# Green DOE based RP-HPLC method for the simultaneous determination of Azelastine and Losartan in spiked human plasma samples

## TL;DR

A green HPLC method was developed to simultaneously determine Azelastine and Losartan in human plasma samples.

## Contribution

A novel green RP-HPLC method using DOE optimization for drug analysis in plasma is introduced.

## Key findings

- The method achieved rapid and accurate quantification of Azelastine and Losartan in spiked plasma.
- The developed approach was validated for linearity and precision according to ICH guidelines.
- Greenness assessment confirmed the environmentally friendly nature of the method.

## Abstract

A green RP-HPLC approach coupled with spectrofluorometric detection was established for the simultaneous estimation of the co-administered drugs Azelastine (AZL) and Losartan (LOS) in spiked human plasma samples. Preliminary trials were carried out for determination of the Critical Method Parameters (CMPs) and Critical Quality Attributes (CQAs). Design of Experiment (DOE) was developed relying on the use of Central Composite Design (CCD) for the optimization of conditions to establish a simple, rapid, cost-effective and environmentally benign approach. The chromatographic separation was based on using a mobile phase composed of methanol: acetonitrile: 0.02M phosphate buffer of pH 3.25 with 0.05% tri-ethylamine (60: 1.5: 38.5, v/v/v) at a flow rate of 1.2 mL/min and injection volume of 10µL. The fluorescence detection was carried out at 245 nm/400 nm and 290 nm/360 nm for estimation of LOS and AZL, respectively. The retention times of AZL and LOS were 8.9 ± 0.2 min and 13.4 ± 0.1 min, respectively. The developed method was validated according to ICH Guidelines with linear relationship in concentration ranges of 0.10–50.0µg/mL for AZL and 0.30–40.0µg/mL for LOS, while in spiked plasma samples the concentration ranges were 0.025–0.07µg/mL for AZL and 0.025–0.10 µg/mL for LOS. The developed approach was successfully applied for the quantitation of both drugs in spiked human plasma samples. Complex MoGAPI and AGREE methodologies were used for greenness assessment.

The online version contains supplementary material available at 10.1038/s41598-026-39426-2.