Nylon filter

After adding hexanal-d₁₂ as an internal standard, the sample, obtained from the exposure assay, was extracted with 2 × 500 µL hexane, filtered through a nylon filter (0.2 µm, Phenomenex, Aschaffenburg, Germany) and subjected to quantitative analysis as reported previously, with minor modifications [21 (link)]. In short, the sample was injected using splitless mode and analysis was performed using a GC-MS (GCMS -QP 2010 Ultra, Shimadzu, Vienna, Austria) with a capillary column (ZB-WAX Zebron, 30 m × 0.25 mm i.d., 0.25 μm film thickness) for separation. Measurement parameters are shown in greater detail by Pignitter et al. [21 (link)]. Stable isotope dilution analysis was applied as quantitation method selecting fragment ions m/z 72 for hexanal and m/z 80 for hexanal-d₁₂ in SIM mode. Limit of detection (LOD = 0.02 µg/mL) was determined by a signal-to-noise ratio of 3, while the limit of quantitation was calculated based on a signal-to-noise ratio of 9.

Concentrations of hesperetin and piperine during solubility, dissolution rate, and permeability studies were measured by high-performance liquid chromatography with the DAD detector (HPLC-DAD). In this study, a Shimadzu Nexera (Shimadzu Corp., Kyoto, Japan) equipped with an SCL-40 system controller, a DGU-403 degassing unit, a LC-40B XR solvent delivery module, a SIL-40C autosampler, a CTO-40C column oven, and a SPD-M40 photodiode array detector was used. For the stationary phase, a Dr. Maisch ReproSil-Pur Basic-C18 100 Å column with 5 µm particle size and 250 × 4.60 mm (Dr. Maisch, Ammerbuch-Entringen, Germany) was used. The mobile phase was methanol:0.1% acetic acid (80:20 v/v). The mobile phase was vacuum-filtered through a 0.45 µm nylon filter (Phenomenex, Torrance, CA, USA). The experimental conditions were as follows: 1.0 mL/min flow rate, wavelengths of 288 nm for hesperetin and 340 nm for piperine, and a column temperature of 30 °C. The injection volume differed depending on the assay. For the solubility study, it was 1 µL, whereas for the dissolution rate and permeability assays, it was 10 µL. The method’s duration was 10 min. The retention times were 4.11 min for hesperetin and 6.77 min for piperine. Chromatograms (Figure S3a,b) and validation parameters (Table S1) were placed in Supplementary Materials.