MSACL 2026 Abstract

Bridging Bioanalysis and Clinical Safety: LC–MS/MS Quantitation of Hydroxychloroquine (HCQ) and Its Metabolites in Whole Blood and Serum

Faraz Rashid and Carmen Gherasim
Department of Pathology and Clinical Laboratories , Michigan Medicine, University of Michigan

Faraz Rashid, Ph.D (Presenter) University of Michigan

Presenter Bio: I am currently working as an Assay Development Scientist and Clinical Technologist Senior at Michigan Medicine, University of Michigan, where I lead the development, validation, and implementation of high-throughput mass spectrometry–based assays (LC-MS and ICP-MS) for clinical toxicology and biochemical genetics in compliance with CLIA and CAP standards. I hold a Ph.D. in Biochemistry from Jamia Hamdard University, New Delhi, India.

I bring over 18 years of combined academic, clinical, and industry experience in mass spectrometry, metabolomics, proteomics, and biomarker discovery. Prior to my current role, I established and managed a metabolomics core facility at Henry Ford Health, where my work integrated metabolomics, immunometabolism, and bioenergetics to investigate neuroinflammatory and oncologic diseases. Earlier in my career, I spent more than a decade in industry at SCIEX, Waters, and Agilent, serving as an omics subject matter expert supporting global biopharma and translational research programs.

I have authored 40+ peer-reviewed publications in high-impact journals, including PNAS, iScience, Molecular Neurobiology, and Scientific Reports, and have served as Principal Investigator on funded research projects. I am widely recognized for my expertise in clinical assay development, high-resolution mass spectrometry, and translational multi-omics research.

Relevant Financial Disclosures (within past 24 months, reported on Apr 21, 2026) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

INTRODUCTION:
Hydroxychloroquine (HCQ) is commonly prescribed for the treatment of Rheumatoid Arthritis (RA), Systemic Lupus Erythematosus (SLE), and other inflammatory rheumatic diseases.

HCQ is metabolized in the liver to form desethylhydroxychloroquine (DHCQ), desethylchloroquine (DCQ), and bisdesethylchloroquine (BDCQ) metabolites. Long-term use of HCQ has been linked to irreversible retinal toxicity and cardiotoxicity by a yet unknown mechanism. Therefore, therapeutic drug monitoring (TDM) of HCQ and its metabolites is necessary to address the tissue associated toxicity. In this study, we developed a rapid and accurate LC-MS/MS method for quantification of hydroxychloroquine and its metabolites in serum and whole blood and assessed the suitability of both sample types for clinical monitoring. This method provides valuable insights into potential long-term toxicity associated with the cumulative HCQ dosage and treatment duration.

OBJECTIVES:
To develop a rapid, LC–MS/MS method suitable to multiple biological matrices for the precise simultaneous quantification of hydroxychloroquine and its metabolites to support clinical toxicology monitoring.

METHODS:
A single and rapid ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for simultaneous quantitation of HCQ and its three metabolites in human blood and serum was developed. Method demonstrates high selectivity enabling complete separation of HCQ and metabolites within 1.5 minutes under isocratic conditions, making it suitable for high-throughput analysis. Separation enables detection of all four analytes using Kinetex (2.6 µm, 2.1 × 100mm) column at the flow rate of 0.45 mL/min with column temperature at 550C. Simple protein precipitation was successfully applied for sample preparation of both blood and serum matrices. HCQ exhibited linearity over the concentration range of 8.0–2000.0 ng/mL, while the three metabolites showed linearity from 4.0 to 2000.0 ng/mL. Regression and statistical analysis yielded a Pearson correlation of 0.995, indicating excellent correlation across the concentration range of all metabolites.

RESULTS:
The method was applied to the simultaneous quantification of hydroxychloroquine (HCQ) and its metabolites in clinical whole blood and serum samples (n = 48) from patients receiving long-term HCQ therapy (2–10 years). Quantitation of HCQ demonstrated that its concentration in whole blood was on average 3.8-fold higher than that in serum with only BDCQ metabolite showing correlation between the levels in whole blood and serum. In our patient cohort, 10.4% of the patients were in therapeutic ranges in serum (TR: 400-600 ng/mL) and 20.8% in whole blood (TR: 750-1200 ng/mL) with the majority being sub-therapeutic 87.5% vs. 68.8% in serum and whole blood, respectively. Seven patient samples exhibited elevated BDCQ concentrations relative to HCQ in whole blood in contrast to serum. The average BDCQ concentration in serum samples was 18.8-fold lower than HCQ, whereas the same patient samples in whole blood showed a reduced ratio of 4.3-fold suggesting a potential association with cumulative exposure and long-term toxicity.

CONCLUSION:
Monitoring of HCQ and its metabolites in whole blood, particularly BDCQ, may substantially improve identification of patients at risk for cumulative toxicity, which may be missed with serum based or parent-drug-only monitoring of HCQ.

REFERENCES:
1.Marmor, M. F. et al. Ophthalmology 2011;118:415–422.
2.Yusuf IH, Sharma S, Luqmani R, Downes SM. Hydroxychloroquine retinopathy. Eye (Lond). 2017 Jun;31(6):828-845.
3.Yusuf IH, Charbel Issa P and Ahn SJ (2023) Hydroxychloroquine-induced Retinal Toxicity. Front. Pharmacol. 14:1196783.