Quantitative Analysis Of Steroid Hormons By UPLC-MS/MS And Its Application In Clinical Detection

Steroid hormones are a kind of non-polar small steroid hormones.They can regulate the metabolic process in vivo through the endocrine system.Clinical steroid hormone detection has developed from radioimmunoassay that can detect up to five steroid hormones at one time,to direct immunoassay that can automatically detect steroid hormones.These methods have the advantages of simple operation and short detection time.Therefore,up to now,they are still common methods for screening hormones in most clinical laboratories.However,because of the cross-interference,matrix interference and low throughput of immunoassay,it cannot meet the need of modern clinical hormone testing as a hormone screening method.The use of liquid chromatography-mass spectrometry(LC-MS)to detect hormones has gradually increased and become popular as the "gold standard" for the detection of steroid hormones.LC-MS/MS is an ultra-high-sensitivity,high-efficiency separation detection system that separates components from a mixture by high-resolution liquid chromatography and then mass spectrometry can quantitative,qualitative the analysis.LC-MS/MS can detect many kinds of sterols at one time,with simple pretreatment simple,fast detection and high accuracy.Due to the calibration of isotope internal standard,the double separation effect of chromatography and mass spectrometry,the matrix effect has been greatly improved.The appearance of false positive results has also become minimal.At present,the clinical application of chromatography and mass spectrometry in China is still in its infancy,and LC/MS technology needs to be further developed and applied to meet the needs of clinical testing and disease diagnosis.In order to study the effects of different pretreatment methods on the sensitivity of steroid hormones,we compared the effects of direct liquid-liquid extraction and liquid-liquid extraction followed by derivatization on the quantitative limitation.HPLC-ESI-Q-TOF MS method was used to complete the methodological development and verification of 10 steroid hormones in human serum.The derivatization and liquid-liquid extraction methods were used in the pretreatment,and the corresponding ion signal response was improved by 2 to 200 times than that of direct liquid-liquid extraction.The derivatization time and temperature of the hydroxylamine solution with the 10 steroid hormones was optimized,the minimum limit of 0.05 ng / m L was obtained,and the liquid chromatography conditions were optimized,so that the total sample detection time was controlled within 5.7 min,which basically satisfied the detection of steroid hormones in human serum.In order to further reduce the sensitivity of the method,the minimum quantitative limitation of the above-mentioned method can not fully meet the detection of human hormones,so we use ultra-high liquid chromatography-electrospray ionization triple quadrupole mass spectrometry(UPLC-ESI-QQQ)method to quantitatively detect 18 steroid hormones in human serum.The pretreatment method directly adopts a liquid-liquid extraction method which is low cost and takes less time,compared to off-line solid phase extraction or derivatization,this pretreatment is performed without lowering the sensitivity(minimum detection limitation 7 pg/m L).The pretreatment time is controlled within 10 min,the detection time is within 6 min.This method has analyzed more hormones than current published literature.The linearity,precision,matrix effect,accuracy and stability of the method meet the requirements of the FDA Biological Sample Detection Guidelines.In addition,a total of 26 patient samples were completed in this chapter,and a patient with adrenal hyperplasia due to 21-hydroxylase deficiency was successfully diagnosed.in addition,we performed quantitative analysis of 20 steroid hormones and their metabolites in human urine by UPLC-ESI-QQQ mass spectrometry.In the detection of steroid hormones in urine,the sample volume is small(190 ?L).The enzymatic used for enzymatic hydrolysis and the solvent used for liquid-liquid extraction are optimized.The minimum quantitative limit(LOQ)can be still as low as 5 pg/m L after the pretreatment of enzymatic hydrolysis and liquid-liquid extraction.The linearity,precision,accuracy and stability of the method also meet the requirements of FDA Biological Sample Detection Guidelines.In addition,we detected not only 17 hormones in the main metabolic pathways of human urine,but also gestrinone,adrenone,11?-hydroxyandrosterone.These hormones are detected for the first time use UPLC-ESI-MS/MS and are important for the diagnosis and treatment of adrenocortical hyperplasia.The method established and validated in this paper can detect 18-20 kinds of steroids in human blood and urine at the same time,including progesterone,androgen,glucocorticoid,salt corticosteroid,estrogen and so on.The method has been applied in the screening and detection of patients with congenital adrenal hyperplasia in our laboratory.Congenital adrenocortical hyperplasia(CAH),Cushing's syndrome,polycystic ovary syndrome,acute or chronic hypofunction of adrenal cortex,systemic lupus erythematosus and other diseases all belong to the scope of clinical steroid hormone detection.LC-MS/MS analysis of these hormones help the diagnosis and treatment of hormone-related diseases by providing reliable experimental data and method.