Simultaneous determination of the novel anti-tumor candidate drug MDH-7 and 5-fluorouracil in rat plasma by LC-MS/MS: Application to pharmacokinetic interactions.

MDH-7 (2,3,9-tri-O-acetyl-5,6-dideoxy-1,10-di-[N4'-pentoxycarbonyl-5'-fluoro cytosine]-4-ulose 1,4: 7,10-difuranose-4,8-pyranose) is a novel anti-tumor drug candidate. To study the pharmacokinetic interaction between MDH-7 and 5-fluorouracil (5-FU), a sensitive and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to simultaneously determine the concentrations of MDH-7 and 5-fluorouracil (5-FU) in rat plasma. Plasma samples were prepared by simple liquid-liquid extraction with ethyl acetate. Chromatographic separation was performed on a Waters XBridge™ C18 column (5 µm, 2.1 mm × 150 mm) with the mobile phase of methanol and H2O (80:20, v/v). The ESI positive and negative ion switch was operated in the multiple reactions monitoring (MRM) mode. The calibration curves showed good linearity (r2 > 0.99) over the ranges of 50-8000 ng/mL for MDH-7 and 10-2000 ng/mL for 5-FU, respectively. The lower limit of quantitations (LLOQs) was 50 ng/mL (MDH-7) and 10 ng/mL (5-FU) with relative standard deviation (RSD) < 13.0%. The proposed method was successfully applied to simultaneous assessment of pharmacokinetic drug-drug interaction between MDH-7 and 5-FU in rats.

Identification of metabolites of the novel anti-tumor drug candidate MDH-7 in rat urine by liquid chromatography coupled with triple quadrupole linear ion trap mass spectrometry.

RATIONALE: Our previous preliminary pharmacokinetic study demonstrated that the novel double pyrimidine tricyclic nucleoside MDH-7 in rats had a very short half-life (<30 min) after oral administration. As a result, the in vivo metabolic profile of MDH-7 should be investigated during early stages of drug development to better select drug candidates. METHODS: In this study, a rapid method was developed to identify the metabolites of MDH-7 in rat urine by means of ultra-performance liquid chromatography (UPLC) coupled with electrospray ionization mass spectrometry (ESI-MS) using a triple quadrupole linear ion trap instrument. MDH-7 and its metabolites were detected and characterized by the combined use of the multiple reaction monitoring-information-dependent acquisition-enhanced product ion (MRM-IDA-EPI) mode and the precursor scan information-dependent acquisition-enhanced product ion (PREC-IDA-EPI) mode. RESULTS: Ten novel metabolites of MDH-7 were identified and characterized in rat urine by LC/ESI-MS and collision-induced dissociation tandem mass spectrometry (CID-MS/MS) analyses. M1 was identified as 5-fluoro-N(4) -[(pentyloxy)carbonyl]cytosine; M2 and M3 were formed by hydroxylation products of M1. Metabolites M4-M10 were formed by a series of degradation reactions such as: deacetylation, hydroxylation, loss of the defluorocytosine base, oxidative-deamination, loss of the defluorouracil base, N-dealkylation and amide hydrolysis. CONCLUSIONS: Based on the profiles of the metabolites, possible metabolic pathways of MDH-7 in rats were proposed for the first time. This study provides new and available information on the metabolism of MDH-7 which is very useful to further understand its in vivo metabolic fate. Copyright © 2016 John Wiley & Sons, Ltd.

Determination and pharmacokinetic study of the novel anti-tumor candidate drug DG-7 in rat plasma by liquid chromatography-tandem mass spectrometry.

DG-7 (11,14-dihydroxy-7,20-epoxy-20-O-derivative of ent-kaurene diterpenoid) is a novel anti-tumor candidate drug. A sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of DG-7 in rat plasma. An aliquot of 50 µL plasma sample was prepared by liquid-liquid extraction with ethyl acetate. Chromatographic separation was accomplished on a Waters XTerra C18 column (2.1 mm × 150 mm, 5 µm) with an isocratic elution system consisting of methanol and water. Detection was performed by multiple reaction monitoring (MRM) mode using electrospray ionization in the positive ion mode. The optimized fragmentation transitions for MRM were m/z 590.1→m/z 260.0 for DG-7 and m/z 180.3→m/z 110.1 for phenacetin (internal standard). The method was linear over the concentration range of 5-2,500 ng/mL. The intra- and inter-day precisions were less than 7.9% and the accuracy was within ± 9.0%. The mean recovery of DG-7 ranged from 76.8% to 79.2%. The validated method has been successfully applied to a pharmacokinetic study in rats after intravenous administration of DG-7.