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In addition, the calibration samples of E1 and E3 were prepared by spiking working solutions into blank matrix. The concentrations of IS in this working solution were 1 ng/ml for E1-d4, E2-d2, and E3-d3, 10 ng/ml for T-d3 and P-d9, 20 ng/ml for DHEA-d6, 5 ng/ml for AD-13C3, and 17-OHP-d8. For LC separation, formic acid in water (0.2% v/v) with ammonium acetate (10 mmol/l) and formic acid in methanol (0.2% v/v) with ammonium acetate (10 mmol/l) were used as mobile phase A and B, respectively. For extraction, samples are allowed to stand under subzero temperature after protein precipitation by ACN.
The chemical structures of testosterone and 11ß-MNT are very similar and cannot be quickly separated by reverse-phase chromatography. Although each fragment has different relative abundance in the CID spectra of 11ß-MNT and testosterone, cross interference is inevitable when 11ß-MNT and testosterone are simultaneously measured by MS/MS. The MRM transitions of analytes and internal standard are testosterone (289→97), 11ß-MNT (289→109), testosterone-2,3,4-13C3 (292→100) and 11ß-MNT-d6 (295→277), respectively. The mobile phases compositions remained the same for 12 minutes, and the columns were re-equilibrated for 3 minutes.
This method will facilitate analysis of steroid hormones in population-based biomonitoring studies. Repeated analysis of similarly fortified urine and serum samples yielded intra-day and inter-day variations of 0–21.7% and 0.16–11.5%, respectively. This paper describes a methodology for simultaneous determination of 19 steroid hormones, viz. However, a specific strategy for the preparation of all biological samples has not yet been fully developed, but recently, some miniaturized devices for on-line sample extraction, and analyte preconcentration have been introduced. The elaborated method could be proposed for clinical application because it is fast, specific, relatively cheap and accurate enough and it may be successfully applied also in the routine investigation of human urine samples.
Calibration standards (0.05, 0.25, 0.5, 1, 4, 10, 25 and 50 ng/ml) and quality control (QC) samples (QC Low 0.1 ng/ml, QC Medium 2.5 ng/ml, and QC High 40 ng/ml) for testosterone and 11ß-MNT were prepared in blank charcoal stripped cynomolgus monkey serum as follows. Here we demonstrate the fast separation and measurement of steroid isomers 11β-MNT and testosterone with high sensitivity by a unique combination of a chiral column in series with a reverse-phase column (LC/LC) in line with tandem MS/MS. A novel chromatographic method to separate testosterone and 11β-MNT would make LC-MS/MS instruments versatile in the analysis of steroids and would find wider application especially in those laboratories without ion mobility-mass spectrometry capabilities. One option is to use ion mobility-mass spectrometry which is a technique that can separate steroid isomers 17–20 and could be a solution to separation and measurement of testosterone and 11β-MNT. Furthermore, a bioanalytical method must be capable of quantitatively measuring trace levels of the steroid because steroids exert biological effects at very low concentrations. In addition, a long-gradient LC-MS/MS method is not practical for most quantitative analyses that involve a large number of samples, including pharmacokinetic studies, which require rapid chromatographic separation.
The accuracy of this method on high-resolution mass spectrometry, OE120, was evaluated against TQ 6500. We transferred this method to a high-resolution mass spectrometer, Orbitrap Exploris 120 (OE120). The epitestosterone peak was present at 2.1 min, which was separated from the testosterone peak and did not interfere with quantitation (Supplementary Figure S3). Deming regression and a Bland–Altman plot showed that the new assay on TQ 6500 correlated well with the reference method (Figure 2).
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