Research article Received: 9 December 2014,
Revised: 9 February 2015,
Accepted: 19 February 2015
Published online in Wiley Online Library: 22 March 2015
(wileyonlinelibrary.com) DOI 10.1002/bmc.3460
UHPLC-MS/MS quantification of buprenorphine, norbuprenorphine, methadone, and glucuronide conjugates in umbilical cord plasma Amy Redmond Kylea, Jennifer Carmicala, Darshan Shahb, Jason Pryorc and Stacy Brownd* ABSTRACT: Opioid use during pregnancy can result in the newborn being physically dependent on the substance, thus experiencing drug withdrawal, termed neonatal abstinence syndrome (NAS). Buprenorphine and methadone are two drugs used to treat opioid withdrawal and are approved for use in pregnancy. Quantification of these compounds in umbilical cord plasma would help assess in utero exposure of neonates in cases of buprenorphine or methadone use during pregnancy. An LC-MS/MS method using solid-phase extraction sample preparation was developed and validated for the simultaneous quantification of methadone, buprenorphine, norbuprenorphine, and glucuronide metabolites in umbilical cord plasma. The average accuracy (percentage error) and precision (relative standard deviation) were 80% and glucuronide metabolites with >75% recovery at multiple concentrations. We also found that, while PPL removal does improve recovery of some analytes (norbuprenorphine, methadone), it does not affect the extraction of the glucuronide conjugates in a significant manner. As far as we know, this is the first validated method for the quantification of buprenorphine and its metabolites from umbilical cord blood plasma. Our data establishes a 2 week maximum freezer storage window in order to achieve the most accurate cord plasma concentrations of these analytes, especially the glucuronide conjugates. Additionally, our study found that the umbilical cord tissue analysis provided by the hospital was less sensitive compared with analysis with umbilical cord blood plasma, indicating that this may
Table 3. Umbilical cord blood plasma concentrations (ng/mL; average ± standard deviation) from patients with Suboxone/Subutex use history during pregnancy (bold lines reflect ‘negative’ cord tissue assay samples) Patient ID
Norbuprenorphine
Buprenorphine–glucuronide
Norbuprenorphine–glucuronide
7.9 ± 0.1 8.0 ± 0.2 13.3 ± 4.3 15.4 ± 5.3 10.7 ± 4.0 11.8 ± 4.1
11.0 ± 3.0 14.8 ± 4.0 9.5 ± 3.0 25.0 ± 9.0 11.6 ± 3.3 14.8 ± 2.3
21.2 ± 2.1 22.8 ± 5.6 20.7 ± 0.8 29.2 ± 7.1 25.0 ± 8.8 Below LOQ
40.3 ± 11.1 49.5 ± 10.9 39.5 ± 3.8 53.5 ± 8.9 Below LOQ 22.9 ± 4.2
Biomed. Chromatogr. 2015; 29: 1567–1574
Copyright © 2015 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/bmc
1573
003 006 012 014 015 018
Buprenorphine
A. R. Kyle et al. be a more appropriate matrix for determinatopm of buprenorphine and its metabolite concentrations. This method was successfully applied to the analysis of cord blood from women with known buprenorphine or methadone use during pregnancy.
Acknowledgments The authors would like to acknowledge the Bill Gatton College of Pharmacy for their ongoing support of student research and funding. They would also like to thank Shimadzu Scientific and the ETSU RDC Major Grant Program for their financial support of this project. Additionally, we wish to thank Johnson City Medical Center ( Johnson City, TN, USA) for allowing access to their patient population and Nilesh Dankhara, MD and Piyuesh Singh, MD for their assistance in sample collection.
