Method development and validation for rapid quantification of hydroxychloroquine in human blood using liquid chromatography-tandem mass spectrometry.

A novel and specific liquid chromatography-tandem mass spectrometric method (LC-MS/MS) was developed and validated for the quantification of hydroxychloroquine in human blood using its stable labeled isotope, hydroxychloroquine-d4 as the internal standard. Chromatographic separation of analytes was achieved using an Agilent ZORBAX Eclipse XDB - C8 analytical HPLC column (50 mm × 2.1 mm, 5 μm). The mobile phase comprising water containing 0.1% formic acid-acetonitrile (94:6, v/v) was delivered isocratically at a flow rate of 0.5 mL/min. The column effluent was detected by API 4000 triple quadrupole mass spectrometer using electrospray ionization (ESI) and monitored by multiple reaction monitoring with positive mode. The precursor to product ion transitions of m/z 336 → 247 and m/z 340 → 251 were used to measure the analyte and IS, respectively. The assay demonstrated a good linear dynamic range of 5-2000 ng/mL for hydroxychloroquine in human blood, with coefficient of determination (r(2)) of =0.9999. The values for intra-day and inter-day precisions of hydroxychloroquine were ≤ 7.86% with the accuracies ranged from 93.8% to 107.6%. The chromatographic run time was 3 min, making it possible to achieve a high throughput analysis. This method was used as a bio-analytical tool in a phase I clinical trial to quantify blood hydroxychloroquine concentrations in patients with non-small cell lung cancer receiving both hydroxychloroquine and gefitinib in their treatment.