Determination of Hydroxychloroquine Concentration in Human Plasma by LC-MS/MS Method

Significance of the topic

The quantification of hydroxychloroquine (HCQ) in human plasma is critical for both therapeutic drug monitoring and pharmacokinetic research, particularly given its widespread use in malaria, autoimmune diseases, and investigational application in COVID-19.

Objectives and overview

This study aimed to establish a rapid, accurate, and robust UHPLC-MS/MS method using the Shimadzu Nexera with LCMS-8050 system for quantifying HCQ in human plasma over a wide concentration range.

Used Instrumentation

• Shimadzu Nexera UHPLC system with LC-30AD pumps, DGU-20A5 degasser, SIL-30AC autosampler, CTO-20AC column oven, CBM-20A controller
• Shimadzu LCMS-8050 triple quadrupole mass spectrometer
• LabSolutions Ver. 5.97 software

Methodology and instrumentation

Plasma samples underwent protein precipitation with acetonitrile. Chromatographic separation used a Shim-pack GIST C18-AQ column (100×2.1 mm, 1.9 μm) under isocratic conditions (20% organic mobile phase B containing methanol/acetonitrile with 0.1% formic acid and 50 mM ammonium acetate in water) at 0.5 mL/min and 40 °C. The mass spectrometer operated in positive ESI mode with MRM transitions (m/z 336.20→247.10 and 336.20→158.15 for HCQ; m/z 320.20→247.10 for internal standard chloroquine).

Main results and discussion

The method demonstrated high specificity with no endogenous interference. Linearity was established from 0.5 to 500 ng/mL (r>0.9988), with a lower limit of quantification of 0.5 ng/mL. Matrix effects ranged from 98.31% to 108.17%. Intra-day precision (RSD) ranged from 1.57% to 8.33%, and accuracy from 97.91% to 106.02%. HCQ remained stable in the autosampler for at least 10 hours at 6 °C.

Benefits and practical applications

This method offers simplicity, speed (10-minute run time), high sensitivity, and reproducibility, making it suitable for clinical pharmacokinetic studies, therapeutic monitoring, and supporting drug development.

Future trends and applications

Future developments could include integration with automated sample preparation for higher throughput, extension to metabolites or multi-analyte panels, and application to other biological matrices to broaden pharmacological investigations.

Conclusion

The validated UHPLC-MS/MS assay on the Shimadzu LCMS-8050 system provides a reliable and efficient approach for the quantification of hydroxychloroquine in human plasma across a broad concentration range.

References

No specific references were provided in the original document.