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.
Matrix effect is a common phenomenon in ESI-LC-MS analysis, produced by ionization enhancement or suppression by matrix components. Reference values were only available for cortisol, progesterone, and testosterone. ALS enzyme was reported to be efficient for the deconjugation of phase II metabolites of various endogenous and exogenous compounds 20,27. However, strong matrix effect following SPE (Figure S2) resulted in poor resolution of pregnenolone and 13C2-D2-pregnenolone.
Working solutions of analytical standards were diluted from stock solutions using acetonitrile (ACN). Individual certified stock solutions of 11-deoxycorticosterone, androstenedione and 13C3-11-deoxycorticosterone (100 µg/mL) with purities of ≥98% were purchased from Sigma-Aldrich (St. Louis, MO, USA). Individual stock solutions of cortisol and estriol (100 µg/mL) were prepared in methanol (MeOH).
Estrone and estradiol were shown separately because of their significantly higher intensities than the other analytes. Concentrations of the target analytes were 100 ng/mL; Injection volume was 10 µL; estrone, estradiol, and estriol were derivatized using dansyl chloride. Nevertheless, androstenedione and 17α-OH-progesterone co-eluted, which, however, was deemed acceptable because of their different MRM transitions (Table 1). Furthermore, the retention times of dansyl-estrone (DC-estrone), DC-estradiol, and DC-estriol were different from all other analytes, indicating improved selectivity (Figure 2 and Figure 3). To improve selectivity and sensitivity of estrogens, we applied an estrogen-specific derivatization step using dansyl chloride, as reported earlier 18,19,25. Addition of formic acid to the standard solution (as an additive) over acetic acid yielded better signal intensity.
The upper and lower layers (20 μl) of all the samples were diluted with IS mixture in ACN (each 0.5 ng/ml, 80 μl), sodium bicarbonate solution (100 μl, 0.1 mol/l in water), and DC solution (10 μl, 10 mg/ml in ACN). Freezing time—after adding water (100 μl), samples were centrifuged (10,000 g) at 4°C for 2 min and stored at −30°C for 5, 10, 30, and 60 min to conduct CIPS. Freezing temperature—after adding water (100 μl), samples were centrifuged (10,000 g) at 4°C for 2 min and stored at −10, −30, or −80°C for 10 min to conduct CIPS. Compared with the mainstream liquid-liquid extraction-based method, this new method exhibits significant progress in throughput, which shortens the time cost of sample preparation from 90 to 40 min. At present, most methods require liquid-liquid extraction or solid phase extraction for sample preparation.
Although chiral separation could eliminate the interference between 11ß-MNT and testosterone as demonstrated in A and B, broad chromatographs limit measurement sensitivity. The imperfection of this chiral separation method was that both analytes had relatively broad peaks in their chromatogram. While 11ß-MNT and testosterone could not be separated with a short retention time using reverse-phase chromatography, separation of the isomers could be achieved with short retention time when an appropriate chiral column was used . These mixtures were centrifuged for 15 minutes at 18,000 g to facilitate separation of the liquid phases, and the organic solvent phase was collected. These calibration standards and QC samples were prepared fresh each day, and standards were analyzed in duplicate; QC samples were analyzed in triplicate on each day of analysis.