Application of HPLC in Quality Analysis of Hydroxychloroquine Sulfate

Significance of the Topic

Hydroxychloroquine sulfate is a critical pharmaceutical agent with antimalarial, immunomodulatory, and emerging antiviral applications, notably in COVID-19 research. Ensuring accurate quantification and impurity profiling is essential to maintain safety and efficacy in clinical use and regulatory compliance.

Objectives and Overview of the Study

This work aims to develop and validate chromatographic methods for hydroxychloroquine sulfate and its related substances according to European Pharmacopoeia (EP) 10.0, and to determine tablet assay content following United States Pharmacopeia (USP) 43. The goal is to achieve robust separation, reliable quantitation, and full compliance with pharmacopeial standards.

Methodology

• EP Assay (UHPLC): Gradient elution on a 1.7 µm C18 column (50 × 2.1 mm), mobile phases methanol/buffer (10:90 to 85:15, v/v), flow rate 0.7 mL/min, column at 40 °C, injector at 6 °C, injection volume 4 µL.
• USP Assay (HPLC): Isocratic elution on a 5 µm C18 column (250 × 4.6 mm) using water/methanol/acetonitrile/phosphoric acid with 96 mg sodium 1-pentanesulfonate, flow rate 1.0 mL/min, column at 40 °C, injector at 6 °C, injection volume 20 µL.
• Sample Preparation: Procedures as specified in EP 10.0 and USP 43 monographs.

Used Instrumentation

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

Main Results and Discussion

The EP method provided resolution values above 3.0 between hydroxychloroquine and Impurity C, and between Impurities B and C, meeting EP criteria. In the USP assay, hydroxychloroquine and chloroquine phosphate were separated with a resolution of 3.728 (>1.8). Reproducibility testing (n=5) yielded RSDs of 0.06% for retention time and 0.03% for peak area, well below the 1.5% USP limit. Analysis of 20 tablet units produced an average content of 99.99% of the label claim (RSD 0.04%), within the USP range of 93–107%.

Benefits and Practical Applications

• Delivers validated, pharmacopeial-compliant methods for routine quality control and impurity monitoring.
• Supports manufacturing QC, stability studies, and batch release operations.
• Enhances confidence in clinical trial supplies and regulatory submissions.
• Adaptable to GMP laboratories and high-throughput environments.

Future Trends and Potential Uses

Advancements in UHPLC, coupling with mass spectrometry, and the adoption of green solvent systems will further improve sensitivity and environmental sustainability. Automation, real-time process analytical technology (PAT), and data-driven impurity profiling using machine learning will streamline workflows and enhance quality assurance.

Conclusion

The established UHPLC and HPLC methods fully satisfy EP 10.0 and USP 43 requirements for separation, reproducibility, and assay of hydroxychloroquine sulfate in tablet forms. These protocols provide reliable and efficient tools for pharmaceutical quality control and regulatory compliance.

Reference

• European Pharmacopoeia 10.0: Monograph for Hydroxychloroquine Sulfate and Related Substances.
• United States Pharmacopeia 43: Monograph for Hydroxychloroquine Sulfate Tablet Assay.
• Shimadzu Application Note No. LC-199: HPLC Analysis of Hydroxychloroquine Sulfate.