Determination of 4-demethoxy-3 '-deamino-3 '-aziridinyl-4 '-methylsulphonyldaunorubicin and its 13-hydroxy metabolite by direct injection of human plasma into a column-switching liquid chromatography system with mass spectrometric detection

A selective, sensitive and fully automated column-switching LC system using direct injection of human plasma followed by mass spectrometry (MS) detect ion was developed and validated to determine the concentrations of 4-demeth oxy-3'-deamino-3'-aziridinyl-4'-methylsulphonylbicin (PNLT-159548) and its 13-hydroxy metabolite (PNU-169884). A 50-mu l human plasma sample was direc tly introduced into a C-4-alkyl-diol silica clean-up column separating anal ytes from proteins and polar endogenous compounds using water and methanol as the mobile phase. The fraction containing PNLT-159548 and its metabolite was back-flushed and transferred to the analytical column. The compounds w ere separated using a Zorbax SE C-8 column (150x4.6 mm, 5 mu m) under gradi ent conditions with the mobile phase containing acetonitrile and 2 mM ammon ium formate, pH 3.5. MS detection was by atmospheric pressure ionisation wi th multiple reaction monitoring in positive ion mode. Linearity was demonst rated over the calibration range of 0.051-10.291 ng/ml for PNU-159548 and 0 .104-10.434 ng/ml for PNU-169884. The assay was validated with respect to a ccuracy, precision and analyte stability. On the basis of the validation da ta, the developed analytical method was found to be suitable for use in Pha se I clinical studies. (C) 1999 Elsevier Science B.V. All rights reserved.