Determination of picropodophyllin and its isomer podophyllotoxin in human serum samples with electrospray ionization of hexylamine adducts by liquid chromatography–tandem mass spectrometry

A liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for determination of the new anticancer agent picropodophyllin (AXL1717) and its isomer podophyllotoxin levels in human serum has been developed. Monitoring of hexylamine adducts rather than proton adducts was used to optimize sensitivity. The chromatography system was an Acquity BEH C18, 2.1 mm × 50 mm 1.7 μm column with gradient elution (mobile phase A: 2.5 mM hexylamine and 5 mM formic acid in Milli-Q water and mobile phase B: methanol). The retention times were 1.4 min for picropodophyllin, 1.5 min for podophyllotoxin and 1.9 min for internal standard deoxypodophyllotoxin. The isomers were base-line separated. The analytes were detected after electrospray ionization in positive mode with selected reaction monitoring (SRM) with ion transitions m/z 516 → 102 for picropodophyllin and podophyllotoxin and m/z 500 → 102 for internal standard. The sample preparation was protein precipitation with acetonitrile (1:3) containing internal standard followed by dilution of the supernatant with mobile phase A (1:1). The limit of quantification (LOQ) was 0.01 μmol/L for picropodophyllin and podophyllotoxin. The limit of detection (LOD) at 3 times the signal to noise (S/N) was estimated below 0.001 μmol/L for picropodophyllin and podophyllotoxin. The quantification range of the method was between 0.01 μmol/L and 5 μmol/L for both isomers. The accuracy was within ±15% of the theoretical value for both picropodophyllin and podophyllotoxin and inter-assay precision did not exceed ±15%, except for the 0.016 μmol/L level of podophyllotoxin, which was 18%. The selectivity of the method was verified by analysis of two different product ions for each analyte and by analysis for interference of seven different batches of blank human serum. The combined recovery and matrix effects were about 83% for picropodophyllin and podophyllotoxin. The new LC–MS/MS method showed sufficient sensitivity and selectivity for determination of picropodophyllin and its isomer podophyllotoxin levels in human serum from subjects receiving therapeutic doses of AXL1717.