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

A sensitive and specific high performance liquid chromatographic (HPLC) met hod with fluorescence detection was developed and validated for the determi nation of topotecan, as lactone form and total lactone plus carboxylate for ms, in human plasma and parotid saliva samples. The sample pretreatment pro cedure involved a simple protein precipitation with cold methanol to quanti fy the lactone form, or a protein precipitation with methanol and acidifica tion 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'-tetramethylet hylenediamine (TEMED) in water (pH 6)-0.1 M hexane-1-sulfonic acid in metha nol-methanol (62:10:28, v/v/v). The detection was performed at 361 nm for excitation and 527 nm for emissio n. 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 t his 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.