Sensitive high performance liquid chromatographic fluorescence determination of topotecan in human plasma and parotid saliva

A sensitive and specific high performance liquid chromatographic (HPLC) method with fluorescence detection was developed and validated for the determination of topotecan, as lactone form and total lactone plus carboxylate forms, in human plasma and parotid saliva samples. The sample pretreatment procedure involved a simple protein precipitation with cold methanol to quantify the lactone form, or a protein precipitation with methanol and acidification with perchloric acid (to convert the lactone ring-opened form into its lactone form quantitatively), to quantify topotecan as a total of lactone and carboxylate forms. The supernatant was analyzed by HPLC using a Zorbax SB-C-18 column and a mobile phase containing 0.01 M N,N,N',N'-tetramethylethylenediamine (TEMED) in water (pH 6)-0.1 M hexane-1-sulfonic acid in methanol-methanol (62:10:28, v/v/v). The detection was performed at 361 nm for excitation and 527 nm for emission. The assay showed linearity in the tested range of 0.1- 75 ng mL(-1). The limit of quantitation was 0.05 ng mL(-1). Precision expressed as %RSD was in the range 0.4 to 17% (limit of quantitation). Accuracy ranged from 85 to 109%. Extraction recovery from plasma or parotid saliva averaged 90%. In this paper, the stability of topotecan as its lactone form in plasma, blood, and methanolic extracts was tested under various conditions. Particularly interesting was the higher stability of the lactone form in the whole blood. The method's ability to quantify topotecan with precision, accuracy, and sensitivity makes it useful in pharmacokinetic studies.