A rapid and reliable method for the quantitation of hydroxychloroquine in serum using turbulent flow liquid chromatography-tandem mass spectrometry

Background: Hydroxychloroquine is routinely used in managing systemic lupus erythematosus and rheumatoid arthritis. Whole blood levels are currently measured in the laboratory but at least one study suggests that serum levels may be equally useful. Moreover, serum samples are the preferred matrix type in the clinical laboratory as a result of their reduced complexity compared to whole blood. These observations suggest that the clinical utility of serum hydroxychloroquine levels needs to be reevaluated using larger studies and more robust assays. We report a turbulent flow LC-MS/MS method we developed for this purpose. Methods: After protein precipitation from serum with 0.33. mol/l perchloric acid, hydroxychloroquine and its deuterated analog were injected onto a Cyclone turbulent flow column for sample cleanup. Analytical separation was accomplished on a HypersilGold C8 column with a gradient of water and methanol, each containing 0.1% formic acid and 10. mmol/l ammonium formate. Analytes were ionized and detected by electrospray ionization mass spectrometry with multiple reaction monitoring. Results: Our method was linear from 15.7 to 2000. ng/ml. Total imprecision at multiple levels was < 5% and accuracy was within ± 15%. The method showed minimal carryover. Our extraction efficiency was 103% and the matrix factor was 101%. Comparison with a reference laboratory method identified constant bias but good correlation between the 2 methods. Conclusions: We present a novel turbulent flow liquid chromatography-tandem mass spectrometry method for quantification of hydroxychloroquine in serum. Our method has comparable sensitivity, selectivity, precision, accuracy, and linearity to previously reported methods. However, it offers simpler sample processing, shorter overall analysis time, and minimal carryover. These characteristics make our method well-suited for efficient analysis of the large number of samples necessary for studies on the clinical utility of serum HQ levels.