Quantifying diclofenac – proven in theory, problematic in practice

AIM: During clinical sample analysis unforeseen problems arose using a previously validated assay for the quantification of diclofenac in the concentration range from 0.0500 to 50.0 ng/mL. Here, we describe the troubleshooting process leading to the identification of the root cause and resulting changes in assay development strategy for robust supported liquid extraction (SLE)-based assays for clinical application. METHODS: Individual steps of the sample preparation workflow and subsequent analysis with liquid chromatography and mass spectrometry (LCMS) were examined. Troubleshooting results were tested using stored validation samples and pooled clinical samples for confirmation. RESULTS: The root cause for altered assay performance could be identified as the SLE plate batch effect attributable to variability in the sorbent material (diatomaceous earth). Reducing the sample loading volume applied to the SLE plates yielded purer extracts and consistent chromatographic performance. Quantitative recovery and signal consistency were restored after the modification. CONCLUSION: Assay robustness was improved by underloading SLE plates. After adjusting the method and re-validation, clinical samples were successfully analyzed. The internal method development strategy was adjusted to avoid full capacity loading of SLE plate with natural sorbents.