Methodological Study On Determination Of Sex Hormones In Human Serum By UPLC-MS/MS

Sex hormones are usually divided into estrogen,androgen and progesterone.They are transformed into each other through the catalytic action of adrenaline and various enzymes in the human body to regulate the physiological functions of the human body.They are important in reproduction,sexual differentiation,pregnancy and immune system regulation.The physiological role.Accurate analysis of its serum concentration level is of great significance for the diagnosis and prevention of infertility,hypogonadism,polycystic ovary syndrome and other related diseases.Ultra performance liquid chromatography-triple quadrupole mass spectrometry(UPLCMS/MS)has strong advantages in detection sensitivity,specificity,analysis speed,and simultaneous detection of multiple indicators.It is a clinically useful method for detecting serum hormones.An important means.This study selected five clinically representative endogenous hormones as target substances,including androgens(androstenedione,testosterone),progestins(progesterone),estrogen(estrone,estradiol).Through the use of liquid-liquid extraction(LLE),combined with UPLC-MS/MS technology,the quantitative analysis methods of testosterone and androstenedione in human serum and the quantitative analysis methods of progesterone,estrone and estradiol in human serum were established.And conduct method validation.The method specificity,linearity,extraction efficiency,method detection limit,quantitation limit,recovery rate,and applied to the detection of serum samples.The main research contents are as follows:1.Established UPLC-MS/MS analysis method of testosterone and androstenedione hormone in human serum,and optimized the pretreatment method,chromatographic conditions and mass spectrometry parameters.After optimization screening,it was confirmed that the target compound was separated using a reversedphase chromatography column.The best mobile phase was acetonitrile-0.01% formic acid water,column temperature was 40℃,flow rate was 0.3 m L/min;after comparison of different extraction solvents,it was found that acetic acid the ethyl acetate/n-hexane(70:30,v/v)solution has the best extraction effect.The method has good specificity and a good linear relationship in the range of 0.025 ng/m L～10 ng/m L.The method detection limit(LOD)of testosterone and androstenedione was 0.01 ng/m L,and the limit of quantification(LOQ)was 0.025 ng/m L;when the experimental addition level was 0.05,0.5,5.0 ng/m L,there was no obvious matrix effect,the extraction recovery rate was from 73.28% to 94.92%,and the method recovery rate ranges was from 89.63% to98.73%;the intra-day RSD was less than 6.5%;the intra-day RSD was less than 7.5%;the stability of the serum sample was examined,and the response value changes was less than 15%.Using this method to measure 35 serum samples,the test results show that in male serum samples,the average testosterone was 4.95 ng/m L and the androstenedione average was 2.23 ng/m L;in female serum samples,the average testosterone was 0.67 ng/m L and the androstenedione average was 1.30 ng/m L,both in normal physiological within range.The method has high sensitivity,accuracy and reliability,and can be used to detect the physiological levels of testosterone and androstenedione in clinical serum samples.2.Established UPLC-MS/MS analysis method of progesterone,estrone and estradiol hormone in human serum,and optimized the pretreatment method,chromatographic conditions and mass spectrometry parameters.The target compound was separated using a reversed-phase chromatography column.The optimal mobile phase was methanol-0.01 m M ammonia,column temperature was 40°C,and the flow rate was 0.3 m L/min.The serum sample was passed through 800 μL ethyl acetate/nhexane(70:30,v/v)and on-machine detection after extraction with n-hexane.The method has good specificity and a good linear relationship in the range of 0.01 ng/m L～ 10 ng/m L.The LOD of progesterone,estrone,and estradiol are 0.005 ng/m L,0.01ng/m L,and 0.01 ng/m L,respectively,LOQ were 0.01 ng/m L,0.025 ng/m L,0.025ng/m L;when the experimental addition levels were 0.05,0.5,5.0 ng/m L,there was no obvious matrix effect,and the extraction recovery rate was between 61.39% and89.55%,The method recovery rate ranged from 86.67% to 105.33%;the intra-day RSD was less than 9.8%;the intra-day RSD was less than 9.66%.The stability of the serum sample was investigated,and the response value changed less than 15%.The serum concentration of 10 premenopausal women was determined by the optimized method.The concentration range of progesterone was 1.80～3.52 ng/m L,and the concentration range of estrone is 0.054～0.189 ng/m L,and the concentration range of estradiol is0.086～0.436 ng/m L,all at normal concentration levels.The method has high sensitivity,accuracy and reliability,and can be used to detect the physiological levels of progesterone,estrone and estradiol in clinical serum samples.