Determination of Progesterone in Beagle Dog Plasma by High Performance Liquid Chromatography-Tandem Mass Spectrometry
Objective To develop a sensitive high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS)method for determination of progesterone in Beagle dog plasma.Methods The separation was accomplished in a Thermo Accucore C18 column(100 mm×2.1 mm,2.6 µm),using 0.1%formic acid-water(A)-acetonitrile(B)as mobile phase of gradient elution.The flow rate was 0.4 ml/min,and the injection volume was 10 μl.The samples were treated with acetonitrile to precipitate the proteins,then the positive ion detection was carried out by the electrospray ion source in the multiple reaction monitoring(MRM)mode.MRM transitions of m/z 315.1→109.2(progesterone)and m/z 271.4→171.9(tolbutamide)were used for quantitative analysis.Results The calibration curve for progesterone was linear over the range of 0.5-100 ng/ml and the lower limit of quantification(LLOQ)was 0.5 ng/ml.The recoveries were 95.97%,105.20%,108.09%respectively and the RSDs of intra-day and inter-day were less than 15%.Conclusion The method is simple,sensitive,accurate and reproducible.It is applicable for determination and pharmacokinetic study of progesterone in Beagle dogs.
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