal agents, over-the-counter medications and food products [21,22]. Less NBUP will be produced following inhibition of the enzyme. Other factors that can inuence BUP metabolism are genetic polymorphisms and environmental factors that can affect CYP3A4 activity and expression of UGTs. The present mean and median concentrations of BUPG were the lowest of any analyte, while that of NBUPG were most abundantconsistent with data of Murphy and Huestis [8] measured from 2 specimens subjects maintained on BUP for over 2 months and of Huang et al. [2], where peak concentrations in plasma collected from 5 subjects on daily sublingual doses of 16 mg BUP and 4 mg naloxone over 2 weeks have been reported. In this study, the maximum NBUP concentration was higher than that of BUP. This is quite surprising as naloxone does not appear to inuence the pharmacokinetics of BUP, whereas HIV protease inhibitors may inhibit its N-desalkylation. Our results are more in

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line with that reported by Murphy and Huestis [8], although a full evaluation of possible factors modulating the activity of CYP3A4 was not feasible in any study. There have been many reports of interactions between BUP and benzodiazepines. Although diazepam (case 16) is metabolised through CYP3A4, it is not considered to inhibit this enzyme. 5. Conclusion LCESIMS/MS provides an efcient method to quantify BUP, NBUP and their glucuronides in human serum samples. Determination of NBUP in addition to BUP is recommended in forensic case work where credibility of statement and assessment of impairment are often scrutinized. Also, determination of glucuronide conjugates may be necessary. Hypotheses detailed above were largely supported by the present ndings. The concentration of NBUP was comparable to that of BUP, and NBUPG was the major metabolite in blood. The concentration of BUPG, however, was partially lower than that of the parent drug. Acknowledgement This research was supported by the Deutsche Forschungsgemeinschaft (Sk 48/3-1). References
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