Benedicte Jørgenrud1, Eline Skadberg1, Julio de Carvalho Ponce2, Håvard Furuhaugen1, Thomas Berg3. 1. Section of Drug Abuse Research, Department of Forensic Sciences, Division of Laboratory Medicine, Oslo University Hospital, P.O. Box 4950 Nydalen, N-0424 Oslo, Norway. 2. Department of Preventive Medicine, School of Medicine, University of Sao Paulo, Av Dr. Arnaldo, 455, Brazil. 3. Section of Drug Abuse Research, Department of Forensic Sciences, Division of Laboratory Medicine, Oslo University Hospital, P.O. Box 4950 Nydalen, N-0424 Oslo, Norway. Electronic address: rmthbe@ous-hf.no.
Abstract
INTRODUCTION: Most bioanalytical LC-MS/MS methods are developed for determination of single drugs or classes of drugs, but a multi-compound LC-MS/MS method that can replace several methods could reduce both analysis time and costs. The aim of this study was to develop a high-throughput LC-MS/MS method for determination of the alcohol biomarker phosphatidylethanol 16:0/18:1 (PEth 16:0/18:1) and 33 other compounds from eight different drug classes in whole blood. METHODS: Whole-blood samples were prepared by 96-well supported liquid extraction (SLE). Chromatographic separations were performed on a biphenyl core shell column with a mobile phase consisting of 10 mM ammonium formate, pH 3.1 and methanol. Each extract was analyzed twice by LC-MS/MS, injecting 0.4 μL and 2 μL, in order to obtain narrow and symmetrical peaks and good sensitivity for all compounds. Stable isotope-labeled internal standards were used for 31 of the 34 compounds. RESULTS: A 96-well SLE reversed phase LC-MS/MS method for determination of PEth 16:0/18:1 and 33 other compounds from eight different drug classes was developed and validated. By using an organic solvent mixture of isopropanol/ methyl tert-butyl ether (1:5, v:v), all compounds, including the polar and ampholytic compounds pregabalin, gabapentin and benzoylecgonine, was extracted by 96-well SLE. DISCUSSION/ CONCLUSION: For the first time an LC-MS/MS method for the determination of alcohol biomarker PEth 16:0/18:1 and drugs and metabolites from several different drug classes was developed and validated. The developed LC-MS/MS method can be used for high-throughput analyses and sensitive determinations of the 34 compounds in whole blood.
INTRODUCTION: Most bioanalytical LC-MS/MS methods are developed for determination of single drugs or classes of drugs, but a multi-compound LC-MS/MS method that can replace several methods could reduce both analysis time and costs. The aim of this study was to develop a high-throughput LC-MS/MS method for determination of the alcohol biomarker phosphatidylethanol 16:0/18:1 (PEth 16:0/18:1) and 33 other compounds from eight different drug classes in whole blood. METHODS: Whole-blood samples were prepared by 96-well supported liquid extraction (SLE). Chromatographic separations were performed on a biphenyl core shell column with a mobile phase consisting of 10 mM ammonium formate, pH 3.1 and methanol. Each extract was analyzed twice by LC-MS/MS, injecting 0.4 μL and 2 μL, in order to obtain narrow and symmetrical peaks and good sensitivity for all compounds. Stable isotope-labeled internal standards were used for 31 of the 34 compounds. RESULTS: A 96-well SLE reversed phase LC-MS/MS method for determination of PEth 16:0/18:1 and 33 other compounds from eight different drug classes was developed and validated. By using an organic solvent mixture of isopropanol/ methyl tert-butyl ether (1:5, v:v), all compounds, including the polar and ampholytic compounds pregabalin, gabapentin and benzoylecgonine, was extracted by 96-well SLE. DISCUSSION/ CONCLUSION: For the first time an LC-MS/MS method for the determination of alcohol biomarker PEth 16:0/18:1 and drugs and metabolites from several different drug classes was developed and validated. The developed LC-MS/MS method can be used for high-throughput analyses and sensitive determinations of the 34 compounds in whole blood.