Development and Validation of an HPLC-MS/MS Method for the Quantitative Determination of Etmaben in Human Blood Plasma

• Published in: Razrabotka i registraciâ lekarstvennyh sredstv (Online) Vol. 13; no. 1; pp. 257 - 271
• Main Authors: Karnakova, P. K., Komarov, T. N., Archakova, O. A., Ivkin, D. Yu, Vetrova, E. S., Terninko, I. I., Shohin, I. E., Narkevich, I. A.
• Format: Journal Article
• Language: English; Russian
• Published: LLC Center of Pharmaceutical Analytics (LLC «CPHA») 01-03-2024
• Subjects: blood plasma; development; etmaben; hplc-ms/ms; pharmacokinetics; quantitative determination; validation
• ISSN: 2305-2066; 2658-5049
• DOI: 10.33380/2305-2066-2024-13-1-1752

Introduction. Etmaben is a promising drug for the treatment of cardiovascular diseases, widely studied in preclinical studies. In order to conduct phase I clinical trials, it is necessary to develop a bioanalytical method for the quantitative determination of etmaben in human blood plasma.
Aim. The aim of the study is to develop and validate a method for the quantitative determination of etmaben in human blood plasma using high-performance liquid chromatography with tandem mass spectrometric detection (HPLC-MS/MS) for the pharmacokinetic study.
Materials and methods. The determination of etmaben in human blood plasma was carried out on a Nexera XR chromatograph with a mass-selective detector LCMS-8040 (Shimadzu Corporation, Japan). Sample preparation: precipitation with acetonitrile. Internal standard: promethazine. Column: Luna C18, 100 Å, 50 × 2.00 mm, 5 µm. Elution in gradient mode at a flow rate of 1.00 mL/min. Mobile phase: 0.1 % formic acid solution in water (eluent A), 0.1 % formic acid solution in acetonitrile (eluent B). Retention time for etmaben and promethazine is approximately 1.18 min and 1.15 min, respectively. Total chromatogram registration time: 4.00 min. Ionization method and mode: electrospray; negative mode for etmaben, positive mode for promethazine. Detection was carried out in the mode of multiple reaction monitoring (MRM): 249.90 → 92.15 m/z; 249.90 → 160.20 m/z (etmaben); 284.95 → 197.95 m/z (promethazine).
Results and discussion. We have developed, for the first time, a method for determining etmaben and performed its full and partial validation according to current regulatory requirements.
Conclusion. The method for determining etmaben in human blood plasma with a confirmed analytical range of 0.250–30.000 µg/mL has been developed and validated. The confirmed analytical range of the method based on the results of the partial validation was 0.040–35.000 µg/mL. The method was successfully applied in phase I clinical trials and can be used for other pharmacokinetic studies of etmaben.