Development and validation of a UPLC-PDA method for quantifying ceftazidime in dried blood spots

Abstract

Bacterial infection is a leading cause of neonatal death. Ceftazidime, commonly used for neonatal infections, is often used off-label. Blood sampling limits pharmacokinetic (PK) studies in neonatal patients. The dried blood spots (DBS) are a potential matrix for microsampling. Herein, we describe an ultra-performance liquid chromatography with a photodiode array (UPLC-PDA) to determine ceftazidime in DBS from neonatal patients in support of pharmacokinetic studies. The Capitainer® device-based DBS samples containing 10 µL blood were extracted in 70% methanol/water (v/v) with acetaminophen as the internal standard (IS). The extraction process was carried out at 20 °C using a block bath shaker at 1000 rpm for 30 min. The extracted ceftazidime was subsequently eluted through an Acquity UPLC HSS T3 column (2.1 × 50 mm, 1.8 µm). Elution was achieved using a water (containing 0.1% trifluoroacetic acid)/acetonitrile linear gradient at a flow rate of 0.5 mL/min, and the analytical time was 3.2 min. The PDA detection wavelength was set at 259 nm. The method underwent thorough validation following the recommendation of the European Bioanalysis Forum (EBF) and the bioanalytical guideline established by the European Medicines Agency (EMA). No interfering peaks were detected at the retention times of ceftazidime and IS. The ceftazidime exhibited a quantification range spanning from 0.5 to 200 µg/mL, and the assay demonstrated good accuracy (intra/inter-assay ranging from 90.1% to 104.8%) and precision (intra/inter-assay coefficient of variations ranging from 4.8% to 11.7%). The method's applicability was demonstrated by analyzing clinical DBS samples collected from neonatal patients.

Keywords: Ceftazidime; Children; Dried blood spots; Method validation; UPLC.