Analysis of Hydroxychloroquine and Metabolites in Human Serum and Plasma Using the Agilent Captiva EMR—Lipid by LC-QQQ

Importance of the Topic

A sensitive and specific method for quantifying hydroxychloroquine and its metabolites in human blood matrices is critical for clinical research, therapeutic drug monitoring, and pharmacokinetic studies. Hydroxychloroquine (HCQ) is widely used for malaria, autoimmune diseases, and has been evaluated in emerging clinical contexts. Accurate measurement in serum and plasma informs dosing, safety, and efficacy assessments.

Objectives and Study Overview

This work aimed to develop and validate a robust LC-MS/MS method for simultaneous quantitation of HCQ and its three primary metabolites (desethylchloroquine, desethylhydroxychloroquine, bisdesethylchloroquine) in 100 µL of human serum or plasma. The study extended previous whole blood assays to serum and plasma matrices, ensuring low sample volume, high throughput, and reliable performance for clinical research applications.

Methodology and Instrumentation

Sample Preparation:

• Protein precipitation of 100 µL sample with 95:5 acetonitrile/methanol containing 5.8 % NH₄OH for basic drug extraction.
• Lipid cleanup using Agilent Captiva EMR—Lipid 96-well plates under positive pressure to remove phospholipids and other matrix interferences.
• Post-elution wash with 80:20 acetonitrile/water containing 5.8 % NH₄OH, followed by vacuum drying.
• Reconstitution in 95:5 acetonitrile/water with 0.1 % formic acid prior to analysis.

Instrumentation:

• Liquid chromatography: Agilent ZORBAX Eclipse XDB-C8 column (2.1 × 50 mm, 3.5 µm) at 40 °C; gradient elution with water and acetonitrile (both 0.1 % FA) at 0.3 mL/min.
• Mass spectrometry: Agilent triple quadrupole LC/MS/MS; electrospray positive ion mode; MRM transitions optimized for each analyte and deuterated internal standard (HCQ-d4).

Key Results and Discussion

• Linearity: 0.4–100 ng/mL with R² > 0.99 for all analytes in serum and plasma.
• Sensitivity: LOQ at 0.4 ng/mL with signal-to-noise ≥ 10 and blank interference < 20 % of analyte signal.
• Recovery: Absolute recoveries between 60–120 % with CV < 15 % at both 10 and 100 ng/mL in serum and plasma.
• Matrix effect: > 50 % matrix effect observed, acceptable given consistent internal standard normalization.
• Accuracy and Precision: Spiked QC samples (1, 10, 100 ng/mL) yielded accuracy within 100 ± 15 % and precision CV < 15 % across all analytes and matrices.

Benefits and Practical Applications

This streamlined workflow reduces sample volume and processing time, minimizes analyte loss and operator error, and supports high-throughput clinical studies. The method enables reliable therapeutic drug monitoring, pharmacokinetic profiling, and safety evaluation of HCQ in diverse patient populations.

Future Trends and Opportunities

• Integration with automated platforms to further increase throughput and consistency.
• Expansion to additional metabolites or structurally related compounds.
• Application to dried blood spot or micro-sampling techniques for remote or pediatric studies.
• Adaptation to novel bioanalytical challenges in emerging infectious diseases or personalized medicine.

Conclusion

The validated LC-MS/MS assay with Captiva EMR—Lipid cleanup delivers a fast, robust, and sensitive solution for quantifying hydroxychloroquine and its metabolites in minimal volumes of human serum and plasma. Its performance meets stringent clinical research requirements and offers a reliable tool for drug monitoring and pharmacokinetic investigations.

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