2487 dual absorbance detector

The shoot and root samples of L. album (1 g) were prepared using a pestle and mortar in liquid nitrogen, followed by extraction in methanol (80% v/v). The samples were then sonicated at room temperature for 1 h. Dichloromethane (4.0 mL) and water (4.0 mL) were added to obtain a partition of compounds between two layers, followed by centrifugation at 5000 rpm for 15 min. The dichloromethane fractions were then collected, dried, and dissolved in 1.0 mL of HPLC-grade methanol for HPLC analysis^{65 (link)}.
The Lignan content was determined using a Waters liquid chromatography system consisting of a 2695 Separations Module (USA) and a 2487 Dual Absorbance Detector (USA). Data acquisition and integration were carried out with Millennium32 software. The chromatographic separations were performed on a 250 × 4.6 mm Eurospher 100–5 C18 column (KNAUER company, Berline, Germany) with a reversed-phase matrix in a gradient system using acetonitrile (solvent A) and distilled water (solvent B) with a 1 mL min⁻¹ flow rate. A UV detector was set at 280 nm. The presence of PTOX, 6-MPTOX, and SECO was identified based on retention time and comparison of UV spectra with the authentic PTOX, 6-MPTOX, and SECO standards purchased from Sigma-Aldrich (Taufkirchen, Germany). Different concentrations of the three compounds (25, 50, 75, and 100 µg mL⁻¹) were used for the calibration curves.

Enzyme-treated and fermented oat drink samples were centrifuged at 14,000×g for 20 min at room temperature (Hettich ROTANTA 460 R, fixed angle rotator). The supernatant was filtered through a 3 kDa molecular weight cut-off filter (Amicon® Ultra-0.5, Merck KGaA, Germany) and diluted with 2 parts of ultrapure water before analysis. Concentrations of sugars (D-(+)-Raffinose pentahydrate and D-(+)-Maltose monohydrate, represented as a marker compound), organic acids and D-(+)-Glucose were measured with a high-performance liquid chromatography (HPLC) system (Alliance 2695 system, Waters Corp., Milford, MA, USA), using a BioRad Aminex HPX-87C (for sugars) or BioRad Aminex HPX-87H (for organic acids) columns (7.8 × 300 mm, 9 μm particle size) (Bio-Rad Laboratories, Inc., CA, USA). A BioRad Micro-Guard Cation C guard column (4.6 × 30 mm, 9 μm particle size) with isocratic elution of ultrapure water at a flow rate of 0.6 mL/min at +85 °C was used for sugar analysis. H guard column (4.6 × 30 mm, 9 μm particle size) with isocratic elution of 5 mM H₂SO₄ at a flow rate of 0.6 mL/min at +35 °C was used for organic acid analysis. Waters 2414 refractive index detector was used for the detection and quantification of substances, which was paired with a Waters 2487 Dual Absorbance Detector for organic acid analysis.

The HPLC method was adapted based on Varaprasad et al. [10 ]. The samples were analyzed in an isocratic HPLC (Waters Corp.) with a C18 column (Symmetry 5 µm, 4.6 x 150 mm). Twenty (20 μl) of the sample was injected using a Rheodyne injection syringe at a flow rate of 0.7 ml/min, maintaining the column at 25°C. Freshly prepared methanol: 0.05 M and phosphate buffer (pH 2.8) (60:40 v/v) were used after degassing and filtering through a 0.45 μM filter. The UV detection wavelength was identified to be 258 nm (Waters 2487 Dual Absorbance Detector). Based on these conditions, the amount of phenytoin bound to the colloidal nanogold was quantified. AuPht (10 ml) was centrifuged and the pellet obtained was resuspended in the mobile phase. Standards in the range of 0.1 mg/ml to 0.3 mg/ml were prepared with the mobile phase as diluents (N=2). Colloidal nanogold was used as a control and analysed under the same conditions. In order to cross-verify the amount of unbound phenytoin, the supernatant was also analysed.