Development and Validation of a Chlorothiazide Whole-Blood Microsampling Assay

BACKGROUND: Chlorothiazide (CLTZ) is among the most commonly prescribed diuretics for preterm infants with bronchopulmonary dysplasia, yet no contemporary pharmacokinetic (PK) data inform its dosing in this population. Traditional plasma assays require blood volumes that are infeasible in neonates, limiting PK research. Volumetric absorptive microsampling (VAMS) enables fixed-volume whole-blood collection at minimal sampling burden. METHODS: We developed and validated a liquid chromatography-tandem mass spectrometry assay for quantification of CLTZ in 10-µL whole-blood samples collected with Mitra VAMS devices. Validation followed FDA bioanalytical guidelines and included accuracy, precision, selectivity, matrix effects, recovery, dilution integrity, hematocrit (HCT) impact, and stability. Clinical feasibility was assessed in infants receiving enteral CLTZ as part of routine care. RESULTS: The assay was linear from 10 to 10,000 ng/mL (R > 0.99). Accuracy and precision met acceptance criteria at all quality control levels, with bias within 2% and maximum coefficient of variation (CV) of 16% at the lower limit of quantification (LLOQ) (10 ng/mL). HCT values from 20% to 60% did not affect performance (bias ≤11%, CV ≤7%). Dilution integrity was preserved with 20-fold dilution, and interference from hydrochlorothiazide was below the LLOQ. CLTZ was stable on VAMS tips for 1 day at room temperature and refrigeration, 63 days at -20°C, 187 days at -80°C, and through 3 freeze-thaw cycles. In clinical samples from 5 infants, concentrations ranged from approximately 200 to 4800 ng/mL and declined as expected after dosing. CONCLUSIONS: This validated VAMS liquid chromatography-tandem mass spectrometry assay accurately quantifies CLTZ from very small blood volumes with minimal HCT sensitivity and robust stability, supporting rigorous PK and exposure-response studies in preterm and critically ill infants.