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

Importance of the Topic

Hydroxychloroquine is widely used for malaria prevention and treatment and has been repurposed for COVID-19 therapy. Accurate quantification of its plasma levels is essential for optimizing dosing, ensuring efficacy and minimizing adverse effects in clinical practice.

Objectives and Study Overview

This study aimed to develop and validate a rapid, sensitive and robust UHPLC-MS/MS method for measuring hydroxychloroquine in human plasma. Key performance parameters including specificity, linearity, limit of quantification, precision, accuracy, matrix effects and stability were systematically evaluated.

Methodology and Instrumentation

Instrumentation Used:

• UHPLC system: Shimadzu Nexera with dual LC-30AD pumps and DGU-20A5 online degasser
• Autosampler: SIL-30AC
• Column oven: CTO-20AC
• System controller: CBM-20A
• Mass spectrometer: Shimadzu LCMS-8050 triple quadrupole

Analytical Conditions:

• Column: Shim-pack GIST C18-AQ, 100 × 2.1 mm, 1.9 µm
• Mobile phase: 0.1 % formic acid with 50 mM ammonium acetate in water (A) and methanol/acetonitrile 1:1 (B)
• Flow rate: 0.50 mL/min, isocratic at 20 % B, column temperature 40 °C
• ESI positive mode; heat block 400 °C; DL 250 °C; probe 300 °C; gas flows: nebulizer 3 L/min N₂, drying 10 L/min N₂, heating 10 L/min air
• MRM transitions: m/z 336.20 > 247.10 (quantifier), 336.20 > 158.15 (qualifier)

Sample Preparation:
Simple protein precipitation with acetonitrile, centrifugation, transfer of supernatant and 2 µL injection.

Main Results and Discussion

• Specificity: no interfering peaks observed in blank plasma at analyte retention times
• Linearity: 0.5–500 ng/mL with correlation coefficient > 0.9988
• Limit of quantification: 0.5 ng/mL
• Precision: intra-day RSD 1.57 %–8.33 %
• Accuracy: 97.91 %–106.02 %
• Matrix effects: 98.31 %–108.17 %
• Stability: HCQ stable in autosampler at 6 °C for 10 hours (RSD < 7.33 %, accuracy 94.72 %–98.01 %)

Benefits and Practical Applications

• Short analysis time (10 min) enables high throughput
• High sensitivity for trace-level detection
• Easy sample preparation reduces hands-on time
• Wide dynamic range covers therapeutic to toxic levels
• Suitable for therapeutic drug monitoring and pharmacokinetic studies

Future Trends and Potential Applications

• Extension to other antiviral and antimalarial compounds
• Automation and integration with robotic sample handling
• Application to alternative biological matrices (urine, tissues)
• Use in clinical trials and personalized medicine initiatives
• Environmental monitoring of pharmaceutical residues

Conclusion

A validated UHPLC-MS/MS method on the Shimadzu Nexera-LCMS-8050 platform delivers reliable, fast and robust quantification of hydroxychloroquine in human plasma, meeting rigorous criteria for specificity, sensitivity, precision and accuracy.

References

• Yang Le Determination of Hydroxychloroquine Concentration in Human Plasma by LC-MS/MS Method Shimadzu Application Note LCMSMS-453 May 2020