Determination of Chlorzoxazone and Its Metabolite in Human Plasma by HPLC-MS

OBJECTIVE To establish a high performance liquid chromatography-electrospray mass spectrometry method for the determination of chlorzoxazone and its metabolite in human plasma.METHODS The HPLC separation was performed on a Thermo C18 column(4.6 mm×150 mm,5 μm)with guard column phenomenex C18.The column temperature was 40 ℃.The mobile phase consisted of acetonitrile-30 mmol·mL-1 ammoniumacetate solution.The flow rate was 1 mL·min-1with a split ratio 3∶1.The compound was ionized in the electrospray ionization(ESI) negative ion source of mass spectrometer and selected ions were at m/z 168.3,170.2(CZX),444.7(IS)and 184.4,186.2(HCZX).Human plasma samples were extracted with methyl t-butyl ether after incubation.RESULTS The linear ranges were 25～10 000 μg·L-1 for chlorzoxazone,10～3 000 μg·L-1 for 6-hydroxychlorzoxazone(r≥0.999 8),and their detect limits were 2.5,2.0 μg·L-1.Recoveries were within 90%～110%,and intra-and inter-day RSDs were all less than 15%.CONCLUSION The methed appeared to be accurate,sensitive,specific and simple.It is suitable for clinical study of chlorzoxazone pharmacokinetics and drug interaction.