Bioanalysis of the novel peptide anticancer drug kahalalide F in human plasma by h.p.l.c. under basic conditions coupled with positive turbo‐ionspray tandem mass spectrometry

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Kahalalide F (KF) is a cyclic depsipeptide derived from the marine mollusc Elysia rufescens. Anti‐cancer activity of KF was observed both in vitro and in vivo especially against prostate cancer cell lines [1]. In order to support the Phase I study a method was developed and validated for the quantitative determination of KF in human plasma using high performance liquid chromatography (h.p.l.c.) coupled with positive turbo‐ionspray tandem mass spectrometry ((h.p.l.c.)‐TISP‐MS/MS). Miniaturized LC combined with mass spectrometric detection was essential to obtain the required selectivity and sensitivity. The use of trifluoroacetic acid, conventionally used in the mobile phase in the LC analysis of peptides, was omitted due to its signal suppressing effects on electrospray ionization. An alternative approach was followed employing a basic mobile phase using a stationary phase that is stable under basic conditions. KF remained positively charged under these conditions while the mobile phase background remained low.The method involves a solid phase extraction (SPE) on C18 columns as sample pre‐treatment followed by LC‐MS/MS analysis. The LC separation was performed on a Zorbax Extend C18 column (150×2.1 mm i.d., particle size 5 µm) with acetonitrile‐10 mm ammonium acetate in water (85 : 15, v/v) as the mobile phase, pumped at a flow‐rate of 0.20 ml min−1. A butyric acid analogue of KF was used as the internal standard.A lower limit of quantitation (LLQ) of 1 ng ml−1 was obtained using a 500 µl sample volume with a linear dynamic range up to 1000 ng ml−1. Accuracies were within 15.1% for the LLQ and within 9.0% for other quality control samples. Precisions did not exceed 9.9%. The drug was found to be stable in the biomatrix for at least 9 months at −20° C and for at least 24 h at ambient temperatures. The results demonstrate that an accurate, selective and sensitive bioanalytical method has been developed for KF analysis in human plasma. The method was successfully applied in a Phase I clinical study of KF. At the starting dose of 20 µg m−2 a Cmax of 1.75 ng ml−1 was obtained.