Acquity uplc photodiode array pda eλ detector

Glucose (enzymatic-colorimetric method, sensitivity: 0.06 mmol/L) and urea (kinetic method, sensitivity: 0.056 mmol/L) concentrations were determined in plasma with an automatic analyzer (Gernon, RAL S.A, Barcelona, Spain). The mean intra- and interassay CV were 1.5% and 1.9% for glucose and 3.2% and 4.8% for urea, respectively. Plasma BHB (kinetic enzymatic method, sensitivity: 0.100 mmol/L) and NEFA (colorimetric method, sensitivity: 0.072 mmol/L) were determined using Randox kits (Randox Laboratories Ltd., Country Antrim, UK). The mean intra- and interassay CV were respectively 3.3% and 3.7% for NEFA and 6.2% in both cases for BHB. Oxidative status was determined using MDA as a biomarker of lipid peroxidation. This indicator was determined by liquid chromatography using an Acquity UPLC H-Class liquid chromatograph (Waters, Milford, MA, USA) equipped with a silica-based bonded phase column (Acquity UPLC HSS PFP, 100 mm × 2.1 mm × 1.8 μm, Waters), an absorbance detector (Acquity UPLC Photodiode Array PDA eλ detector, Waters) and a fluorescence detector (2475 Multi λ Fluorescence Detector, Waters). The quantification of MDA was done by fluorescence detection at ʎ_excitation = 530 nm and ʎ_emission = 550 nm following the chromatographic conditions described in Bertolín et al. (2019) (link). The mean intra- and interassay CV were 4.6% and 7.3%, respectively.

UPLC analysis were all carried out on a Waters ACQUITY UPLC system, including a quaternary solvent deliver system, an ACQUITY UPLC Photodiode Array (PDA) eλ Detector (Waters Co., Milford, CT, USA), and an autosampler (Waters Co., Milford, CT, USA). A reverse-phase ACQUITY UPLC column (100 mm × 2.1 mm, ID, 1.7 μm; Waters Co., Milford, CT, USA) was applied for the UPLC analyses. We used methanol-water (methanol: 0–10 min, 10%–90%) as the mobile phase. The effluent was continuously monitored at 220 nm and the flow rate was 0.5 mL/min. The preparative HPLC separation was performed using an YMC-Pack ODS-A column (250 mm × 20 mm ID, 5 μm; YMC, Kyoto, Japan). A Waters 600 system (Waters) was carried out for the preparative HPLC separation of streptochlorin. The mobile phase was methanol-water (65%:35%) at a flow rate of 9 mL/min, and the detection wavelength was 220 nm. The chemical structures of the peak fractions were identified by ESI-MS, ¹H-NMR, and ¹³C-NMR analysis and compared with the published literatures.

Tocopherols and retinol content were determined following the methodology described by Bertolín et al. [13 (link)]. A 0.2 g sample of freeze-dried meat was saponified with 3 mL of saponification solution (10% w/v potassium hydroxide in a 50:50 mixture of ethanol and distilled water v:v) overnight under an inert N2 atmosphere. Subsequently, the analytes were extracted twice with 5 mL of n-hexane: ethyl acetate (9:1, v:v) with 5 µg mL⁻¹ of BHT mixture and evaporated in a vacuum evaporator for 30–40 min at 40 °C. A 1 Ml quantity of acetonitrile: dichloromethane: methanol (75:10:15) was added to dissolve the dry residue, then filtered through a PTFE filter into a 2 mL HPLC* vial. Tocopherols and retinol analyses were performed using an ACQUITY UPLC H-Class liquid chromatograph (Waters, Milford, MA, USA) equipped with a silica-based column (Acquity UPLC HSS T3, 1.8 µm × 2.1 mm × 150 mm column, Waters), an absorbance detector (Acquity UPLC Photodiode Array PDA eλ Detector, Waters), and a silica-based bound phase column. Tocopherols were detected by measuring the fluorescent emission at λexc = 295 nm and λemi = 330 nm and retinol by the absorbance at 325 nm. Quantification of tocopherols and retinol was performed using a five-point calibration curve from pure standards. Tocopherols and retinol values are expressed in μg/g of DM.