Development and Validation of QbD-Driven Bioanalytical LC-MS/MS Method for the Quantification of Paracetamol and Diclofenac in Human Plasma

Abstract

The present work describes Quality by Design (QbD)-based development and validation of a LC-MS/MS method for estimation of paracetamol and diclofenac in human plasma using gabapentin as an internal standard (IS). Sample preparation and extraction was carried out by protein precipitation method. The processed samples were subjected to chromatographic analysis. The calibration range was observed between concentrations 205-13060 ng/ml for paracetamol and 40-2555 ng/ml for diclofenac. The developed method was optimized by Box-Behnken design for critical factors like mobile phase composition, flow rate and pH, thus evaluating design for peak area and retention time as the responses. The experimental design approach indicated significant decrease in method variability due to the influential method variables during optimization along with enhanced method robustness. Validation studies revealed that developed method fulfilled all the criteria like linearity, accuracy, precision, selectivity, sensitivity and stability for estimation of paracetamol and diclofenac in human plasma. Moreover, the stability studies performed in human plasma also showed no change in chemical nature of the drug. Overall, the studies indicated that the developed method is simple, robust and economical for its routine applicability utility in pharmacokinetic and bioequivalence studies.

Keywords:

• Quality by design
• method validation
• box-behnken design
• mass spectrometry
• robustness optimization