References Bonfiglio R, King RC, Olah TV and Merkle K. The effects of sample preparation methods on the variability of the electrospray ionization response for model drug compounds. Rapid Communications in Mass Spectrometry 1999; 13: 1175–1185. Burns L and Mattick RP. Using population data to examine the prevalence and correlates of neonatal abstinence syndrome. Drug and Alcohol Review 2007; 26: 487–492. Castro A, Concheiro M, Shakleya DM and Huestis MA. Development and validation of a liquid chromatography mass spectrometry assay for the simultaneous quantification of methadone, cocaine, opiates and metabolites in human umbilical cord. Journal of Chromatography B 2009; 877: 3065–3071. Ceccate Am Klinkenberg R, Hubert P and Streel B. Sensitive determination of buprenorphine and its N-dealkylated metabolite norbuprenorphine in human plasma by liquid chromatography coupled to tandem mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis 2003; 32: 619–631. Concheiro M, Shakleya M and Huestis MA. Simultaneous quantification of buprenorphine, norbuprenorphine, buprenorphine–glucuronide and norbuprenorphine–glucuronide in human umbilical cord by liquid chromatography tandem mass spectrometry. Forensic Science International 2009; 188: 144–151. Gray T and Huestis M. Bioanalytical procedures for monitoring in utero drug exposure. Analytical and Bioanalytical Chemistry 2007; 288: 1455–1465. Jones HE, Johnson RE, Jasinski DR, O’Grady KE, Chisholm CA, Choo RE, Crocetti M, Dudas R, Harrow C, Huestis MA, Jansson LM, Lantz M, Lester BM and Milio L. Buprenorphine versus methadone in the treatment of pregnant opioid-dependent patients: effects on the neonatal abstinence syndrome. Drug and Alcohol Dependence 2005; 79: 1–10. Kackinko SL, Jones HE, Johnson RE, Choo RE, Concheiro-Guisan M and Huestis MA. Urinary excretion of buprenorphine, norbuprenorphine,
buprenorphine–glucuronide, and norbuprenorphine–glucuronide in pregnant women receiving buprenorphine maintenance treatment. Clinical Chemistry 2009; 55(6): 1177–1187. Kellogg A, Rose CH, Harns RH and Watson WJ. Current trends in narcotic use in pregnancy and neonatal outcomes. American Journal of Obstetrics and Gynecology 2011; 259: e1–e4. Lin HR, Chen CL, Huang CL, Chen ST and Lua AC. Simultaneous determination of opiates, methadone, buprenorphine and metabolites in human urine by superficially porous liquid chromatography tandem mass spectrometry. Journal of Chromatography B 2013; 925: 10–15. Liu AC, Lin TY, Su LW and Fuh MR. Online solid-phase extraction liquid chromatography–electrospray–tandem mass spectrometry analysis of buprenorphine and three metabolites in human urine. Talanta 2008; 75: 198–204. Luthi G, Blangy V, Eap CB and Ansermot N. Buprenorphine and norbuprenorphine quantification in human plasma by simple protein precipitation and ultra-high performance liquid chromatography tandem mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis 2013; 77: 1–8. Mercolini L, Mandrioli R, Conti M, Leonardi C, Gerra G and Raggi MA. Simultaneous determination of methadone, buprenorphine and norbuprenorphine in biological fluids for therapeutic drug monitoring purposes. Journal of Chromatography B 2007; 847: 95–102. Nanovskaya TN, Deshmukh S, Brooks M and Ahmed MS. Transplacental transfer and metabolism of buprenorphine. Journal of Pharmacology and Experimental Therapeutics 2002; 300: 26–33. Nekhaeva IA, Nanovskaya TN, Deshmukh S, Zharikova OL, Hankins GDV and Almed MS. Bidirectional transfer of methadone across human placenta. Biochemical Pharmacology 2005; 69: 187–197. Oechsler S, Skopp G. Buprenorphine and major metabolites in blood specimens collected for drug analysis in law enforcement purposes. Forensic Science International 2010; 195: 73–77. Rodrigues-Rosas ME, Lofwall MR, Strain EC, Siluk D and Wainer IW. Simultaneous determination of buprenorphine, norbuprenorphine and the enantiomers of methadone and its metabolites (EDDP) in human plasma by liquid chromatography/mass spectrometry. Journal of Chromatography B 2007; 850: 538–543. Welle-Strand GK, Skurtveit S, Jones HE, Waal H, Bakstad B, Bjarko L and Ravndal E. Neonatal outcomes following in utero exposure to methadone or buprenorphine: A National Cohort Study of opioid-agonist treatment of pregnant women in Norway from 1996 to 2009. Drug and Alcohol Dependence 2013; 127: 200–206. Winhusen T, Wilder C, Wexelblatt SL, Theobald J, Hall ES, Lewis D, Hook JV and Marcotte M. Design considerations for point-of-care clinical trials comparing methadone and buprenorphine treatment for opioid dependence in pregnancy and for neonatal abstinence syndrome. Contemporary Clinical Trials 2014; 39: 158–165. Yue H, Borenstein MR, Jansen SA and Raffa RB. Liquid chromatography– mass spectrometric analysis of buprenorphine and its N-dealkylated metabolite norbuprenorphine in rat brain tissue and plasma. Journal of Pharmacological and Toxicological Methods 2005; 52: 314–322.
