Abstract
High-performance liquid chromatography (HPLC) is the most common analytical method practiced in various fields and used for analysis of almost all drug compounds in the pharmaceutical industries. During drug development, an evaluation of potential drug interaction with cytochrome P450 (CYP) is essential. A “cocktail” approach is often used in drug development to evaluate the effect of a drug candidate on multiple CYP enzymes in a single experiment. So far, simultaneous analysis of multiple CYP substrates, which have greatly different structure and physicochemical properties, has required organic solvents and mobile phase gradient methods. However, despite the recent emphasis on environmental protection, analytical methods that use only aqueous solvents without the use of organic solvents for separation have not been studied well. This study sought to develop the simultaneous analysis of multiple CYP substrates by using poly(N-isopropylacrylamide) (PNIPAAm)-based temperature-responsive chromatography with only aqueous solvents and isocratic methods. Good separation of multiple CYP substrates was achieved without using organic solvents and any gradient methods by temperature-responsive chromatography utilizing a P(NIPAAm-co-n-butyl methacrylate (BMA))- and P(NIPAAm-co-N-acryloyl L-tryptophan methyl ester (L-Trp-OMe))-grafted silica column. Overall, PNIPAAm-based temperature-responsive chromatography represents a remarkably simple, versatile, and environmentally friendly bioanalytical method for CYP substrates and their metabolites.
Highlights
Since Anastas and Warner outlined the twelve principles of green chemistry over two decades ago[1], green chemistry has become a standard concept among scientists in the pharmaceutical and chemical industries as well as academia
A “cocktail” approach is often used in drug development for in vitro and clinical drug interaction studies to evaluate the effect of a drug candidate on multiple cytochrome P450 (CYP) enzymes in a single experiment
Some studies have reported a simultaneous analytical method for multiple CYP substrates using organic solvents and mobile phase gradient methods[13,14,15], until now, there have been no reports of a simultaneous analytical method that is performed with only aqueous solvents and isocratic methods
Summary
Since Anastas and Warner outlined the twelve principles of green chemistry over two decades ago[1], green chemistry has become a standard concept among scientists in the pharmaceutical and chemical industries as well as academia. N-butyl methacrylate (BMA) as a co-monomer leads to increased hydrophobicity, and the use of an aromatic amino acid co-monomer, such as N-acryloyl L-phenylalanine methyl ester (L-Phe-OMe), leads to enhanced molecular recognition[21,22,23] By utilizing these characteristics, temperature-responsive chromatography with PNIPAAm-grafted stationary phase has been developed[24]. Three different experiments using PNIPAAm-based temperature-responsive chromatography were conducted to demonstrate the simultaneous analysis of: (1) a cocktail of multiple CYP substrates for in vitro drug interaction assays, (2) CYP substrates and their metabolites, and (3) a cocktail of multiple CYP substrates for clinical drug interaction studies This PNIPAAm-based temperature-responsive chromatography represents a remarkably simple and environmentally friendly analytical method
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