Measurement of anti-cancer agent methoxyamine in plasma by tandem mass spectrometry with on-line sample extraction

In this work, we present the development and validation of a tandem mass spectrometry method for the quantitative determination of methoxyamine (CH3ONH2), a potential new chemotherapeutic agent, in human and mouse plasma. Methoxyamine together with the internal standard (I.S.) methoxyl-D3-amine was directly derivatized in plasma sample with a novel chemical agent 4-(N,N-diethylamino)benzaldehyde. The product solution was injected into an on-line Oasis® HLB extraction column (2.1 mm×20 mm) for analyte extraction. After the elution of extractives, the derivatized analytes were monitored by the positive-electrospray-ionization mass spectrometry (ESI-MS-MS). The structures of derivatized analytes were elucidated by fragmentation. Quantitation of plasma methoxyamine was carried out by the multiple reaction monitoring (MRM) mode. This method had a linear calibration range of 1.00–1000 ng/ml with a correlation coefficient of 0.9999 for methoxyamine in both human and mouse plasma. The limit of detection (LOD) and limit of quantification (LOQ) for methoxyamine in plasma were 0.150 and 0.500 ng/ml, respectively. It was demonstrated that the method had high recovery and accuracy (90.1–94.7 and 90.1–96.3%), as well as excellent intra- and inter-assay precision (2.2 and 3.7%), at three concentration levels (5.00, 50.0, 500 ng/ml). This method has been used to analyze the plasma levels of methoxyamine in samples obtained from male CD1 mice after bolus intraperitoneal injection of 2, 5 and 20 mg methoxyamine hydrochloride (CH3ONH2.HCl) per kilogram mouse.