The mass spectrometry-based methods offer high sensitivity and specificity of the analysis and enable a metabolomics approach by scoring a panel of molecules in a single run. This is primarily because of the simplicity and cost effectiveness of the test. Despite several flaws, such as cross-reactivity, high variability between different commercial kits, and only a “single hormone per assay” approach, immuno-based assays are the most frequently used method in clinical practice. Currently, two different methods are used for the analysis of steroid hormones in both clinical and basic research practice: Immunoassays and tandem mass spectrometry-based methods. The precise and rapid determination of their concentration is crucial for diagnostics and monitoring of many disorders, such as Cushing’s syndrome, Addison’s disease, and congenital adrenal hyperplasia, as well as personalized treatment. Steroid hormones play an essential role in many regulatory systems, such as the immune system, stress response, mineral balance, and in the development of sexual characteristics. In conclusion, in this method article, we describe a simple, sensitive, and cost-effective 2D-LC/MS/MS method suitable for the routine analysis of a complex of steroid hormones allowing high analytical specificity and sensitivity despite minimal sample processing and short throughput times.
For all the analytes, the lowest calibration point relative standard deviation was less than 10.8%, indicating a good precision of the assay within the lowest concentration of interest. The method detection limit for 15 steroid hormones ranged between 0.008 nmol/L (2.88 pg/mL) for aldosterone and 0.873 nmol/L (0.252 ng/mL) for DHEA. The absolute recovery for each analyzed steroid hormone ranged between 101.6% and 116.5%. Our results indicate the linearity of the method for all steroid hormones with squared regression coefficients R 2 ≥ 0.995, within-run and between-run precision (RSD < 6.4%), and an accuracy of 92.9% to 106.2%.
The optimized method was applied to a set of human plasma samples, including chylous. In addition, we developed an independent analytical run for estradiol, significantly increasing the assay accuracy while taking an additional 10 min to perform an analytical run of a sample. The following analytical run is 18 min long for all steroid hormones. In our arrangement, the preparation of one sample takes only 10 min and can accommodate 40 samples per hour when tested in series. For this purpose, we designed a procedure based on the 2D-liquid chromatography-tandem mass spectrometry with a minimalistic sample pre-treatment. The aim of the current research was to develop a simple and rapid mass spectrometry-based assay for the determination of 15 steroid hormones in human plasma in a single run, which would be suitable for a routine practice setting.
0 kommentar(er)
