The sensitivity (as LLOQ) was determined at 0.01 ng/ml for E1, E2, and E3, 0.015 ng/ml for T, 0.025 ng/ml for 17-OHP, 0.05 ng/ml for AD, 0.04 ng/ml for P, and 0.25 ng/ml for DHEA. The coefficient of determination (R2) was at least 0.9886, which demonstrated excellent linearity. In consequence, storage at −30°C for 10 min was regarded as the optimal condition for CIPS in this study. Moreover, partition coefficients, enrichment factors, and absolute recoveries of all the targets were investigated as the freezing time increased from 10 to 60 min. The volume of upper phase was only less than 50 μl in this case. According to the results from experiment III, after storing for 5 min at −30°C, CIPS cannot be completely induced. In human urine, steroid hormones are present almost exclusively as phase II metabolites. To calculate the method limit of detection (LOD) and limit of quantification (LOQ) of steroid hormones in urine, six replicates of synthetic urine were fortified with each target analyte at 1 ng/mL, a concentration that yielded signal-to-noise (S/N) ratios of 6.4–62.4. Precision was assessed as intra-day and inter-day variations in measured concentrations in three pooled urine or serum samples fortified at 10, 20 and 200 ng/mL and was calculated as the coefficient of variation (CV%). Pooled human serum fortified with target analytes (at concentrations of 10, 20, and 200 ng/mL) was used for method development and validation. Synthetic urine and pooled human urine fortified with target analytes (at concentrations of 10, 20, and 200 ng/mL) were used for method development and validation. However, due to the low volatilities of steroid hormones, analysis by GC-MS requires derivatization, a laborious and time consuming step 16,17. The methodologies used to determine steroid hormones such as corticosteroids, androgens, progestagens and their metabolites are of great importance in the field of biomedical analysis. Comparison of pregnenolone in serum following extraction with LLE, Bond Elut C18, Bond Elut Plexa, and Bond Elut NEXUS. The method was validated for accuracy, precision, sensitivity, linearity, and through the analysis of certified plasma standard reference material. Mean concentrations were used in the calculation of the percent contribution. In human serum, the LODs ranged from 0.11 ng/mL (estrone) to 0.35 ng/mL (pregnenolone), and the LOQs were between 0.38 ng/mL (estrone) and 1.18 ng/mL (pregnenolone) (Table 3). The influence of matrix effects was less than 15% (Tables S3–1 to S3–4) , which justifies the measurement of normal serum samples with analytical standards prepared in charcoal-striped serum. The LLOQ is defined as the lowest concentration that can be measured yielding method accuracy values of ± 20% of the nominal value, and method precision values of percent coefficient of variation %CV ≤ 20%. Chromatographic parameters were tuned for the coupling of LC/LC and MS/MS to achieve separation and high detection sensitivity for trace level analysis of 11ß-MNT and testosterone. The LC/LC method measures total testosterone and does not differentiate testosterone (17β-hydroxyandrost-4-en-3-one) from epitestosterone (17α-hydroxyandrost-4-en-3-one; refer to S1), whose separation and measurement methods have been previously described 36–37. In consequence, CIPS realized extraction and purification of sex hormones by simply cooling down the can-containing system for several minutes. After inductively coupled plasma–optical emission spectrometry analysis, the residual sodium concentration in the upper phase was less than 2% of the lower phase. During the first CIPS, all the hydrophobic sex hormones were spontaneously apportioned into the ACN-rich layer on the top, whereas the endogenous hydrophilic interference was excluded to the water-rich phase at the bottom. Moreover, four samples were also detected by a traditional LLE-based method for method comparison. The results exhibited no obvious differences (apparent recovery of 97.4–106.8% for all the targets), which demonstrated that at least three dozens of samples can be run in a batch without compromising method robustness. Sex hormones can be successfully quantified in all these samples. And the apparent recovery did not obviously change in these samples (98–104% for 0 min, 92–105% for 2 min, and 94–101% for 5 min). (A) MRM transition 289➔109 is designated to the 11ß-MNT measurement, (B) MRM transition 289➔97 is designated to the testosterone measurement, (C) A total ion chromatogram of 11ß-MNT and testosterone. (A) MRM transition 289➔109 is designated to 11ß-MNT measurement, (B) MRM transition 289➔97 is designated to testosterone measurement, (C) A total ion chromatogram of 11ß-MNT and testosterone. Chromatograms of 11ß-MNT and testosterone separated by chiral chromatography within a short period of time. Therefore, it was necessary to enhance the sensitivity of chromatographic signals by narrowing the chromatographic peaks to measure trace levels of the steroids. A total ion chromatogram (TIC) of 11ß-MNT and testosterone separated by a Pursuit PFP column (3 μm, 100 × 2 mm) via 2-hour long gradient elution.
