Rapid determination of gefitinib and its main metabolite, O-desmethyl gefitinib in human plasma using liquid chromatography-tandem mass spectrometry.

A novel, rapid and specific liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the simultaneous quantification of gefitinib and its predominant metabolite, O-desmethyl gefitinib in human plasma. Chromatographic separation of analytes was achieved on an Alltima C18 analytical HPLC column (150 mm × 2.1 mm, 5 µm) using an isocratic elution mode with a mobile phase comprised acetonitrile and 0.1% formic acid in water (30:70, v/v). The flow rate was 300 µL/min. The chromatographic run time was 3 min. The column effluents were detected by API 4000 triple quadrupole mass spectrometer using electrospray ionization (ESI) in positive mode. Linearity was demonstrated in the range of 5-1000 ng/mL for gefitinib and 5-500 ng/mL for O-desmethyl gefitinib. The intra- and inter-day precisions for gefitinib and O-desmethyl gefitinib were ≤10.8% and the accuracies ranged from 89.7 to 104.7% for gefitinib and 100.4 to 106.0% for O-desmethyl gefitinib. This method was used as a bioanalytical tool in a phase I clinical trial to investigate the possible effect of hydroxychloroquine on the pharmacokinetics of gefitinib. The results of this study enabled clinicians to ascertain the safety of the combination therapy of hydroxychloroquine and gefitinib in patients with advanced (Stage IIIB-IV) non-small cell lung cancer (NSCLC).

Simultaneous determination of raltegravir and raltegravir glucuronide in human plasma by liquid chromatography-tandem mass spectrometric method.

Raltegravir is a highly efficacious inhibitor of HIV integrase. Large pharmacokinetic variability has been reported in clinical trials and this could be due to glucuronidation of raltegravir, the only reported metabolism pathway. In order to precisely evaluate and monitor the raltegravir and raltegravir glucuronide simultaneously, a novel, sensitive and robust liquid chromatography-tandem mass spectrometric method was developed and validated for simultaneous determination of raltegravir and raltegravir glucuronide in human plasma. A simple protein precipitation with acetonitrile was utilized for plasma sample preparation prior to analysis. Baseline chromatographic separation was achieved on a ZORBAX Eclipse XDB-C8 using gradient elution mode. The run time was 9 min at a constant flow rate of 0.4 ml/min. The mass spectrometer was operated under a positive electrospray ionization condition. Excellent linearity (r(2) ≥ 0.9997) was achieved for raltegravir and raltegravir glucuronide in the range of 2-2000 nmol/l. The average recovery of raltegravir and raltegravir glucuronide was 105.8% and 102.2%, respectively. The precision (coefficient of variation) was 1.6-6.6% for raltegravir and 2.1-6.9 for raltegravir glucuronide, respectively. The accuracy was 98.6-106.1% for raltegravir and 96.3-100.3% for raltegravir glucuronide. The plasma samples were tested to be stable after nine freeze-thaw cycles and exposure to room temperature for 24 h. This well-validated assay was applied for the quantification of raltegravir and raltegravir glucuronide in plasma samples within 24 h after a single oral dose of 400 mg raltegravir in six healthy subjects.