DEVELOPMENT AND VALIDATION OF AN LC-MS/MS METHOD FOR SIMULTANEOUS DETERMINATION OF METFORMIN AND SEMAGLUTIDE IN HUMAN PLASMA

JAGAPATHI RAJU VATSAVAYI; NALANDA BABY REVU

doi:10.22159/ajpcr.2025v18i9.55110

Authors

JAGAPATHI RAJU VATSAVAYI Department of Pharmaceutical Analysis, GITAM School of Pharmacy, GITAM (Deemed to be University), Visakhapatnam, Andhra Pradesh, India. https://orcid.org/0009-0003-0747-1948
NALANDA BABY REVU Department of Pharmaceutical Analysis, GITAM School of Pharmacy, GITAM (Deemed to be University), Visakhapatnam, Andhra Pradesh, India. https://orcid.org/0000-0002-9314-2459

DOI:

https://doi.org/10.22159/ajpcr.2025v18i9.55110

Keywords:

Metformin, semaglutide, Electro spray ionization, Method validation, mass spectrometer

Abstract

Objective: To develop a method capable of simultaneous quantification of metformin and semaglutide studied as a potential combination therapy for treating diabetes.

Methods: A refined protein precipitation extraction technique was used with warfarin as an internal standard for metformin and semaglutide. The two compounds were separated on a Kinetex Polar C18 (50 mm × 2.1 mm, 5 μ particle size) column, with a positive polarity electro spray ionization on a Liquid chromatography with Tandem Mass Spectrometry instrument. The estimation was done through a multiple reaction monitoring method and a gradient program utilizing acetonitrile and 0.1% formic acid in water as mobile phases to achieve a separation in 2.2 min.

Results: The established method performed linearly over a working range of 10.0–10,000 ng/mL for metformin (r2>0.98) and 1.00–1000 ng/mL for semaglutide (r2>0.98) in human plasma. The specificity, selectivity, precision, accuracy, recovery, matrix effects, and stability were within acceptable limits as necessitated by the guideline on bioanalytical method validation, as mentioned in the International Council for Harmonisation of Technical Requirements of Pharmaceuticals for Human Use M10.

Conclusion: This highly selective and sensitive method, in which 10.0 ng/mL was employed for metformin and 1.00 ng/mL was employed for semaglutide as the Lower Limit of Quantification (LLOQ), can be utilized for estimation in human plasma and will facilitate further application to bioequivalence and population pharmacokinetics studies. This method has the advantage of a lower LLOQ over other existing methods.

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