Accucore aq c18

With the aim of confirming the chemical composition of the aqueous fraction, we proceeded to an HPLC analysis. The analysis was performed on a Dionex UHPLC U3000RS system equipped with a LPG-3400RS quaternary pump, a RSLC WPS-300T RS automated injector, a TCC-300SD column oven, and a UHPLC+ DAD-3000 diode array detector (ThermoFisher SA, Voisins le Bretonneux, France). The system was fitted with an Accucore aQ C18 (15 cm × 3 mm i.d., 2.6 µm particle size) column, itself protected by a Securiguard (Thermofisher). The mobile phases were solvent A 0.1% trifluoroacetic acid (TFA) in water and solvent B acetonitrile. The gradient was set as follows: The initial acetonitrile content was 0%, it was raised to 10% in 5.24 min, 15% in 0.32 min, 20% in 0.31 min, and 100% in 0.32 min and was maintained for 4 min. Flow rate was set at 0.8 mL/min, column temperature was set at 40 °C, and injection volume was 8 μL. A HPLC chromatogram from Hibiscus sabdariffa AF was recorded at 280 and 520 nm [29 (link)].

Ceftobiprole was obtained from Basilea Pharmaceutica International Ltd. (Basel, Switzerland), manufactured on March 2017 and stored in a freezer at −20 °C). Deionised water was obtained from a Labconco System by Millipore (Bedford, MA, USA). Acetonitrile (ACN, gradient grade for HPLC, ≥99.9%), ACN for LC-MS (≥99.9%), methanol (MeOH, gradient grade for HPLC, ≥99.9%) and DMSO (for HPLC, 99.7%) were obtained from Honeywell (Charlotte, NC, USA). Sodium hydroxide (≥98.8%), ammonium hydrogencarbonate (≥99.0%) and ammonium formate (≥99.0%) were delivered by Chempur (Piekary Śląskie, Poland); HCl (pure p.a., 35–38%) and H₂O₂ (pure p.a., 30%) were bought from POCH (Gliwice, Poland); acetic acid (for LC-MS, 99.8%) and ammonium acetate (reag. Ph. Eur., ≥98.0%) were purchased from Merck (Darmstadt, Germany); acetic acid (pharma grade, 99.9%) was obtained from AppliChem (Darmstadt, Germany); and ammonium acetate (for LC-MS, ≥99.0%) and formic acid (reag. Ph. Eur., ≥98%) were manufactured by Sigma-Aldrich (Darmstadt, Germany).
The following chromatographic columns were used: Kinetex C18 (150 × 3 mm, 2.6 μm), Kinetex PS C18 (150 × 2.1 mm, 2.6 μm) and Kinetex Biphenyl (150 × 2.1 mm; 1.7 μm) from Phenomenex (Torrance, CA, USA), as well as Accucore AQ C18 (150 × 4.6 mm, 2.6 μm) from Thermo Fisher Scientific (Waltham, MA, USA).

Free versus associated pollutants were separated by centrifugation of the formulations on a 10kDa NanoSep^® (Pall, France) at 5.000g for 5 min. The amount of pollutants in the filtrates were quantified by high-performance liquid chromatography (HPLC) using calibration curves of free pollutants. The percentage of association efficiency (AE) was calculated as follows:
$\%AE= \left[\frac{theoretical pollutant amount-calculated pollutant in the filtrate}{theoretical pollutant amount}\right]\times 100$
The chromatographic analyses were performed on an Ultimate 3000 Autosampler (ThermoScientific, France) and chromatographic data were collected and processed with the Chromeleon software.
The analytical column (Accucore aQ C18, 150x3 mm, 2.6μm particle size) was purchased from ThermoScientific, France. The flow rate was 0.5 mL.min-1 and the injection volume was 100μL. For naphthalene the mobile phase consisted of methanol/water (70:30 v/v) with a detection at 219nm and a retention time around 5 min. For benzo(a)pyrene, the mobile phase consisted of methanol/water (90:10,v/v) with a detection at 254nm and a retention time around 8 min. For DEHP, the mobile phase was a gradient of water/acetonitrile (15:85 v/v) progressively shifting to 100% acetonitrile (from 3 to 6.5 min) The detection was at 225nm with a retention time of around 9 min.