Quantification of topotecan and its metabolite N-desmethyltopotecan in human plasma, urine and faeces by high-performance liquid chromatographic methods

Sensitive high-performance liquid chromatographic (HPLC) methods have been developed and validated for the simultaneous determination of the antitumor drug topotecan and its metabolite N-desmethyltopotecan in human plasma, ur ine and faeces. Both compounds are reversibly hydrolysed to their hydroxyca rboxylate forms at physiologic pH. Separate HPLC systems have been develope d for the determination of lactone and total (lactone plus hydroxycarboxyla te forms) concentrations in plasma. The instability of the analytes in plas ma requires immediate protein precipitation with ice-cold methanol. The lac tone forms of the analytes were stable in the methanol extracts for at leas t 15 months when stored at -70 degrees C. For the determination of the tota l levels, the plasma extracts were acidified with 25 mM phosphoric acid to convert the compounds into their lactone forms quantitatively. The sample p retreatment procedure for urine included dilution in methanol while the fae cal samples were homogenized in distilled water and then extracted twice wi th an acetonitrile-ammonium acetate mixture. Separation was achieved on rev ersed-phase columns (Zorbax SB-C18) and detection was performed fluorimetri cally at 380/527 nm. Within-run and between-run precisions were less than 1 0% and average accuracies were between 90 and 110%. The methods were used i n a mass balance study in patients with malignant solid tumors to determine the disposition and routes of elimination of topotecan and N-desmethyltopo tecan. (C) 1999 Elsevier Science B.V. All rights reserved.