Validation and Application of a Dried Blood Spot Amoxicillin Assay

Dried blood spot (DBS) antibiotic assays can facilitate pharmacokinetic (PK) investigations in situations where venous blood sampling is logistically and/or ethically challenging. The aim of this study was to establish, validate and demonstrate the application of a DBS amoxicillin assay for PK studies in vulnerable populations. The matrix effect, process efficiency (84–104%) and recovery (85–110%) of the liquid chromatography–mass spectrometry (LC–MS/MS) assay for amoxicillin in DBS was determined at 1, 10 and 100 µg/mL, and three different haematocrits. Thermal stability studies of amoxicillin in DBS were performed and a bridging study comprising 26 paired plasma and DBS samples was conducted in four healthy individuals. The limits of detection and quantification were 0.02 and 0.05 µg/mL for plasma and DBS amoxicillin assays, respectively. Accuracy and interday precision of amoxicillin in DBS (0.1–100 µg/mL) were 88–103% and 4.5–9.2%, respectively. At room temperature (22 °C) and 4 °C, amoxicillin was stable in DBS for ≈4 and 26 h, respectively. There was no degradation of amoxicillin in DBS at −20 °C for > 6 months. When comparing DBS and plasma collected from healthy volunteers, the slope of the Deming regression was 0.74. Amoxicillin CL/F estimates from DBS and plasma concentration data were 40.8 and 30.7 L/h/70 kg, respectively; V/F was 43.2 and 37.4 L/70 kg, respectively. In conclusion, amoxicillin can be reliably assayed from DBS in research studies but may have limited application in therapeutic drug monitoring. Due to poor stability at room temperature, amoxicillin DBS samples should be promptly dried and placed in frozen storage.