Simultaneous Qualitative and Quantitative Analysis of Tyloxapol in Tobramycin Eye Drops by LC-Q-TOF and HPLC-ELSD

Abstract

This study employed a reverse engineering approach to evaluate and estimate the key excipients in the Reference Listed Drug (RLD), thereby supporting the development of cost-effective generic pharmaceutical products. By combining Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometer (LC-Q-TOF) with high-performance liquid chromatography–evaporative light scattering detection (HPLC–ELSD), both qualitative and quantitative analysis of tyloxapol in the formulation was achieved. Mass spectrometry analysis showed that the tyloxapol reference standard, tobramycin eye drops, and excipient tyloxapol 03 exhibited essentially consistent retention times,charge states, m/z ratios, and molecular weight distributions. The spectral characteristics of excipient tyloxapol 01 and excipient tyloxapol 02 were essentially consistent, as reflected in their retention times and three sets of mutually matched characteristic mass spectral peaks. Based on these qualitative results, a quantitative HPLC-ELSD method using a TSKgel G2000SWXL column was established for the content analysis of tyloxapol. Methodology validation demonstrated that the method exhibits a strong linear relationship within the concentration range of 243.9-731.7 µg/mL (R2=0.999), with favorable accuracy (recovery rate 99%-106%), precision (repeatability RSD ≤ 2.2%, intermediate precision RSD ≤ 1.6%), specificity, and solution stability. The combination of Q-TOF MS and HPLC-ELSD can be applied for the qualitative and quantitative analysis of excipients in pharmaceutical formulations, supporting the reverse engineering process in generic drug development.