Determination of testosterone in deer blood by solid phase extraction coupled with liquid chromatography-mass spectrometry
HU Chun-lan12 CHEN Yuan-yuan12 WANG Chun-min12 REN Gui-qi12▲
1.Jingfukang Pharmaceutical Group Co.Ltd.,Hebei Province,Chengde 067000,China;
2.Hebei Traditional Chinese Medicine New Auxiliary Material Technology Innovation Center,Hebei Province,Chengde 067000,China
Abstract：Objective To establish a method for the determination of testosterone in deer blood by solid phase extraction coupled with liquid chromatography-mass spectrometry for the quality control of deer blood.Methods Firstly,the samples were pretreated,and then the testosterone content in deer blood was determined by using fixed quantum ion(109.06)under the conditions of liquid chromatography and mass spectrometry.Build sample preparation: acetonitrile precipitation deer blood protein,ethyl acetate extracted first testosterone,the solution to the activation of SAX(Maximum loading volume was 500 mg,volume was 3 mL)solid phase extraction column,the liquid obtained by nitrogen blow to nearly dry,N-hexane extraction,three times to add to the activated Florisil(maximum loading volume was 500 mg,volume was 3 mL)solid phase extraction column,abandon the filtrate,ethyl acetate,N-hexane(60∶40,V/V)elution pillars,nitrogen blow dry.The solution was dissolved in 50% methanol and passed through 0.22 μm microporous filtration membrane.High performance liquid chromatography condition:shim-Pack XR-ODS column(2.0 mm×75 mm,2.2 μm).The mobile phase consisted of methanol-0.1% formic acid-water and gradient elution.Column temperature was 40℃.Flow rate was 0.3 mL/min.Sample size was 20 μL.The mass spectrum condition:de-clustering voltage(DP)was 60.0 V,collision energy(CE)was 10.0 eV.Results In the linear range of 1.5165×10-4 to 3.033×10-1 μg/mL,the R of the fixed quantum ion 109.06 was 0.9995.The average recovery was 66.62%(n=6), RSD was 3.44%.The content of testosterone in four batches of deer blood was 3.30×10-4 to 3.48×10-4 μg/mL,with RSD <3%.Conclusion The method for the determination of testosterone in deer blood by solid phase extraction coupled with liquid chromatography-mass spectrometry was established.The linear range and recovery rate of solid phase extraction coupled with liquid chromatography-mass spectrometry are in line with the requirements of laws and regulations,and could provide reference for the quality evaluation of deer blood.
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