Simultaneous determination of four anthracyclines and three metabolites in human serum by liquid chromatography–electrospray mass spectrometry

Abstract

A sensitive and very specific method, using liquid chromatography–electrospray mass spectrometry (LC–ES-MS), was developed for the determination of epirubicin, doxorubicin, daunorubicin, idarubicin and the respective active metabolites of the last three, namely doxorubicinol, daunorubicinol and idarubicinol in human serum, using aclarubicin as internal standard. Once thawed, 0.5-ml serum samples underwent an automated solid-phase extraction, using C 18 Bond Elut cartridges (Varian) and a Zymark Rapid-Trace robot. After elution of the compounds with chloroform–2-propanol (4:1, v/v) and evaporation, the residue was reconstituted with a mixture of 5 m M ammonium formate buffer (pH 4.5)–acetonitrile (60:40, v/v). The chromatographic separation was performed using a Symmetry C 18, 3.5 μm (150×1 mm I.D.) reversed-phase column, and a mixture of 5 m M ammonium formate buffer (pH 3)–acetonitrile (70:30, v/v) as mobile phase, delivered at 50 μl/min. The compounds were detected in the selected ion monitoring mode using, as quantitation ions, m/ z 291 for idarubicin and idarubicinol, m/ z 321 for daunorubicin and daunorubicinol, m/ z 361 for epirubicin and doxorubicin, m/ z 363 for doxorubicinol and m/ z 812 for aclarubicin (I.S.). Extraction recovery was between 71 and 105% depending on compounds and concentration. The limit of detection was 0.5 ng/ml for daunorubicin and idarubicinol, 1 ng/ml for doxorubicin, epirubicin and idarubicin, 2 ng/ml for daunorubicinol and 2.5 ng/ml for doxorubicinol. The limit of quantitation (LOQ) was 2.5 ng/ml for doxorubicin, epirubicin and daunorubicinol, and 5 ng/ml for daunorubicin, idarubicin, doxorubicinol and idarubicinol. Linearity was verified from these LOQs up to 2000 ng/ml for the parent drugs ( r≥0.992) and 200 ng/ml for the active metabolites ( r≥0.985). Above LOQ, the within-day and between-day precision relative standard deviation values were all less than 15%. This assay was applied successfully to the analysis of human serum samples collected in patients administered doxorubicin or daunorubicin intravenously. This method is rapid, reliable, allows an easy sample preparation owing to the automated extraction and a high selectivity owing to MS detection.