1574 wileyonlinelibrary.com/journal/bmc
Copyright © 2015 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2015; 29: 1567–1574
Revised: 9 February 2015,
Accepted: 19 February 2015
Published online in Wiley Online Library: 22 March 2015
(wileyonlinelibrary.com) DOI 10.1002/bmc.3460
UHPLC-MS/MS quantification of buprenorphine, norbuprenorphine, methadone, and glucuronide conjugates in umbilical cord plasma Amy Redmond Kylea, Jennifer Carmicala, Darshan Shahb, Jason Pryorc and Stacy Brownd* ABSTRACT: Opioid use during pregnancy can result in the newborn being physically dependent on the substance, thus experiencing drug withdrawal, termed neonatal abstinence syndrome (NAS). Buprenorphine and methadone are two drugs used to treat opioid withdrawal and are approved for use in pregnancy. Quantification of these compounds in umbilical cord plasma would help assess in utero exposure of neonates in cases of buprenorphine or methadone use during pregnancy. An LC-MS/MS method using solid-phase extraction sample preparation was developed and validated for the simultaneous quantification of methadone, buprenorphine, norbuprenorphine, and glucuronide metabolites in umbilical cord plasma. The average accuracy (percentage error) and precision (relative standard deviation) were 80% and glucuronide metabolites with >75% recovery at multiple concentrations. We also found that, while PPL removal does improve recovery of some analytes (norbuprenorphine, methadone), it does not affect the extraction of the glucuronide conjugates in a significant manner. As far as we know, this is the first validated method for the quantification of buprenorphine and its metabolites from umbilical cord blood plasma. Our data establishes a 2 week maximum freezer storage window in order to achieve the most accurate cord plasma concentrations of these analytes, especially the glucuronide conjugates. Additionally, our study found that the umbilical cord tissue analysis provided by the hospital was less sensitive compared with analysis with umbilical cord blood plasma, indicating that this may
Table 3. Umbilical cord blood plasma concentrations (ng/mL; average ± standard deviation) from patients with Suboxone/Subutex use history during pregnancy (bold lines reflect ‘negative’ cord tissue assay samples) Patient ID
Norbuprenorphine
Buprenorphine–glucuronide
Norbuprenorphine–glucuronide
7.9 ± 0.1 8.0 ± 0.2 13.3 ± 4.3 15.4 ± 5.3 10.7 ± 4.0 11.8 ± 4.1
11.0 ± 3.0 14.8 ± 4.0 9.5 ± 3.0 25.0 ± 9.0 11.6 ± 3.3 14.8 ± 2.3
21.2 ± 2.1 22.8 ± 5.6 20.7 ± 0.8 29.2 ± 7.1 25.0 ± 8.8 Below LOQ
40.3 ± 11.1 49.5 ± 10.9 39.5 ± 3.8 53.5 ± 8.9 Below LOQ 22.9 ± 4.2
Biomed. Chromatogr. 2015; 29: 1567–1574
Copyright © 2015 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/bmc
1573
003 006 012 014 015 018
Buprenorphine
A. R. Kyle et al. be a more appropriate matrix for determinatopm of buprenorphine and its metabolite concentrations. This method was successfully applied to the analysis of cord blood from women with known buprenorphine or methadone use during pregnancy.
Acknowledgments The authors would like to acknowledge the Bill Gatton College of Pharmacy for their ongoing support of student research and funding. They would also like to thank Shimadzu Scientific and the ETSU RDC Major Grant Program for their financial support of this project. Additionally, we wish to thank Johnson City Medical Center ( Johnson City, TN, USA) for allowing access to their patient population and Nilesh Dankhara, MD and Piyuesh Singh, MD for their assistance in sample collection.
