High pressure liquid chromatography system 1260 infinity 2

Chromatographic analyses were performed using an Agilent High-Pressure Liquid Chromatography system 1260 Infinity II equipped with a quaternary pump, an autosampler, and a photodiode array detector (Agilent Technologies, Santa Clara, CA, USA). The system control and data processing were performed using an OpenLab CDS LC ChemStation from Agilent. Separation was carried out on a reverse-phase Poroshell 120 EC-C18 (3 × 150 mm, 2.7 µm) with guard EC-C18 (3 mm) maintained at 20 °C. The injection volume was 1 µL. The compounds in standard and sample solutions were separated using a mobile phase consisting of 50 mM of potassium hydrogen phosphate (100%, pH 6.80) at 0.6 mL/min. The working solutions were prepared daily by appropriate dilutions with the mobile phase. The absorbance was monitored at 254 nm.

This second acellular assay measures the depletion of ascorbic acid (AA). Briefly, a range of particle concentrations from 0.56 to 56 µg/mL was prepared in a synthetic respiratory tract lining fluid (RTLF) containing 0.9% NaCl at pH 7.4 and AA (200 µM). Solutions were incubated for 4 h at 37 °C, then centrifuged for 10 min at 15,000 rpm and 4 °C. An aliquot of each supernatant was diluted 1:5 in the mobile phase (Potassium hydrogen phosphate, 10 mM, pH 3). Solutions were filtered before the injection with a Nylon 0.45 µM syringe filter (Cloup, Champigny-sur-Marne, France). Finally, samples were injected into an Agilent High-Pressure Liquid Chromatography system 1260 Infinity II, equipped with a DAD detector (Agilent Technologies, Santa Clara, CA, USA). The chromatography was equipped with a Poroshell 120 EC-C18 (3 × 150 mm, 2.7 µm) with a guard EC-C18 (3 mm) maintained at 25 °C. The flow rate of the mobile phase (100%) was 0.6 mL/min. UV data was collected at 220 nm. This method was validated to meet the ICH requirements. The percentage of AO depletion was calculated according to the following equation [18 (link)]:

ATP and ADP quantification were performed with the Agilent High-Pressure Liquid Chromatography system 1260 Infinity II equipped with a DAD detector (Agilent Technologies, Santa Clara, CA, USA). Stock solutions of 10 mM ATP (adenosine 5′-triphosphate disodium salt hydrate > 99%), ADP (adenosine 5′-diphosphate sodium salt > 98%), and AMP were prepared by dissolving the appropriate amounts in ultrapure water. The above stock solutions were stored at −20 °C, and the working solutions were prepared daily by appropriate dilution in the mobile phase. The mobile phase consisted of 50 mM potassium hydrogen phosphate (pH 6.80). Analytes were separated on a Poroshell 120 EC-C18 (3 × 150 mm, 2.7 µm) with a guard EC-C18 (3 mm) at 254 nm. The chromatographic separation was carried out at 20 °C with 0.6 mL/min as the flow rate. The identification of peaks was based on the comparison of retention times and diode-array spectra of chemical standards and biological samples. The analytical method was validated by assessing the linearity, limits of quantification, and detection and accuracy to meet the ICH requirements.