Quantification of Buprenorphine, Norbuprenorphine and 6-Monoacetylmorphine in Urine by Liquid Chromatography-Tandem Mass Spectro

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Yuan et al., J Chromat Separation Techniq 2013, 4:3 Chromatography http://dx.doi.org/10.4172/2157-7064.1000174 Separation Techniques

Research Article Article OpenOpen Access Access Quantification of Buprenorphine, Norbuprenorphine and 6-Monoacetylmorphine in Urine by Liquid Chromatography-Tandem Mass Spectrometry Chao Yuan, Katherine Lembright, Courtney Heideloff and Sihe Wang* Department of Clinical Pathology, Cleveland Clinic, Cleveland, USA

Abstract Monitoring pain management medications and illicit drugs in urine is commonly used to assess patient compliance. Previously, we developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to measure 19 analytes important for pain management. In the current report, we validated this method for two additional drugs, buprenorphine and heroin. For buprenorphine, we quantified both the parent drug and its major metabolite, norbuprenorphine. For heroin, we monitored its unique metabolite, 6-monoacetylmorphine (6-MAM). Urine samples were subjected to enzymatic hydrolysis prior to turbulent flow online extraction and LC-MS/MS analysis. No matrix effect or interference was found. Lower limits of quantifications were 9.7, 9.6, and 4.9 ng/mL for buprenorphine, norbuprenorphine and 6-MAM, respectively. Within the linear range, analytical recovery was 80.5-113.0% for all analytes. Intra-assay and total coefficient of variations were between 0.2% and 10.3%. This method demonstrated consistent patient results (n=40) with the independent LC-MS/MS methods offered by two other laboratories. Percentage of glucuronide conjugation of 6-MAM varied from 0 to 45% in 8 patient urine samples positive for 6-MAM. In conclusion, we have successfully expanded current pain management panel to include buprenorphine and heroin with high sensitivity, specificity, and precision.

Keywords: LC-MS/MS; Buprenorphine; Norbuprenorphine; Heroin; Materials 6-Monoacetylmorphine Standard solutions of buprenorphine, norbuprenorphine, Introduction 6-MAM, buprenorphine glucuronide, norbuprenorphine glucuronide, buprenorphine-d4, norbuprenorphine-d3, 6-MAM-d3, were from Monitoring the use of prescribed pain medications and illicit drugs Cerilliant (Round Rock, TX). Deuterium-labeled standards were used is routinely used to assess the compliance of patients enrolled in pain as internal standards (IS). Drug-free urine (Liquichek urine toxicology management programs [1]. Liquid chromatography-tandem mass negative control, Bio-Rad, Hercules, CA) was used to prepare calibrators spectrometry (LC-MS/MS) methods have been increasingly used due to (25, 50, 250, 500, and 1000 ng/mL). Glucuronidase used for hydrolysis the high specificity and sensitivity, relatively simple sample preparation, was isolated from Patella vulgate (Sigma-Aldrich, St. Louis, MO). All and ability of analyzing a large panel of analytes simultaneously [2]. other reagents were the same as our previous report [3]. Previously, we developed an LC-MS/MS method to monitor the usage of 15 drugs important for pain management [3]. In the current study, Methods our goal was to add two more drugs, buprenorphine and heroin, Sample preparation including enzymatic hydrolysis, on-line to this panel to meet the clinical needs. Buprenorphine is an opioid extraction with turbulent flow chromatography and analytical methods medication used to treat chronic pain, and it has less potential for abuse were described in our previous report [3]. In this work, a 2-channel when combined with naloxone, an opioid antagonist [4]. Heroin is a multiplexed LC system (Aria TLX2 from ThermoFisher Scientific, commonly abused street drug in the US with increasing trend among Waltham, MA) was used, and the analytical time between injections persons aged 12 or older [5]. was reduced from 23 to 12 minutes. The mass spectrometer (TSQ In humans, buprenorphine is metabolized to norbuprenorphine. Ultra from ThermoFisher Scientific) was operated in the positive Both the parent drug and the metabolite undergo extensive conjugation electrospray ionization mode for all the added analytes and IS. Multiple with glucuronide prior to excretion in urine [6]. A hydrolysis step is reaction monitoring (MRM) transitions for each analyte are listed in needed when measuring total concentrations of buprenorphine and (Table 1). Method validation was performed using the same protocol norbuprenorphine [7,8]. Enzymatic hydrolysis of these conjugates is commonly used, and the choice of glucuronidase and incubation conditions has significant impact on the hydrolysis efficiencies [9]. *Corresponding author: Sihe Wang, LL3-3, 9500 Euclid Ave, Cleveland, OH Heroin is rapidly metabolized (half-life is 3 minutes in plasma) first 44195, Tel: 216-445-2634; Fax: 216-445-0212; E-mail: [email protected] to 6-monoacetylmorphine (6-MAM), and further to morphine. Received January 23, 2013; Accepted March 23, 2013; Published March 26, Although the morphine to codeine concentration ratio can be helpful 2013 in differentiating heroin users from codeine users, presence of 6-MAM Citation: Yuan C, Lembright K, Heideloff C, Wang S (2013) Quantification of in urine indisputably confirms heroin use [10]. Presence of conjugated Buprenorphine, Norbuprenorphine and 6-Monoacetylmorphine in Urine by Liquid 6-MAM in urine has been indicated, [11] but its relative amount has Chromatography-Tandem Mass Spectrometry. J Chromat Separation Techniq 4: not been reported. In this study, we aimed to measure buprenorphine, 174. doi:10.4172/2157-7064.1000174 norbuprenorphine, and 6-MAM in urine after enzymatic hydrolysis. We Copyright: © 2013 Yuan C, et al. This is an open-access article distributed under intended to use the same sample preparation and analytical methods the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and that we previously developed [3]. source are credited.

