Api4000 triple quadrupole lc ms ms

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.