LC-MS/MS Method Development and Validation for Clinical Pharmacokinetics and Therapeutic Drug Monitoring of Potassium-Competitive Acid Blocker Vonoprazan-Based Triple Therapy for H. pylori in Human Plasma

<b>Background:</b> A novel triple therapy regimen for <i>Helicobacter pylori</i> eradication, recently approved by the U.S. FDA, comprises vonoprazan (VPN), a potassium-competitive acid blocker, in combination with amoxicillin (AMX) and clarithromycin (CMN). This study presents the development and full validation of a rapid, selective, and sensitive LC-MS/MS method for the simultaneous quantification of these three drugs in spiked human plasma. <b>Methods:</b> Sample preparation was performed using a simple and efficient liquid-liquid extraction (LLE) technique. Chromatographic separation was achieved within 5 min using a Phenomenex Kinetex C18 column (100 × 4.6 mm, 2.6 µm) and a gradient elution system consisting of 0.1% formic acid in water and acetonitrile. Moreover, diazepam was used as an internal standard. The mass spectrometric detection was conducted in multiple reaction monitoring (MRM) mode using positive electrospray ionization. <b>Results:</b> The method exhibited excellent linearity over the investigated concentration ranges (2, 5, 10, 20, 50, and 100 ng/mL for amoxicillin and clarithromycin and 5, 10, 20, 30, 50, and 100 ng/mL for vonoprazan). Intra- and inter-day precision and accuracy values met FDA bioanalytical method validation guidelines, with relative standard deviations and relative errors below 15%. Mean absolute recoveries were above 93% for all analytes. <b>Conclusions:</b> The developed method was fully validated, rapid, selective, and sensitive LC-MS/MS and was assessed using the AGREE tool as a greenness assessment approach, confirming its environmental friendliness and alignment with green analytical chemistry principles.