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Tolbutamide,an oral sulfonylurea oral hypoglycemic drug used in the treatment of Tyre Ⅱ diabetes mellitus was selected as the representative probe substrate for cytochrome P450 2C9 in vitro and in vivo.To investigate the pharmacokinetics of tolbutamide and its metabolite 4-Hydroxytolbutamide,a sensitive and selective methord of high performance liquid chromatography for the determination of tolbutamide and its metabolite 4-Hvdroxvtolbutamide in human plasma was developed and validated.