Development and validation of a chromato-mass-spectrometric method for quantitative determination of apigenin, apigetrin and luteolin in mice plasma

Introduction. Currently, the practical interest of researchers is aimed at studying the biomedical potential of secondary plant metabolites in order to create new drugs. Flavonoids are a large class of natural phenolic compounds with a wide range of biological activity. Particular attention is paid to the ability of some flavones (apigenin and luteolin, their glycoside apigetrin) to suppress the growth of malignant tumors. However, low concentrations of these polyphenols in plant matrices do not allow reliable assessment of pharmacokinetics in the human body. In this regard, an urgent bioanalytical task is the development of accurate and reproducible methods using high-tech hybrid methods of analysis.

Purpose of the study. Development and validation of chromato-mass-spectrometric methodology of the quantitative determination of apigenin, apietrin andlLutheololin in the blood plasma of mice.

Material and methods. To develop the methodology of quantitatively determined flavones, a highly effective liquid chromatograph Agilent Technologies 1260 Infinity II and the mass spectrometer Ab Sciex Qtrap 3200 MD was used. The chromatographic separation was carried out on the analytical column Agilent Infinitylab Poroshell 120 EC-C18 with a gradient mobile phase containing 0.1 % of ant and acetonitril. For detecting, analytes and the internal standard warfarin were ionized by electrical exposure in the mode of negative ions using multiple reactions monitoring. The sampling of plasma of the blood of mice was precipitating with methanol.

Results. The optimal conditions of the convertible-phase chromatographic separation of apigenin, apigerin, lutheololin and varfarin with times of retention from 5,3 to 7,8 minutes were selected. Each compound in optimal conditions of electrical ionization is protoned with the formation of the composition of the composition [М–Н]^- and fragmentation on two characteristic ion-products. The methodology of simultaneous quantitative HPLC-MS/MS determination of flavones in the plasma of mice, characterized by a linearity in a wide range of concentrations and a low limit of quantitative determination of 1.0 ng/ml, values of the degree of extraction of analyts in the range of 90,8-100,4 %. The validation characteristics of the methodology satisfy the relevant regulatory requirements of selectivity, the influence of the matrix effect, intraulinary accuracy and precision.

Conclusion. The results of validation tests indicate the suitability of the developed express and rather accurate bioanalytic methodology of the quantitative determination of the three structurally related flavons in the joint presence of mice in the blood plasma. The metrological characteristics of the methodology allow you to use it to conduct the analytical part of pharmacokinetic studies of drugs containing both individual flavonoids and plant extracts.