Securityguard c18 column

Quantitation of testosterone was performed in selected reaction monitoring (SRM) mode. Mass transitions and optimized MS/MS parameters are given in Table S5. Analyst® software v1.4.1 (AB SCIEX) was used for SRM, peak integration, and analyte quantitation. Standard curves were prepared for testosterone in both tissue culture media and water in the range 0–20 ng/ml and the limit of detection (LOD) and lower limit of quantitation (LLOQ) were established in both matrices. The concentration of testosterone was measured in FM, SDM and APSCE media. Peak areas for these samples were quantified against the external calibration curves of testosterone (Table S6). Reverse phase chromatographic separation of testosterone was achieved using a Perkin Elmer Series 200 LC (Beaconsfield, UK) equipped with a Luna C8(2) column (3 μm; 20 × 4 mm i.d.) and SecurityGuard C18 column (4 × 3 mm) (Phenomenex, UK) maintained at 30 °C and a flow rate of 0.5 ml min⁻¹ using the conditions in Table S7. An API4000 triple quadrupole LC/MS/MS (Applied Biosystems, USA) was used for analysis with electrospray ionization (ESI) performed in positive ion mode using nitrogen gas with source parameters found in Table S8.

Two kinds of mass spectrometry were used in this study. One was Agilent 1260 LC system coupled with an Agilent 6540 quadrupole/time of flight mass spectrometry (LC-Q-TOF/MS, Agilent Technologies, Palo Alto, CA, United States). Chromatographic separation was carried on a Hedera ODS-2 C18 analytical column (150 mm × 2.1 mm, 5 μm; Hanbon Science and Technology, Huai’an, China) with a security Guard-C18 column (4 mm × 2.0 mm, 5 μm; Phenomenex, Torrance, CA, United States). The flow rate was 0.3 mL/min and the mobile phase of methanol (solvent A) and water containing 0.1% acetic acid (solvent B) in a linearly gradient program was conducted. The gradient program was as the following condition: 0–3 min, 95% B; 3–15 min, 95–30% B; 15–20 min, 30–10% B; 20–25 min, 10% B; 25–26 min, 10–95% B; 26–32 min, 95% B. The column temperature was maintained at 55°C. The injection volume was 10 μL. The mass spectrometry was equipped with an electrospray ionization source and operated in the positive mode with the full scan range of m/z 100–m/z 1300. Besides, auto MS/MS and targeted MS/MS modes were chosen for obtaining more fragmented ion information of analytes. Drying gas temperature of 350°C with N₂ gas flow at 10 L/min, nebulizer pressure of 45 psi, capillary voltage of 4000 V, fragmentor of 135 V and collision energy of 25 eV were set.