2.5. LC‐MS/MS analysis

LC‐MS/MS analysis was carried out on an Agilent 1290 series chromatographic system and an Agilent 6460 triple quadrupole mass spectrometer (Agilent Technologies) with electrospray ionization (ESI). Separation of negatively charged hormones was achieved in a Waters Xbridge BEH C18 column (2.1 mm × 150 mm, 3.5 μm), whereas separation of positively charged hormones was achieved in a Phenomenex Gemini C18 column (2.0 mm × 100 mm, 3.0 μm). The mobile‐phase gradient was composed of solvent A (0.1% ammonia) and solvent B (acetonitrile) with a flow rate of 300 μl/min and was performed as follows: 0–0.5 min (80% A, 20% B), 1–6 min (60% A, 40% B), 6–7 min (55% A, 45% B), and 7–9 min (10% A, 90% B). The injection volume was 15 μl, and the column was set at 40°C. For mass spectrometric analysis, the ESI settings were as follows: capillary voltage 3.0 kV; desolvation gas flow rate 9 L/min at 350°C; and nebulizer pressure 45 psi. MS‐MS acquisition was performed in multiple‐reaction‐monitoring (MRM) mode. The optimized settings for product ions of each hormone are shown in Table ​Table11.

Mass spectrometry parameters for each hormone using multiple‐reaction‐monitoring (MRM) mode