After incubation (10 min) at 65°C, CIPS was performed again at −30°C for 10 min. Then, protein precipitation was conducted by centrifugation (10,000 g) for 2 min at 4°C. Initial ACN-water proportion—different volumes of water (0, 50, 100, 200, and 300 μl) were added to create a gradient of ACN content (67, 57, 50, 40, and 33%).
Total testosterone within-day and between-day variations on two instruments. Specimens were extracted three times and analyzed on two unit-resolution instruments. Our goal was to develop a routine quantitation method with the lower AMR around 2 ng/dL . Testosterone was eluted at 1.5 min, and examples of extracted ion chromatograms are shown in Figure 1 for the lowest calibrator, 2 ng/dL (0.069 nmol/L). The divert valve was set to collect data in the window of 1–3 min. The product ion spectrum collected from a triple quadrupole instrument with an ESI source showed that the dominant fragment ions were m/z 97 and m/z 109 14,15.
Androgens and progestogens were detected in their original forms, whereas estrogens were quantified as DC-modiXfied products (naming as E1-DC, E2-DC, and E3-DC). To diminish contamination, LC fluid for the first 1 min was discarded by using exchange value. The flow rate and column temperature were set as 0.5 ml/min and 65°C. The gradient was set as 0–0.2 min 60% B, 0.2–3.5 min 60–100% B, 3.5–4.5 min 100% B, 4.5–4.6 min 100–60% B, and 4.6–6 min 60% B. An Ekspert ultraLC 100-XL system coupled with AB SCIEX 4500 QTRAP mass spectrometer was utilized to perform the detection of sex hormones. The whole study was supervised under the Ethics Committee of Renmin Hospital of Wuhan University and abided by the Declaration of Helsinki principles.
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.
We used isocratic LC/LC chromatography with a mobile phase that consisted of 50% B for 5 minutes and a fast gradient to 98% B in 0.75 minutes. Mobile phases were 10 mM formic acid in water (A) and 10 mM formic acid in acetonitrile (B). The sample extract was re-dissolved in another buffer solution (150 μl, 0.2 M ammonium carbonate, pH 9.8), the sample solution was extracted twice using hexane (600 μl each), and the combined organic layers were evaporated. The sample and internal standard were mixed well before a buffer solution (150 μl, 0.5 M sodium acetate, pH 5.5) was added, and then the sample solution was vortexed on a plate vortexer for 2 hours. A primary stock solution of 11ß-MNT and testosterone was prepared at 1 mg/ml each in acetonitrile. Initially, neat solutions of testosterone and 11ß-MNT were prepared by dissolution in acetonitrile. Acetonitrile, water of UHPLC/MS grade, formic acid of GC/MS grade, sodium acetate, ammonium carbonate, testosterone-2,3,4-13C3 (Cerilliant analytical reference standards) and epitestosterone were from Sigma-Aldrich (St. Louis, MO).
Then, profiting from CIPS extraction and IS normalization, carry-over and matrix effect was proved acceptable. The newly developed CIPS-coupled LC-MS/MS method was carefully validated according to the latest guidelines from the Food and Drug Administration and Clinical and Laboratory Standards Institute. They were injected both before and after the real samples. During the detection, two medium-QC samples were also enrolled. In result, although the deviation from losing targets during storage could be normalized by IS, the upper phase after CIPS was suggested to be collected in 2 min at room temperature to prevent potential adverse impact on detection. According to the results, after standing for 10 min, it was difficult to find any boundary in the solution.

Gender: Female

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