J Chromat Separation Techniq ISSN:2157-7064 JCGST, an open access journal Volume 4 • Issue 3 • 1000174 Citation: Yuan C, Lembright K, Heideloff C, Wang S (2013) Quantification of Buprenorphine, Norbuprenorphine and 6-Monoacetylmorphine in Urine by Liquid Chromatography-Tandem Mass Spectrometry. J Chromat Separation Techniq 4: 174. doi:10.4172/2157-7064.1000174

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Analyte MRM Transitiona LLOQ HLOQ Accuracy Carryover Precision (level 1/2/3, n=30) (parent→quan, qual) (ng/ml) (ng/ml) range (%) Limit (ng/ml) Mean (ng/ml) Total CV (%) Intra CV (%) Buprenorphine 468.3→396.3, 414.3 9.7 5647 80.5-113.0 10160 40/378/747 6.4/6.4/4.8 5.0/0.5/4.1 Norbuprenorphine 414.3→187.1, 340.1 9.6 4317 83.8-96.0 10443 32/326/613 3.3/3.3/10.3 2.2/0.2/9.9 6-MAM 328.1→211.1, 193.1 4.9 4800 96.0-100.4 8681 10/107/207 13.3/4.3/3.7 4.9/2.4/3.6 aTwo MRM transitions, a quantifier (quan) and a qualifier (qual) were monitored for each analyte. The quantifier to qualifier ratio was used for peak identification based on criteria set forth by the CLSI C50-A guideline Table 1: MRM transitions and validation results.

3500000 3 4 a 3000000 9 1000000 Buprenorphine 2500000 7 10 (468.3→396.3) Patient 2000000 800000 18 1500000 6 8 11 20 Blank Intensity 12 5 14 17 2 15 600000 1000000 13

