Determination of vitamin K1 concentration in plasma of beagle dogs by LC-MS/MS and its application
OBJECTIVE To establish and validate a simple,rapid and sensitive LC-MS/MS method for the determination of cis/trans-isomers of vitamin K,in dog plasma.METHODS Vitamin K1-d7 was utilized as internal standard(IS).After a pre-treatment of methanol:isopropanol(50∶50,V/V),plasma sample was separated on a ChromCore C30(15 cm × 4.6 mm,3 μm)column.Isocratic elution with water(containing 0.1％formic acid)and methanol(containing 0.1％formic acid)(1∶99,V/V)was performed at a flow rate of 1.0 mL·min-1 and a column temperature of 25 ℃.The analytes and IS were quantified by multiple reaction monitoring mode(MRM)in atmospheric pressure chemical ionization source(APCI+)with ion pairs of m/z 451→m/z 187(vitamin K1)and m/z 458→m/z 194(vitamin K1-d7).RESULTS The linear ranges of vitamin K,trans/cis-isomers were 5.00-5 000 and 5.00-1 000 ng·mL-1(r≥0.996 4),respectively.Intra/inter-day precision was within 12.50％and rela-tive error(RE％)of accuracy within±11.38％.No obvious matrix effect was observed for analytes or IS.Selectivity,sensitiv-ity,extraction recovery and stability all fulfilled the requirements of the guidance for biological sample analysis.AUC0-t values of trans/cis-isomers of vitamin K,were(3 950±1 198)and(537.2±205.7)ng·h·mL-1,respectively.CONCLUSION The vali-dated LC-MS/MS method is simple,rapid,specific and sensitive.It has been successfully applied for studying the pharmacokinet-ics of cis/trans-isomers of vitamin K1 in dogs after an intravenous injection of vitamin K1.
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