A liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based assay to profile 20 plasma steroids in endocrine disorders

Abstract

BackgroundLiquid chromatography-tandem mass spectrometry (LC-MS/MS)-based assays are employed in more and more clinical laboratories to quantify steroids. The steroid quantification by LC-MS/MS shows great value in screening or diagnosing endocrine disorders; however, the number of functional steroids included in the LC-MS/MS methods is still limited.MethodsHere, we describe the performance and validation of a 20-steroid plasma panel by LC-MS/MS. The panel included progestogens (including mineralocorticoids and glucocorticoids), androgens and estrogens biosynthesized in steroid metabolic pathways. The LC-MS/MS method was validated according to guidance documents, and subsequently employed to profile steroid changes in endocrine disorders.ResultsUsing LC-MS/MS, 20 steroids were separated and quantified in 8 min. Coefficients of variation (CVs) of the 20 analytes at the lower limit of quantification (LLoQ) were all less than 15% (ranging from 1.84% to 14.96%). The linearity of the assay was demonstrated by all the R2 values greater than 0.995. Individual plasma steroids changed significantly in patients with subclinical Cushing’s syndrome (SCS) and polycystic ovary syndrome (PCOS) – 17-hydroxypregnenolone (17-OH-PR), testosterone (T) and dihydrotestosterone (DHT) were significantly decreased in SCS patients, while in PCOS patients, pregnenolone, corticosterone (CORT), androstenedione (A4) and T were significantly increased and DHT was decreased.ConclusionsThe LC-MS/MS method we developed for the quantification of 20 plasma steroids is clinical practicable. The steroid profiling data using this assay indicate its screening value for endocrine disorders. To further explore the value of the assay, more investigations are however needed.

Simultaneous quantitation of four androgens and 17‐hydroxyprogesterone in polycystic ovarian syndrome patients by LC‐MS/MS

“…2,37,38 In Guo et al's recent study, with a smaller patient cohort, also confirmed that T, A4, and DHT together might contribute to the more accurate diagnosis of PCOS. 39 Similarly, in present study, the However, according to their logistic regression study using forward stepping models, it was found that their large steroid panel was no According to the current PCOS diagnosis guideline, 38 it is required and necessary to exclude the possibility of NCAH mimicking the symptoms of PCOS. In a recent review article comparing PCOS and NCAH, 41 on average, 87% of NCAH patients and 25% of PCOS patients showed elevated serum 17-OHP levels, which was the only recommended screening assay to differentiate the two distinct diseases requiring different therapeutic treatments.…”

“…In Guo et al's recent study, with a smaller patient cohort, also confirmed that T, A4, and DHT together might contribute to the more accurate diagnosis of PCOS 39 . Similarly, in present study, the combinational use of the four‐androgens provided optimum PCOS discrimination with the ROC analyses with the best sensitivity and specificity, suggesting their potential diagnostic value as a testing panel, although the cutoff values for each analyte needs to be further validated in independent cohorts.…”

Differential expression of peptides serves as an indicator of IgA nephropathy in pediatric patients

“…An internal standard for the peptide group was not used in the current study; search parameters were strictly controlled to ensure the accuracy of data. Filter parameter setting were as follows: 0≤ retention time ≤50; feature significance, ≥0; feature fold change, ≥1; 1≤ charge ≤10; with peptide ID: True; protein significance, ≥20; protein fold change, ≥2; significance method: PEAKSQ; confident unique supports, ≥1; normalization use Total Ion Chromatography; search parameter settings: Quantification type: Label free quantification; mass error tolerance, 20.0 ppm; retention time shift tolerance, 4.0 min; dependent on PID ( 25 ): 20; False Discovery Rate threshold, 1%.…”

Mass spectrometry: Future opportunities for profiling and imaging steroids and steroid metabolites

“…Testosterone and cortisol, being abundant, were relatively swiftly switched to MS analysis in clinical biochemistry and research settings, but estrogens, particularly in lower ranges (Figure 1), presented challenges, for example assessing effectiveness of aromatase inhibitors in women recovering from breast cancer, where residual hormone may promote tumour regrowth [19]. Although the resolving power of LC is not as good as GC, ongoing improvements in LC technology are enabling multi-steroid profiling, albeit some steroids having poorer sensitivity and isomer separation remains challenging [20,21]. A further benefit of LC over GC is the ability to include conjugates in the analyte profile [22].…”