1 Intensity 500000 16 19 400000 0 7.5 9.5 11.5 13.5 15.5 17.5 200000

Retention Time (min) 0 8 10 12 14 16 18 Figure 1: Chromatogram of a spiked urine specimen. Peak identity and concentration (ng/mL) are listed below. 1: morphine (250); 2: oxymorphone Retention Time (min) (250); 3: hydromorphone (250); 4: dihydrocodeine (250); 5: codeine (250); 6: amphetamine (250); 7: O-desmethyltramadol (250); 8: benzoylecgonine b (250); 9: Oxycodone (250); 10: 6-MAM (250); 11: methamphetamine (250); 160000 12: hydrocodone (250); 13: norfentanyl (250); 14: tramadol (250); 15: norbuprenorphine (250); 16: THCA (1000); 17: buprenorphine (1000); 18: Patient fentanyl (25); 19: methadone (25); 20: EDDP (25). The three newly added 120000 analytes are highlighted with underscores. Blank Norbuprenorphine (414.3→187.1) as our previous report [3]. Major assay characteristics included matrix Intensity 80000 effects, interference, analytical measurement range (AMR), carryover, precision, and method comparison. Usage of the leftover patient 40000 samples in this study was approved by the Institutional Review Board of Cleveland Clinic. 0 Results and Discussion 8 10 12 14 16 18 Retention Time (min) Buprenorphine, norbuprenorphine and 6-MAM were readily c detected without any change to our previous sample preparation and HPLC methods [3]. The measurement of the original 19 analytes was 2000000 not affected. In November 2011, propoxyphene was withdrawn from Patient 1500000 US market due to its serious toxicity to the heart. Since then, we had Blank found that the positive rate of propoxyphene declined sharply, and the same trend has been reported by others [12]. We stopped monitoring Intensity 1000000 6-MAM propoxyphene use in September 2012. The chromatogram of the (328.1→211.1) remaining analytes and the 3 newly added ones is shown in (Figure 1). 500000 The efficiency of enzymatic hydrolysis was estimated using blank urine samples spiked with known amounts (450 ng/mL) of glucuronide- conjugated standards, and complete hydrolysis (>95%) was achieved 0 for both buprenorphine and norbuprenorphine within 1 hour of 8 10 12 14 16 18 incubation at 60°C. In practice the hydrolysis time was kept at 16-20 Retention Time (min) hours to ensure the complete hydrolysis of codeine-glucuronide [3]. Figure 2: Evaluation of matrix effects. Six negative patient samples (3 male and 3 female) were prepared without adding the IS. Upon injection, a constant The recovery of on-line turbulent flow extraction was assessed by flow (5 µL/min) of each analyte (1 µg/mL prepared in methanol) was infused comparing the peak areas from injecting spiked blank urine specimens post-column into the mobile phase using a T junction. MRMs of each analyte (300 ng/mL, n=3) onto turbulent column with those from injecting the were monitored for the entire LC gradient. A representative chromatogram of a patient (solid line) and a blank injection of water (dashed line) monitored for the same specimens directly onto the analytical column. The mean recovery primary transition are shown. Retention time of each analyte is indicated with was determined to be 16.4%, 95.9% and 84.7% for buprenorphine, an arrow. norbuprenorphine, and 6-MAM, respectively. The relative recovery after correction by IS varied from 97.9% to 114.5%. No obvious matrix No interference was found in five commercial urine controls which effect was observed in the post-column T infusion experiments in were Lyphochek urine toxicology control level 3 (Bio-Rad), Lyphochek which 6 negative patient samples (3 males and 3 females) were injected quantitative urine controls (both normal and abnormal, Bio-Rad), while a constant infusion of the analytes was introduced (Figure 2). MAS Urichem TRAK liquid assayed urine controls (Levels 1 and 2,

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Buprenorphine (ng/ml) Norbuprenorphine (ng/ml) quantification (LLOQ) was determined by lowest concentration tested Patient CCF NMS Diff. (%) CCF NMS Diff. (%) with <20% CV and 100 ± 20% accuracy. The LLOQ was found to be 9.7, 1 905 970 -6.9 827 970 -15.9 9.6, and 4.9 ng/mL for buprenorphine, norbuprenorphine and 6-MAM, 2 427 440 -3.0 469 440 6.4 respectively (Table 1). Carryover was assessed by extracting two levels 3 261 220 17.0 233 220 5.7 (low and high) of spiked patient samples in triplicate and by running

4 173 180 -4.0 174 180 -3.4 each set in the sequence of low1-high-low2, where low2 is a reinjection

