Acclaim organic acid column

Samples (500 μL) were collected from the outlet, centrifuged (17000 g, 7 min, 4°C) and utilized for HPLC analysis (Dionex Ultimate 300, Thermo Fisher Scientific). To detect ε‐caprolactone, 6‐hydroxyhexanoic acid, and adipic acid, the samples were acidified with 1 M HCL before HPLC analysis with a ratio of 1:5. A Dionex Ultimate 300, Thermo Fisher Scientific separation module equipped with a variable wavelength detector operating at 210 nm was used. An injection volume of 20 μL onto an Acclaim organic acid column (150 cm length, 3.0 mm diameter, 3 μm particle size, ThermoFisher Scientific) was applied. The mobile phase consisted of MilliQ water (A), 100 mM Na₂SO₄ (B) (pH 3, adjusted with methanesulfonic acid), and pure acetonitrile (C). The column oven was kept constant at a temperature of 60°C. The column was equilibrated for 2 min at the initial eluent composition of 95% B and 5% C. A binary elution gradient with 0.4 mL min^–1 over 7 min was applied from 95 to 20% mobile phase B with subsequent regeneration of the column with 20% B for 1 min and washing the column with a 95% mobile phase B for 2 min.

Metabolite analyses were performed with an HPLC system (Elite LaChrom, VWR, Pennsylvania, USA) equipped with an organiser, a diode array detector (L-2455), a thermostatted column oven (L-2300), an autosampler (L-2200) and a pump (L-2130). An Acclaim organic acid column (5 μm, 4.0 × 150 mm, Thermo Scientific, USA) was used to separate acetate from other analytes. HPLC carrier solvent (100 mM Na₂SO₄, pH 2.65/H₂SO₄) and MQ-water was prepared just prior the analyses and filtered through a membrane filter (NC45-0.45 μm, Whatman, UK). Samples were diluted 1:10 with carrier solvent at a final volume of 600 μl and adjusted to a pH 2 by adding 6 μl 20% H₂SO₄.
To remove particles, the samples were centrifuged (16,000×g, 5 min, 4 °C) and the supernatant was transferred into HPLC-vials. 10 mM solutions of lactate, acetate, pyruvate, l-cysteine, cystine and formate served as standards and a sample containing only medium served as a control. 10 μl samples were applied onto the 30 °C-thermostatted column at a flowrate of 0.6 ml × min⁻¹ and a run time of 15 min. The detection of the organic acids occurred at 210 nm.