Uv vis 2489

The total amount of carotenoids and β-carotene content were determined by HPLC method according to [17 (link)], with slight modifications. The oil samples (1.0 ± 0.01 g) were extracted with a 10 mL of n-hexane containing 1% butylhydroxytoluene (BHT), then filtered through a 0.45 mm polyvinylidene fluoride (PVDF) syringe filter (Millipore, Burlington, MA, USA). The total carotenoids and β-carotene contents were analyzed using the HPLC method on a Waters HPLC system consisting of 2695 liquid separation module, UV–Vis detector UV–Vis 2489 (Waters Corporation, Milford, MA, USA), and equipped with an RP-C30 column, (5 μm, 4.6 × 250 mm, YMC™ Europe, Dinslaken, Germany) connected to a C30 guard column (5 μm, 10 × 4.0 mm, YMC Europe, Dinslaken, Germany). The flow rate was 0.65 mL/min, column temperature was 22 °C, and β-carotene was detected at 450 nm. The mobile phase consisted of methanol (solvent A) and methyl-tert-butyl ether (solvent B). The samples were injected at 1% B (held 1 min), and the gradient then changed to 100% B (1−90 min) and again to 1% B in 5 min (held 5 min). For quantification, a calibration curve was produced using an authentic all-trans-β-carotene standard (concentration range was from 0.1 to 5.0 mg/100 mL).

Metabolite analysis was performed using a WATERS e2695 separations module equipped with an Aminex HPX-87H column (Bio-Rad) and 5 mM H₂SO₄ as eluent. Detection of peaks occurred simultaneously by a refractive index detector (WATERS 2414) and a dual-wavelength detector (WATERS UV/Vis 2489). Data processing was done with Empower Pro software (Empower 2 Software, copyright 2005–2008, Waters Corporation, Milford, Massachusetts, USA).

Metabolite analysis was performed using a WATERS e2695 Separations Module equipped with an Aminex HPX-87H column (Bio-Rad) and 5 mM H₂SO₄ as eluent. Detection of peaks occurred simultaneously by a refractive index detector (WATERS 2414) and a dual-wavelength detector (WATERS UV/Vis 2489). Data processing was done with Empower Pro software (Empower 2 Software, copyright 2005–2008, Waters Corporation, Milford, MA, USA).

MOX concentration in all samples was quantified using HPLC, Alliance Waters e2695 separations module, and a Waters 2489 UV/Vis dual absorbance detector. A detection wavelength (λ_max) of 254 nm was set. The mobile phase consisted of a mixture of phosphate buffer (18 mM) containing 0.1% v/v triethylamine (pH 2.8, adjusted with dilute phosphoric acid) and methanol (60:40 v/v) at a flow rate of 1.0 mL/min [29 (link)]. Chromatographic separation was achieved within 10 min using a Phenomenex Luna^® C₁₈ column (250 × 4.6 mm, 5 μ) as a stationary phase with a retention time of 7.1 ± 0.2 min. The samples were analyzed through a Waters chromatography data system and Empower software. The HPLC method was found to be linear over the MOX base concentration range of 1.0–100 μg/mL, with a limit of detection (LOD) and limit of quantitation (LOQ) of 0.8 and 2.4 µg/mL, respectively.

The PTX was analyzed by modifying the HPLC method reported in the literature [34 (link),35 (link)]. The HPLC system comprised a Waters Alliance e2695 separations module and Waters 2489 UV/Vis dual absorbance detector. A C₁₈ column (4.6 × 250 mm, 5 µm particle size), λ = 227 nm, mobile phase consisting of acetonitrile and water in a 60:40 ratio, with a flow rate of 1.2 mL/min was used [34 (link),36 (link)]. The samples were analyzed through Empower software, and the standard plot (0.1–20 µg/mL) was constructed using linear regression by plotting the AUC against the concentrations. The equation for the line and fit of the linear model (R²) was calculated. The assay, entrapment efficiency and release study samples were determined from the standard plot.