5 96 82 15.7 89 82 8.2 of low1. Analyte concentration in the low sample was 10-20 ng/mL. There would be no carryover if the difference between low and low Table 2: Method comparison using patient specimens spiked with unconjugated 1 2 buprenorphine and norbuprenorphine. Five negative patient samples were was within 20% of low1 and that the mean of low2 was within 3 standard spiked with different concentrations of buprenorphine and norbuprenorphine, deviations of the mean of low1. Analyte concentration of the passing and analyzed by the reported method (CCF) and an LC-MS/MS method offered high sample was determined after dilution and is listed in (Table 1). at NMS Labs (Willow Grove, PA). The measured concentrations and the percent differences are shown. Precision was evaluated based on CLSI EP10-A3 guideline at three concentration levels, run twice a day over 5 days. The total CV was 3.3- 13.3% across the concentration levels tested (Table 1). Measurement a of buprenorphine and norbuprenorphine was compared with an LC-

Scatter Plot (Buprenorphine) Bland-Altman Plot (Buprenorphine) MS/MS method offered by NMS Labs (Willow Grove, PA) using 40 leftover patient samples. Buprenorphine and norbuprenorphine were 1500 Slope=1.108 (1.056-1.160) 80 Mean bias= 25.2% Intercept= 20.3 ng/ml (5.0-35.7) 2 SD= 27.8% detected by both methods in all 40 samples. As shown in (Figures 3a r= 0.9898 60 N= 40 and 3b), a positive bias was observed for both buprenorphine (+25.2%) 1000 40 and norbuprenorphine (+15.3 %). Interestingly, such a bias was not noticed in a later comparison using negative urine samples (n=5) 20 500 (%) Difference LC-MS/MS (ng/ml) LC-MS/MS spiked with unconjugated buprenorphine and norbuprenorphine 1:1 line 0 Deming regression (Table 2). Therefore, the bias was hypothetically due to differences in 0 -20 hydrolysis efficiencies. Measurement of 6-MAM was compared with 0 500 1000 1500 0 400 800 1200 Reference LC-MS/MS (ng/ml) Average (ng/ml) an independent LC-MS/MS method (ARUP, Salt Lake City, UT) using 40 leftover patient samples. Seven samples with low concentrations b (5.0, 7.4, 9.0, 10.0, 12.2, 14.4, and 21.0 ng/mL by this method) were not quantifiable by the ARUP method. Quantitative comparison of the Scatter Plot (Norbuprenorphine) Bland-Altman Plot (Norbuprenorphine) remaining 33 samples is shown in (Figure 3c). The obvious positive bias 2000 Slope=1.146 (1.103-1.190) Intercept= -0.5 ng/ml (-27.1-26.2) Mean bias= 15.3% (+28.8%) was likely due to the lack of a hydrolysis step in the ARUP 50 2 SD= 18.2% r= 0.9939 method. In order to estimate the percentage of glucuronide-conjugated 1500 N= 40 40

30 6-MAM in patient urine, we analyzed eight 6-MAM positive samples 1000 20 (68-877 ng/mL) with and without performing the hydrolysis step to

Difference (%) Difference determine the total and free 6-MAM concentrations. The percentage

LC-MS/MS (ng/ml) LC-MS/MS 10 500 1:1 line 0 of conjugated 6-MAM was calculated as [total-free]/total, and varied Deming regression 0 -10 from 0 to 45%. To the best of our knowledge, this is the first report 0 500 1000 1500 2000 0 500 1000 1500 2000 on the proportion of glucuronide-conjugated 6-MAM in urine. It is Average (ng/ml) Reference LC-MS/MS (ng/ml) noteworthy that the majority of 6-MAM positive samples (37 out 40) contained both morphine (7,000-90,000 ng/mL) and codeine (80-4,500 c ng/mL), and their morphine to codeine ratios were greater than 10. Scatter Plot (6-MAM) Bland-Altman Plot (6-MAM) This pattern is consistent with heroin use [10]. 8000 Slope=1.409 (1.363-1.455) 80 Intercept= -34.6 ng/ml (-101.6-32.4.2) Mean bias= 28.8% r= 0.9960 2 SD= 26.2% Conclusion 6000 N= 33 60 Buprenorphine, norbuprenorphine, and 6-MAM have been 4000 40 successfully added to an existing LC-MS/MS test panel for pain

