Establishment And Application Of LC-MS/MS Analysis Method For Four Steroid Hormones In Human Serum

Objective: Steroids are a class of lipid soluble small molecule hormones that are catalyzed by cholesterol through a series of enzymes,including androgens,estrogen,progesterone,and adrenal cortical hormones.Elevated and decreased levels of steroid hormones are associated with many diseases,such as congenital adrenal cortical hyperplasia,which leads to insufficient synthesis of cortisol(COL)and precursor substance 17α-Hydroxyprogesterone(17-OHP),Progesterone(P)and bypass product testosterone(T)increase,so the determination of steroid hormones plays an important role in the diagnosis and treatment of clinical diseases.The purpose of this study is to establish a method for simultaneous determination of four types of steroid hormones in human serum [testosterone(T),progesterone(P),17α-Hydroxyprogesterone(17-OHP),cortisol(COL)] was used as a high performance liquid chromatography tandem mass spectrometry(LC-MS/MS)method to provide a reliable reference method for clinical hormone related diseases diagnosis and treatment.Research methods: A liquid chromatography tandem mass spectrometry(LC-MS/MS)was used to establish an analytical methodology for the determination of four types of steroid hormones(T,P,17-OHP,COL)in human serum.The optimal method is determined by optimizing the mass spectrometry conditions(charge ratio of the substance to be measured,energy parameters),chromatographic conditions(mobile phase selection,column temperature,flow rate,etc.),sample pretreatment(extraction solvent selection,extraction solvent proportion and volume),and then the performance of this methodology is verified.400 human serum samples were actually measured,and the serum samples were from Shenyang Kangwei Medical Laboratory.80 samples were randomly selected from the 400 samples and compared with the results of commercially available reagent kits for correlation analysis.When the correlation coefficient is greater than 0.99,the accuracy of this method is verified to be very good.Using Infinity Lab Poroshell 120 EC-C18 chromatographic column(2.7μm,4.6×50 mm);Column temperature: 40℃;Mobile phase: A phase: formic acid+water(100 μL+100 m L);Phase B: Acetonitrile(pure);The flow rate is 0.5m L/min;The method of liquid-liquid extraction(methyl tert butyl ether)is used.Conclusion: This study established a method that can simultaneously detect four types of steroid hormones(T,P,17-OHP,COL)in human serum.Compared with other reported literature methods,this study has the advantages of low cost,less serum required,simple operation,and short elution time.This method can achieve integrated detection of T,P,17-OHP,and COL at the same time,avoiding the drawbacks of repeated and large blood draws in clinical patient testing,Improved patient compliance and clinical applicability.