Hypersil gold c8

The assay for EPO of the samples was determined using Vanquish UHPLC system (Thermo Fisher Scientific, USA). In short, the samples and reference product Eprex^® (20 µL of each) were applied in RPC column (Hypersil GOLD C8, 175 Å, 2.1 × 100 mm, 1.9 µm) (Thermo Fisher Scientific, USA) for analysis. The formulation buffer was considered as baseline reference. A gradient of mobile phase A (water with 0.1% FA) and mobile phase B (90% ACN in water with 0.1% FA) was used as carrier solvent at a flow rate of 0.3 mL/min. The impurity profiles of the samples were analyzed using SEC in reference to Eprex^®. Analyses were performed in an Ultimate 3000 RSLC system (Thermo Fisher Scientific, USA) using 50 μL samples in a Biobasic SEC-300 column (300 mm × 7.8 mm, 5 µm) (Thermo Fisher Scientific, USA). Phosphate buffer (pH 7.4) was used as mobile phase at a flow rate of 1.0 mL/min. The column temperature was maintained at 70 °C, and run time was 25 min for both methods. The signals were detected at 280 nm, and chromatograms were recorded.

The sample injection volume was 5 μL, and peak separation was performed on a Hypersil GOLD C₈ (2.1 × 150 mm, 5 μm, Thermo Scientific, Waltham, MA) maintained at 30°C. The analysis was performed to gradient condition using Agilent 1290 infinity II system with autosampler, column oven, and binary pump (HPLC water containing 0.01% (v/v) formic acid, A; 100% methanol, B), and flow rate was 0.2 mL/min. LC-MS/MS data were acquired with an Applied Biosystems SCIEX 4000 QTRAP hybrid triple quadrupole-linear ion trap mass spectrometer equipped with a Turbo V ionization source. The detection was conducted using multiple reaction monitoring (MRM) of the transitions of m/z 89 > 43 for lactate, m/z 132 > 88 for aspartate, and m/z 157 > 112 for ¹³C₅, D₅, ¹⁵N-glutamate (ISTD) in the negative ion mode. Acquisition and analysis data were performed with Analyst® software (ver.1.6.2; Applied Biosystems, Foster City, CA, USA).