Chemstation software

Vitamin C was quantified as the sum of ascorbic (AA) and dehydroascorbic acids extracted through ultrasound-assisted extraction, as described by [1 (link)]. Briefly, 0.5 g of each sample was homogenized in an aqueous solution with 8% acetic acid and 3 mL of 3% H₃PO₄ and sonicated using an Elmasonic E15H (Elma, Singen, Germany) bath for 30 min. Afterwards, the obtained solution was centrifuged for 10 min at 400× g in an Eppendorf 5804 centrifuge (Eppendorf, Germany, Hamburg). The supernatant was filtered and 20 µL was injected into the HPLC-DAD-ESI-MS system equipped with a quaternary pump, autosampler, DAD detector, and coupled to an MS-detector single-quadrupole Agilent 6110 (Agilent-Technologies). An XDB C18 Eclipse column (4.5 × 150 mm) with a flow rate of 0.5 mL/min was used for separation and identification of the compounds, with 1% formic acid:acetonitrile (95:5) in distilled water (v/v) (A) and 1% formic acid in acetonitrile (B) as binary gradients. For MS fragmentation, a scanning range of 100–600 m/z in the ESI (+) mode was conducted with a capillary voltage of 3000 V, a temperature of 300 °C, and a nitrogen flow rate of 7 L/min. Chromatograms were recorded at a wavelength of 240 nm, and data acquisition was done using Agilent ChemStation software (Rev B.04.02 SP1, Palo Alto, CA, USA). The analysis was conducted in triplicate.

Bruker Esquire HCT mass spectrometer (Bruker Technologies, Bremen, Germany) equipped with a 1200 Series liquid chromatograph (Agilent Technologies, Waldbronn, Germany) and controlled by ChemStation software (Version B.01.03 [204], Agilent Technologies, Waldbronn, Germany) was used.
Chromatographic separation was achieved on a 150 mm × 2.1 mm, 5 μm particle, Agilent Zorbax SB-C18 column at 30°C. A gradient elution programme was conducted for chromatographic separation with mobile phase A (0.1% formic acid in water) and mobile phase B (acetonitrile) as follows: 0–4.0 min (10-80% B), 4.0–8.0 min (80-80% B), 8.0–9.0 min (80-10% B), and 9.0–13.0 min (10-10% B). The flow rate was 0.4 mL/min.
The quantification was performed by the peak area method. The determination of target ions was performed in selective ion monitoring mode (m/z 240 for bupropion, m/z 268 for metoprolol, m/z 326 for midazolam, m/z 180 for phenacetin, m/z 198 for omeprazole, m/z 271 for tolbutamide, and m/z 237 for IS) and positive ion electrospray ionization interface. Drying gas flow was set to 7 L/min and temperature to 350°C. Nebuliser pressure and capillary voltage of the system were adjusted to 25 psi and 3,500V, respectively.

The remaining two freeze-dried exudate pellets were resuspended in 1 mL of LC-MS grade water and transferred into new 2.5 mL microcentrifuge tubes. At this point, equal aliquots (5 μL) from all samples were combined in a new tube, per each sample type (leachates, and hydroponics), to be used as quality control (QC) sample, followed by addition of 100 μL of internal standard (0.2 mg mL⁻¹ of succinic-d₄ acid, and glycine-d₅) to all samples before being lyophilised overnight using a speed vacuum concentrator (Concentrator 5301, Eppendorf, Cambridge, UK). All dried extracts were derivatized by oximation followed by a silylation step, using methoxyamine-hydrochloride in pyridine and N-Methyl-N-(trimethylsilyl) trifluoroacetamide, respectively. Metabolomics data were acquired using a 7890 B GC coupled to a 5975 series MSD quadrupole mass spectrometer, equipped with a 7693 auto-sampler and piloted by Chemstation software (Agilent, Technologies, UK).

SCFA analysis was performed using Headspace sampler-gas chromatography-Flame ionization detector (HSS-GC-FID) analysis. To provide a brief overview, all SCFAs were extracted from 0.1 g of mice fecal samples in 1 mL of distilled H₂O. These samples were vortexed and then centrifuged at 13,000 rpm for 3 min at room temperature. The resulting supernatant was transferred to a vial containing a GC buffer solution, and 2-ethylbutyric acid was added as an internal standard^{23 (link)}. The samples were analyzed using an Agilent 7890B GC system equipped with a 7697A headspace sampler and FID (Agilent Technologies, USA). Data acquisition and processing were performed using ChemStation software from Agilent Technologies.

The release of the curcumin from the membranes was determined as early described with some modifications [73 (link)]. Briefly, square pieces of membranes (ca. 2 cm²) were weighed and placed into individual amber vials. Considering the very low solubility of the curcumin in aqueous solutions, the release kinetic was performed at 37 °C in PBS/EtOH (90:10 v/v). At predetermined time intervals (every hour for 6 h, then at 12, 24, 48 and 72 h), supernatants were withdrawn, and the same amount of fresh solution was added back to the release medium. The curcumin concentration was measured using HPLC-UV with a linear elution gradient consisting of mobile phase A (0.1% acetic acid), B (Acetonitrile), and C (Methanol). The detection wavelength was set at 420 nm and Curcumin quantitation was based on a standard curve in PBS/EtOH (90:10 v/v). System control and data acquisition were performed using ChemStation software (Agilent Technologies). The results were presented in terms of cumulative release (percentage with respect to the loaded drug) as a function of time.