Establishment Of A UPLC-MS/MS-based Method For The Detection Of 19 Steroid Hormones In Dried Blood Spots

Steroid hormones are a class of fat-soluble small molecule compounds,mainly synthesized in gonads and adrenal glands,and play an important role in various vital activities of human body.However,due to their low content in the body,it is difficult to detect them,and the development of a simple and accurate detection technique has become an important task in the field of medical testing.Ultra performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS)method with its advantages of high throughput,high sensitivity and high specificity has been more widely used for the detection of steroid hormones in recent years.At the same time,dried blood spots have unique advantages over traditional venous blood smears and have high application value in newborns and some special groups.In this study,a series of work was carried out for the detection of 19 steroid hormones by UPLC-MS/MS method in dried blood spots.In this study,a UPLC-MS/MS method was developed for the determination of 19 steroid hormones in dried blood spots and the method was validated.Based on this method,the steroid hormones in blood cells were investigated to establish a traceable steroid hormone standard curve for dried blood samples,and finally,the distribution range of the 19steroid hormones in four healthy humans(two males and two females)was determined.In this study,a BEH C8 column and UPLC-MS/MS were used to separate the samples,and the extraction process was simple and accurate.The samples were analyzed by positive ionization mode(ESI~+)and negative ionization mode(ESI~-)and recorded in multiple reaction monitoring mode(MRM).We used a background correction method to remove endogenous hormone interference to achieve traceable analysis of steroid hormones in dried blood spot samples,and subjected the steroid hormone standard curve dried blood spots to the same pre-treatment as natural samples to avoid matrix effects on steroid hormone measurements in dried blood spots.Ultimately,the linear correlation coefficients r~2of the steroid hormone standard curves of dried blood spots we obtained were all≥0.997,with good linearity.Meanwhile,over the entire analytical range,the method showed matrix effects in the range of 87.1%to 131.3%,inter-assay coefficient of variation<12.07%,intra-assay coefficient of variation<18.16%,spiked recoveries in the range of 67.33%to 135.00%,carryover contamination rates in the range of-0.0917%to 5.4030%,and the distribution of the 19steroid hormones within the 4 healthy humans(2 males and 2 females)was within the distribution range reported in previous reports.