A systematic stepwise optimization framework for rapid multi-analyte UPLC-MS/MS plasma analysis with integrated...

Area of Science:

• Analytical Chemistry
• Bioanalytical Chemistry
• Mass Spectrometry

Background:

• Bioanalytical method development for multiple analytes using Ultra-Performance Liquid Chromatography-tandem Mass Spectrometry (UPLC-MS/MS) faces recurring limitations.
• Current workflows can lack structure, reproducibility, and consideration for environmental impact.

Purpose of the Study:

• To propose a stepwise optimization guidance model for rapid multi-analyte UPLC-MS/MS method development.
• To establish a structured, reproducible, and ecologically sustainable analytical workflow for bioanalysis.

Main Methods:

• A six-stage model encompassing physicochemical profiling, extraction strategy, chromatographic and mobile phase optimization, method validation, and greenness evaluation.
• Demonstration using Rifaximin, Ciprofloxacin, and Fluconazole in human plasma with Ibuprofen as internal standard.
• Environmental applicability assessment using Analytical Greenness Score Assessment (AGSA) and Environmental Protection Performance Index (EPPI).

Main Results:

• Physicochemical profiling informed ionization mode and solubility predictions.
• Liquid-liquid extraction with dichloromethane and rapid isocratic separation (C18 column, methanol/water 95:5 v/v) were successfully implemented.
• Method validation yielded excellent linearity (r² ≥ 0.999).
• Greenness evaluations confirmed environmental applicability.

Conclusions:

• The proposed sequential framework provides a methodological reference for UPLC-MS/MS users.
• It facilitates structured and reproducible bioanalytical workflows.
• The model advances ecological sustainability in multi-analyte UPLC-MS/MS applications.