Simultaneous determination of 12 steroids by isotope dilution liquid chromatography-photospray ionization tandem mass spectrometry
Introduction
The steroid hormones, derived from cholesterol and synthesized in the adrenal cortex, the gonads, and the placenta, are of great clinical importance and act as biological messengers [1], [2]. Secreted into and transported by blood to their target cells, the steroids are in equilibrium between their protein-bound and free forms. 25-hydroxyvitamin D3 is the most stable and abundant metabolite of vitamin D and an important regulating factor of calcium metabolism. Serum concentration of 25-hydroxyvitamin D3 is considered to be a good marker for Vitamin D deficiency.
Clinically important endogenous steroids can be measured by numerous techniques which include HPLC with UV detection [3], [4], and various immunoassays (IAs) [5], [6], [7], [8]. HPLC–UV based methods have difficulties in separating multiple analytes of interest from contaminants in plasma or serum and generally lack sensitivity. IAs have been widely used in many laboratories and hospitals because of their simplicity, speed, and sensitivity. Major drawbacks of immunoassays for the steroids include their lack of specificity [9], [10], [11] and the fact that you need a different assay for each steroid.
A number of tandem-mass spectrometry based methods for the measurement of steroids and Vitamin D have been reported in the literature [12], [13], [14], [15], [16], [17], [18], [19]. However, most of these assays measure only one or a couple of the analytes of interest. We previously reported a nine-steroid profile approach employing tandem mass spectrometry utilizing the API-3000 with atmospheric pressure photoionization source [11]. Steroid profiles for example in congenital adrenal hyperplasia (CAH) allow for the acquisition of more clinically useful data than can be obtained through the measurement of a single steroid alone.
Section snippets
Chemicals
Androstenedione, aldosterone, testosterone, DHEA, DHEAS, cortisol, corticosterone, 11-deoxycortisol, progesterone, 17α-hydroxyprogesterone, 17β-estradiol, 25-hydroxyvitamin D3, ammonium acetate, and bovine albumin (96%) were from Sigma-Aldrich (St. Louis, MO). Deuterium labeled internal standard: testosterone-1,2-d2 (testosterone-d2), cortisol-9,11,12,12-d4 (cortisol-d4), and estradiol-2,4,16,16-d4 (estradiol-d4) were from Cambridge Isotope Laboratory, Inc. (Andover, MA);
Results and discussion
The HPLC separation was performed on a C-8 column which greatly shortened the analysis time. The gradient was optimized to provide the maximum separation possible in a minimum time period. The eluant and gradient conditions used are detailed in Table 3. As aldosterone and cortisol are both avidly bound to cortisol binding globulin it was found necessary to add an aliquot of cortisol equivalent to 30 μg/dl to the serum to prevent precipitation of the low amounts of aldosterone with the proteins.
Conclusions
We have developed a 2nd generation steroid profile assay employing an isotope dilution LC-APPI-MS/MS-based method for the analysis of 12 steroids in serum samples with an 11-min run time. The method requires 200 μl serum. This method correlates very well with the other MS/MS-based methods employed at the Mayo Clinic. The combined attributes of organic solvent protein precipitation coupled with LC-APPI-MS/MS techniques under MRM mode provide a unique combination of analytical capability which
Acknowledgements
This work was supported by grant M01-RR13297 from the General Clinical Research Center Program of the National Center for Research Resources, National Institutes of Health, Department of Health and Human Services, Bethesda, MD, USA. It was also supported in part by grant 1 U10HD45993-02 of the National Institute of Child Health and Development, Bethesda, MD.
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Development and validation of a sensitive LC-MS/MS method for simultaneous quantification of thirteen steroid hormones in human serum and its application to the study of type 2 diabetes mellitus
2021, Journal of Pharmaceutical and Biomedical AnalysisCitation Excerpt :The method development for the endogenous analytes with similar structures has been a challenge due to possible interference from unknown compounds in biological matrix and environmental impurities. To date, a number of different LC–MS/MS assays for steroid profiling in the serum have been reported [3,10,11,18–21]. Taylor et al. [3] proved that serum steroid paneling by LC–MS/MS was useful for discriminating adrenocortical carcinoma from other adrenal lesions, and additional serum markers related to disease could be discovered by exploratory steroidomics studies.
Steroid metabolomics: machine learning and multidimensional diagnostics for adrenal cortical tumors, hyperplasias, and related disorders
2019, Current Opinion in Endocrine and Metabolic ResearchCitation Excerpt :Although there have been advances in GC technology, including coupling to different detection systems, the demands of cumbersome sample preparation and low sample throughput make the technology most suitable as a discovery tool rather than as a part of routine diagnostics [6]. The simpler preparation and higher throughput of biological specimens for liquid chromatography (LC) with tandem MS (LC–MS/MS) provides advantages over GC-based methods, leading to increasing use of LC–MS/MS for steroid profiling [13–19]. Although LC–MS/MS can offer relatively accurate measurements, the method is not free from interferences [20].