References Bonfiglio R, King RC, Olah TV and Merkle K. The effects of sample preparation methods on the variability of the electrospray ionization response for model drug compounds. Rapid Communications in Mass Spectrometry 1999; 13: 1175–1185. Burns L and Mattick RP. Using population data to examine the prevalence and correlates of neonatal abstinence syndrome. Drug and Alcohol Review 2007; 26: 487–492. Castro A, Concheiro M, Shakleya DM and Huestis MA. Development and validation of a liquid chromatography mass spectrometry assay for the simultaneous quantification of methadone, cocaine, opiates and metabolites in human umbilical cord. Journal of Chromatography B 2009; 877: 3065–3071. Ceccate Am Klinkenberg R, Hubert P and Streel B. Sensitive determination of buprenorphine and its N-dealkylated metabolite norbuprenorphine in human plasma by liquid chromatography coupled to tandem mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis 2003; 32: 619–631. Concheiro M, Shakleya M and Huestis MA. Simultaneous quantification of buprenorphine, norbuprenorphine, buprenorphine–glucuronide and norbuprenorphine–glucuronide in human umbilical cord by liquid chromatography tandem mass spectrometry. Forensic Science International 2009; 188: 144–151. Gray T and Huestis M. Bioanalytical procedures for monitoring in utero drug exposure. Analytical and Bioanalytical Chemistry 2007; 288: 1455–1465. Jones HE, Johnson RE, Jasinski DR, O’Grady KE, Chisholm CA, Choo RE, Crocetti M, Dudas R, Harrow C, Huestis MA, Jansson LM, Lantz M, Lester BM and Milio L. Buprenorphine versus methadone in the treatment of pregnant opioid-dependent patients: effects on the neonatal abstinence syndrome. Drug and Alcohol Dependence 2005; 79: 1–10. Kackinko SL, Jones HE, Johnson RE, Choo RE, Concheiro-Guisan M and Huestis MA. Urinary excretion of buprenorphine, norbuprenorphine,
buprenorphine–glucuronide, and norbuprenorphine–glucuronide in pregnant women receiving buprenorphine maintenance treatment. Clinical Chemistry 2009; 55(6): 1177–1187. Kellogg A, Rose CH, Harns RH and Watson WJ. Current trends in narcotic use in pregnancy and neonatal outcomes. American Journal of Obstetrics and Gynecology 2011; 259: e1–e4. Lin HR, Chen CL, Huang CL, Chen ST and Lua AC. Simultaneous determination of opiates, methadone, buprenorphine and metabolites in human urine by superficially porous liquid chromatography tandem mass spectrometry. Journal of Chromatography B 2013; 925: 10–15. Liu AC, Lin TY, Su LW and Fuh MR. Online solid-phase extraction liquid chromatography–electrospray–tandem mass spectrometry analysis of buprenorphine and three metabolites in human urine. Talanta 2008; 75: 198–204. Luthi G, Blangy V, Eap CB and Ansermot N. Buprenorphine and norbuprenorphine quantification in human plasma by simple protein precipitation and ultra-high performance liquid chromatography tandem mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis 2013; 77: 1–8. Mercolini L, Mandrioli R, Conti M, Leonardi C, Gerra G and Raggi MA. Simultaneous determination of methadone, buprenorphine and norbuprenorphine in biological fluids for therapeutic drug monitoring purposes. Journal of Chromatography B 2007; 847: 95–102. Nanovskaya TN, Deshmukh S, Brooks M and Ahmed MS. Transplacental transfer and metabolism of buprenorphine. Journal of Pharmacology and Experimental Therapeutics 2002; 300: 26–33. Nekhaeva IA, Nanovskaya TN, Deshmukh S, Zharikova OL, Hankins GDV and Almed MS. Bidirectional transfer of methadone across human placenta. Biochemical Pharmacology 2005; 69: 187–197. Oechsler S, Skopp G. Buprenorphine and major metabolites in blood specimens collected for drug analysis in law enforcement purposes. Forensic Science International 2010; 195: 73–77. Rodrigues-Rosas ME, Lofwall MR, Strain EC, Siluk D and Wainer IW. Simultaneous determination of buprenorphine, norbuprenorphine and the enantiomers of methadone and its metabolites (EDDP) in human plasma by liquid chromatography/mass spectrometry. Journal of Chromatography B 2007; 850: 538–543. Welle-Strand GK, Skurtveit S, Jones HE, Waal H, Bakstad B, Bjarko L and Ravndal E. Neonatal outcomes following in utero exposure to methadone or buprenorphine: A National Cohort Study of opioid-agonist treatment of pregnant women in Norway from 1996 to 2009. Drug and Alcohol Dependence 2013; 127: 200–206. Winhusen T, Wilder C, Wexelblatt SL, Theobald J, Hall ES, Lewis D, Hook JV and Marcotte M. Design considerations for point-of-care clinical trials comparing methadone and buprenorphine treatment for opioid dependence in pregnancy and for neonatal abstinence syndrome. Contemporary Clinical Trials 2014; 39: 158–165. Yue H, Borenstein MR, Jansen SA and Raffa RB. Liquid chromatography– mass spectrometric analysis of buprenorphine and its N-dealkylated metabolite norbuprenorphine in rat brain tissue and plasma. Journal of Pharmacological and Toxicological Methods 2005; 52: 314–322.
1574 wileyonlinelibrary.com/journal/bmc
Copyright © 2015 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2015; 29: 1567–1574