Difference (%) Difference management service. The quantification of these analytes was proven

LC-MS/MS (ng/ml) LC-MS/MS 2000 20 1:1 line sufficient for clinical use with high sensitivity, specificity, and precision. Deming regression 0 0 We have analyzed over 3000 specimen in the past 6 months without any 0 1000 2000 3000 4000 5000 6000 0 1000 2000 3000 4000 5000 problems, and successfully passed the College of American Pathologists Reference LC-MS/MS (ng/ml) Average (ng/ml) proficiency tests. Figure 3: Method comparison. Leftover patient samples were split and analyzed References by the reported method and an independent LC-MS/MS method. Results obtained from both methods were quantitatively compared using Deming 1. Reisfield GM, Salazar E, Bertholf RL (2007) Rational use and interpretation regression (left panels). The determined slope, Y-intercept, and correlation of urine drug testing in chronic opioid therapy. Ann Clin Lab Sci 37: 301-314. coefficient (r) are shown. Numbers in parenthesis are 95% confidence intervals. Bias was analyzed using Bland-Altman plot (right panels) in which dashed line 2. Porter WH. Clinical toxicology. In: Burtis CA, Ashwood EA, Bruns DE, Eds. represents mean percentage bias, and dotted lines represent ± 2SD. 2006. 3. Yuan C, Heideloff C, Kozak M, Wang S (2011) Simultaneous quantification of 19 drugs/metabolites in urine important for pain management by liquid ThermoFisher Scientific). In the AMR study, a specimen prepared by chromatography-tandem mass spectrometry. Clin Chem Lab Med 50: 95-103. spiking a pool of blank patient urines with high levels of the analytes was serially diluted with the same blank urine pool. The resulting 4. Johnson RE, McCagh JC (2000) Buprenorphine and naloxone for heroin dependence. Curr Psychiatry Rep 2: 519-526. specimens were processed and analyzed in triplicate. The lower limit of

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5. Administration SAaMHS. Results from the 2011 national survey on drug use 9. Elsohly MA, Gul W, Feng S, Murphy TP (2005) Hydrolysis of conjugated and health: Mental health findings. In: SERVICES HAH, Ed. Rockville, MD2012. metabolites of buprenorphine II. The quantitative enzymatic hydrolysis of 6. Huang W, Moody DE, McCance-Katz EF (2006) The in vivo glucuronidation of norbuprenorphine-3-beta-D-glucuronide in human urine. J Anal Toxicol 29: buprenorphine and norbuprenorphine determined by liquid chromatography- 570-573. electrospray ionization-tandem mass spectrometry. Ther Drug Monit 28: 245- 251. 10. Moriya F, Chan KM, Hashimoto Y (1999) Concentrations of morphine and codeine in urine of heroin abusers. Leg Med (Tokyo) 1: 140-144. 7. Feng S, ElSohly MA, Duckworth DT (2001) Hydrolysis of conjugated metabolites of buprenorphine. I. The quantitative enzymatic hydrolysis of buprenorphine-3- 11. Baselt RC (2004) Disposition of toxic drugs and chemicals in man. Seventh beta-D-glucuronide in human urine. J Anal Toxicol 25: 589-593. edition Foster City, CA: Biomedical Publications.

8. Kronstrand R, Selden TG, Josefsson M (2003) Analysis of buprenorphine, 12. Puet B, DePriest A, Knight J, Heltsley R, Black DL, et al. (2013) Urine drug norbuprenorphine, and their glucuronides in urine by liquid chromatography- testing of chronic pain patients. V. Prevalence of propoxyphene following its mass spectrometry. J Anal Toxicol 27: 464-470. withdrawal from the United States market. J Anal Toxicol 37: 1-4.

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J Chromat Separation Techniq ISSN:2157-7064 JCGST, an open access journal Volume 4 • Issue 3 • 1000174