Quantification of morphine, morphine 6-glucuronide, buprenorphine, and the enantiomers of methadone by enantioselective mass spectrometric chromatography in whole blood.

Abstract

Deaths among drug addicts are frequently caused by intoxication with methadone and/or morphine. These drugs are often used in combination with other drugs, such as buprenorphine. In addition, methadone is generally used as a mixture of R- and S-enantiomers. To date, a method for separation and quantitation of these specific drugs has not been developed. The aim of this study was to develop a sensitive enantioselective method for quantitation of morphine, its active metabolite morphine 6-glucuronide, buprenorphine, and R- and S-methadone, in a single analytical run. Whole blood samples were diluted with 0.5 mol/L ammonium carbonate buffer and extracted on a Bond Elut C18 solid-phase extraction column with an automatic solid-phase extraction system. Chromatographic separation was performed on a chiral alpha-1-acid glycoprotein column with an acetonitrile/ammonium acetate buffer (10 mmol/L, pH 7.0, 22:78 v/v) mobile phase. The whole blood concentrations of the drugs were quantified by mass spectrometry using their stable isotope-labeled compounds as internal standards. The method was validated with respect to specificity, linearity, precision, limits of detection, and quantification and matrix effects. The precision (coefficient of variation) was below 15%, and the accuracy was between 90 and 115%. This method will be useful for routine analyses in forensic laboratories where blood samples are frequently analyzed for drugs of abuse. In some cases, sudden death from methadone overdose is caused by the enantiomeric form of the methadone, which makes the enantiomer separation capability of this method important.