2.7. HPLC Analyses

Glucose, lactate and phosphate concentrations were measured with an ion-exclusion liquid chromatographic method using a sulfonated polystyrene divinyl benzene column (Aminex HPX-87H, Bio-Rad, Hercules, CA, USA) in an Agilent 1200 series HPLC system (Agilent, Santa Clara, CA, USA). A 0.01 N H₂SO₄ solution was used as the mobile phase with a flow rate of 0.45 mL/min [29]. All measurements were performed with an AZURA UV/VIS detector (Knauer, Berlin, Germany) with a refractive index detector temperature of 35 °C. The standard deviation of the technique was determined as 0.31% for glucose, 0.26% for lactate and 1.01% for phosphate measurement. Phosphate uptake rate was calculated taking into consideration the amount of phosphate present in the medium and also the volume of H₃PO₄ added for pH control.

Amino acid concentrations were determined by HPLC after derivatization in a reversed-phase Eclipse Plus C18 column (Agilent) at 40 °C according to manufacturer’s instructions (Agilent). The flow rate was adjusted to 0.64 mL/min and two solvents (solution A and B) were used in the mobile phase. Solution A consisted of 10 mM K₂HPO₄ and 10 mM K₂B₄O₇ and solution B of a 45/45/10% v/v/v mix of acetonitrile, methanol and water, respectively [29]. Amino acids were detected at 266/305 nm for fluorenylmethoxycarbonyl derivates and at 450 nm for o-phthalaldehyde derivates. The final amino acid concentration was quantified using an internal standard calibration. The standard deviation associated with the measurement of amino acid concentration was 4 ± 1%.