In vitro cytochrome P450 activity: development and validation of a sensitive high performance liquid chromatography-tandem mass spectrometry method for the quantification of six probe metabolites after derivatization with pyridine-3-sulfonyl chloride in an aqueous environment
| Autor: | Sylvie M.N. Mulliez, Koen Boussery, Lies De Bock, Jan Van Bocxlaer, Sofie Vande Casteele |
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| Rok vydání: | 2010 |
| Předmět: |
Male
Electrospray Pyridines Electrospray ionization Mass spectrometry Tandem mass spectrometry Biochemistry High-performance liquid chromatography Sensitivity and Specificity Analytical Chemistry chemistry.chemical_compound Cytochrome P-450 Enzyme System Drug Stability Liquid chromatography–mass spectrometry Tandem Mass Spectrometry Animals Humans Least-Squares Analysis Rats Wistar Derivatization Chromatography High Pressure Liquid Chromatography Chemistry Organic Chemistry Selected reaction monitoring Reproducibility of Results General Medicine Sulfinic Acids Rats Pharmaceutical Preparations |
| Zdroj: | Journal of chromatography. A. 1218(6) |
| ISSN: | 1873-3778 |
| Popis: | For the determination of the in vitro cytochrome P450 activity in microsomes, a quantification method for the probe metabolites, formed during incubation, is required. Due to insufficient sensitivity of a previously developed high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method for some of the metabolites, a fast and easy derivatization method with pyridine-3-sulfonyl chloride (PS) is described. Acetaminophen (CYP1A2), dextrorphan (CYP2D6), hydroxy-chlorzoxazone (CYP2E1) and hydroxy-mephenytoin (CYP2C19) can be derivatized because of the presence of a phenolic OH, whereas hydroxy-midazolam (CYP3A4) and hydroxy-tolbutamide (CYP2C9) remain unchanged. As PS improves the ionization efficiency in the positive electrospray ionization (ESI) mode, the sensitivity of the detection is improved significantly and meets requirements for the activity determination. Native negative electrospray type molecules, moreover, become positive ESI candidates. The direct derivatization in the aqueous incubation medium, without any other sample pre-treatment steps, such as evaporation or extraction, makes this procedure easy to perform. The method using 20 s microwave irradiation was shown to equal a 10 min reaction in a 60 °C heating block, consequently simplifying and shortening the process. Collision induced fragmentation of the derivatives resulted in at least one native compound, rather than derivative, specific product ion, thereby improving the selectivity of the method in the multiple reaction monitoring mode. The HPLC–MS/MS method was validated, and was demonstrated to be sensitive, selective, precise and accurate. The absence of a relative matrix effect was established, notwithstanding that an absolute matrix effect was observed. The analysis of a sample after microsomal incubation, from which some of the metabolites could not be quantified using the method without derivatization, proved the usefulness of the method. |
| Databáze: | OpenAIRE |
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