LC-MS/MS Method for Determination of Hydroxychloroquine and Metabolites: Application in a Pharmacokinetic Study

Hydroxychloroquine (HCQ) was originally used as an antimalarial and immunomodulation drug. We developed and validated a simple and sensitive ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for simultaneous quantitation of HCQ and its three metabolites in rat blood, and reported their pharmacokinetic parameters. The chromatographic separation and detection of analytes were achieved within 4 min on ZORBAX SB-C₈ (3.5 μm, 2.1 × 150 mm) column with gradient elution, and the flow rate was 0.25 mL/min. Simple protein precipitation was successfully applied for sample pretreatment. The HCQ displays a good linearity in the range of 2.0-5000.0 ng/mL, and the three metabolites also show good linearity ranging from 1.0 to 2500.0 ng/mL, with all correlation coefficients (R ²) better than 0.98. In conclusion, this rapid, sensitive method was successfully developed, validated, and then applied to a pharmacokinetic study of HCQ in rat model in high dose. The results of the pharmacokinetic study presented an average half-life time 21.14 ± 10.31 h (mean ± SD) of HCQ, which is much shorter in human compared to that in mice. For the three metabolites, longer half-life times (approximately 100 h) were shown in rat.