Application of HPLC in Quality Analysis of Hydroxychloroquine Sulfate

Importance of the Topic

Ensuring the purity and correct dosage of hydroxychloroquine sulfate is crucial for its safe use in malaria, autoimmune diseases and COVID-19 clinical trials. High-performance liquid chromatography (HPLC) remains a key technique for quality control of pharmaceuticals, providing accurate quantitation of active ingredients and the detection of related impurities.

Study Objectives and Overview

The study developed and validated two chromatographic methods for hydroxychloroquine sulfate analysis. The first follows the European Pharmacopoeia (EP10.0) for the separation and quantification of hydroxychloroquine and its related substances. The second adheres to the United States Pharmacopeia (USP43) for assay of tablet formulations to verify dosage compliance.

Methodology and Instrumentation

• EP method (UHPLC): C18 column (50×2.1 mm, 1.7 µm); gradient elution with methanol/buffer mixtures; flow rate 0.7 mL/min; injection 4 µL; column temperature 40 °C; autosampler at 6 °C.
• USP method (HPLC): C18 column (250×4.6 mm, 5 µm); isocratic mobile phase water/methanol/acetonitrile/phosphoric acid with sodium 1-pentanesulfonate; flow rate 1.0 mL/min; injection 20 µL; column 40 °C; autosampler 6 °C.

Used Instrumentation

• Shimadzu UHPLC system LC-30A
• Shimadzu HPLC system LC-2040C 3D
• LabSolutions DB ver 6.87 chromatography workstation

Main Results and Discussion

EP assay achieved baseline separation with resolution values exceeding 3.0 for hydroxychloroquine sulfate versus Impurity C and for Impurity B versus Impurity C. Total impurity levels remained below pharmacopeial limits.
USP assay demonstrated a resolution of 3.728 between hydroxychloroquine sulfate and chloroquine phosphate, surpassing the USP requirement of >1.8. Method reproducibility was excellent, with peak area RSD of 0.03% (n=5). Analysis of commercial tablets yielded 99.99% of the labeled content, meeting the USP criteria of 93–107%.

Benefits and Practical Applications

These validated methods provide reliable quality control tools for pharmaceutical manufacturers and analytical laboratories. They support regulatory compliance, ensure batch-to-batch consistency, and underpin clinical trial safety by confirming drug purity and dosage.

Future Trends and Applications

Advancements may include coupling these methods with mass spectrometry for trace-level impurity profiling, automation for high-throughput screening, and integration into process analytical technology (PAT) frameworks. Adaptation to bioanalytical matrices could expand monitoring in pharmacokinetic studies.

Conclusion

The EP10.0 UHPLC and USP43 HPLC methods demonstrated robust separation, high resolution of impurities, and repeatable quantitation of hydroxychloroquine sulfate. Both assays fulfill current pharmacopeial standards and are suited for routine quality control of tablets.

Reference

• European Pharmacopoeia, 10.0
• United States Pharmacopeia, 43