Empower 2.3 system

A volume of 10 μL of the filtrate was injected into the HPLC system and the chromatographic analysis was carried out using a Waters Alliance system (Waters Corporation, Milford MA, USA), with a 2695 separation module and a 2487 UV detector (Waters, Milford). The system included a Supelcogel H column (250 mm × 4.6 mm) fitted with Supelcogel H Guard Column (50 mm × 4.6 mm) for the analytic separation, using 0.1% H₃PO₄ as mobile phase and0.17 mL/min flow rate. The UV detection was performed at 210 nm. Data were collected and analyzed with the Empower 2.3 system (Waters, Milford). The organic hydroxy-acids were identified according to their retention times: 10.7 min for citric acid, 11.2 min for gluconic acid, 15.1 min for lactic acid. The compounds peak area was used for quantification, based on a calibration curve obtained by injecting different volumes of a standard solution containing 30 ng/µL lactic acid, 25 ng/µL citric acid and 77 ng/µL gluconic acid. LOD/LOQ were 1.2/3.6 ng/µL for lactic acid, 1.1/2.2 ng/µL for citric acid and 1.9/5.7 ng/µL for gluconic acid. All used reagents were HPLC grade reagents, purchased from EMD Millipore, Merck Group.