Development of a capillary electrophoresis–mass spectrometry method for the analysis of metformin and its transformation product guanylurea in biota

Abstract

A method with capillary electrophoresis coupled to mass spectrometry was optimized to determine the uptake of metformin and its metabolite guanylurea by zebrafish (Danio rerio) embryos and brown trout (Salmo trutta f. fario) exposed under laboratory conditions. Metformin was extracted from fish tissues by sonication in methanol, resulting in an absolute recovery of almost 90%. For the extraction of guanylurea from brown trout, solid-phase extraction was implemented with a recovery of 84%. The use of a mixture of methanol and glacial acetic acid as a non-aqueous background electrolyte was vital to achieve robust analysis using a bare fused-silica capillary with an applied voltage of +30 kV. Problems with adsorption associated with an aqueous background electrolyte were eliminated using a non-aqueous background electrolyte made of methanol/acetic acid (97:3) with 25 mM ammonium acetate (for zebrafish embryos) or 100 mM ammonium acetate (for brown trouts), depending on the sample complexity and matrix influences. High resolution and high separation selectivity from matrix components were achieved by optimization of the ammonium acetate concentration in the background electrolyte. An extensive evaluation of matrix effects was conducted with regard to the complex matrices present in the fish samples. They required adapting the background electrolyte to higher concentrations. Applying this method to extracts of zebrafish embryos and brown trout tissue samples, limits of detection for both metformin and guanylurea in zebrafish embryos (12.2 μg/l and 15 μg/l) and brown trout tissues (15 ng/g and 34 ng/g) were in the low μg/l or ng/g range. Finally, metformin and guanylurea could be both quantified for the first time in biota samples from exposure experiments.

Effects of Metformin and its Metabolite Guanylurea on Fathead Minnow (Pimephales promelas) Reproduction

“…Although plasma concentrations were not directly measured in the present study, a low octanol/water partition coefficient (log K OW ; −1.4) and high dissociation constant (pK a ; 11.5) indicate that metformin would not be expected to bioaccumulate to any significant degree. In addition, prior research has shown metformin uptake in zebrafish embryos and larvae (Ussery et al, 2018) and in brown trout larvae with a maximal reported bioconcentration factor of 0.2 L/kg in whole larvae (Knoll et al, 2020). Thus, the exposure concentration used for the 96-h time course would produce plasma concentrations considerably lower than human therapeutic plasma concentrations, which average 10-20 µM (Hess et al, 2018;Owen et al, 2000).…”

Synthesis of Molecularly Imprinted Polymers for Selective Extraction Followed by Solid Phase Determination of Metformin in Pharmaceutical Preparation and in Human Serum

“…As a result, the process of recognition in MIPs can be characterized in resemblance to enzyme-proven mechanismssubstratum-Complex is formed in the (lock and key) model 7,8 . Certain MIP applications were prepared in SPE 10,11 .…”