(W1230-06-39) Simultaneous LC-MS/MS Method for the Quantitation of Azithromycin, Hydroxychloroquine and its Metabolites In SARS-CoV-2(-/+) Populations Using Dried Blood Spots

Purpose: In the context of the SARS-CoV-2 pandemic, there is a pressing need for rigorous clinical trials on hydroxychloroquine (HCQ), HCQ major metabolites (Desethylchloroquine (DCQ), Bisdesethylchloroquine (BDCQ) and Monodesethylhydroxychloroquine (DHCQ)), and azithromycin (AZI) as potential treatments for COVID-19. To facilitate at-home biospecimen collection by study participants and reduce the risk of SARS-CoV-2 exposure for both participants and clinical staff, we developed and validated a sensitive LC-MS/MS method for the simultaneous quantitation of HCQ, its metabolites, and AZI from dried blood spots (DBS). This approach aligns with COVID-19 home quarantine guidelines issued in numerous US jurisdictions during the first year of the pandemic. The goal of this study is to enable the reliable and accurate measurement of drug concentrations in DBS samples collected by study participants, which will support therapeutic drug monitoring of HCQ, its metabolites, and AZI in the context of the COVID-19 pandemic.

Methods: A Shimadzu Scientific LC-MS/MS 8060 system was used for method development and validation. The chromatographic separation of all analytes were achieved on a reversed phase AQUASIL C18 (5 μm, 50 x 4.6 mm) protected with an AQUASIL C18 guard column. Mobile phase was 0.2 % v/v formic acid in water and 0.1% formic acid in methanol at a gradient elution with flow rate of 0.5 mL/min. Whatman 903 filter paper was used for blood sample collection, preparation of calibration standards and quality control samples. Phree 96 well plate protein precipitation extraction was utilized for analytes extraction. HCQ-d4, BDCQ-d4, and AZI-d3 are used as Internal Standards (IS) due to similar analyte chromatographic behaviour. Hematocrit (Hct) effect was evaluated over a range of Hct values (0.2, 0.4, and 0.6) for both Lower Quality Control (LOQ) and High Quality Control (HQC). The blood:plasma (Kb/p) ratio was determined at a concentrations of 1 µM for all analyte , after 0, 30- and 60-min incubations at 37 °C.

Results: The developed LC-MS/MS method was fully validated in accordance with the 2018 Food and Drug Administration (FDA) guidelines and regulation for Bioanalytical Method Validation. Analytes were separated and quantitated using MS/MS with an positive electrospray ionization (ESI+) source in multiple reaction monitoring (MRM) mode of AZI, HCQ, DCQ, BDCQ, DHCQ, HCQ-d4, BDCQ-d4, and AZI-d3 with the following precursor ion > product ion combination of m/z 749.50 >591.45, 336.10 >247.2, 292.1 0 >114.2, 292.30 >114.45, 264.0 >179.00, 308.15 >179.0, 340.1 >251.20, 268.10 >179.0 and 754.50 >596.45 , respectively. The MS/MS response was linear over the concentration range of 1–2000 ng/mL for all analytes with a correlation coefficient (R2) of 0.98 or better and high on column sensitivity range of 0.11 to 217.39 pg for all analytes. Carryover effect was negligible with no co-eluting analyte peak ( < 20% of the LLOQ) or IS peak ( >5% of the IS) in any samples injected after the HQC samples. The intra- and inter-day precision and accuracy were within the acceptable limits per FDA guidelines. All samples utilized two (3mm each) DBS punches, which is equivalent to ~10µL of blood, for sample preparation. Absolute extraction recovery (AER) and matrix effect (ME) showed reproducible and efficient sample extraction method with AER >88% and ME >88%. All analytes and metabolites were found to be stable and within ± 15% of the actual concentration at bench-top (20 ℃, 50 days), auto-sampler (4 ℃, 36 hrs) and long-term neat stock (–80 ± 5 ℃, 8 months). Hct effect were negligible with the detection accuracy across all concentration levels within an acceptable ±15% limits. Kb/p ratio indicate high haematocrits partition compared to the plasma for all analytes. The validated method was successfully applied to the quantification of AZI, HCQ and its metabolites concentrations for more than 2000 DBS samples as part of two randomized multi-center clinical trials (NCT04328961 and NCT04354428). The DBS sample concentrations for AZI and HCQ ranged from 1.1-141.1 ng/mL and 0.8-24941.1 ng/mL, respectively. The developed method was utilized to evaluate variability in drug concentrations following prescribed fixed dosing and build pharmacokinetic models to help understand the drug ADME properties in humans.

Conclusion: We developed and validated the first robust, sensitive, and reproducible LC-MS/MS method for quantitating AZI, HCQ, and its metabolites (DCQ, BDCQ, and DHCQ) in human blood utilizing a DBS sample collection technique. The DBS sampling method was advantageous with simple sample collection, a limited amount of blood sample required, easy transfer and storage conditions, and exhibited excellent drug stability. The DBS extraction method was both reproducible and efficient, resulting in high analyte extraction yields, while using significantly less plastic and organic solvents compared to traditional methods, making it an environmentally friendly option. The method showed superior selectivity and sensitivity, with nominal concentrations as low as 1 ng/mL. The presented LC-MS/MS method provides a valuable tool in evaluating the pharmacokinetic/pharmacodynamic properties of AZI, HCQ, and its metabolites as a single agent or in combination with other drugs.

Acknowledgements: We would like to acknowledge the valuable discussion and scientific input from Dr. Mark Wener. The work was supported in part by the Fred & Pamela Buffett Cancer Center Support Grant from the National Cancer Institute under award number P30 CA036727, grants INV-017062 and INV-01620 from the Bill and Melinda Gates Foundation and an Award from the University of Washington (UWSC11894 and UWSC12049). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.