Acquity uplc h class liquid chromatograph

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.

In every meat sample, lipid oxidation was determined by measuring the concentration of malondialdehyde (MDA) by liquid chromatography, using the procedure described by Bertolín et al. [31 (link)]. Briefly, 10 g of the meat samples were homogenized with 10 mL of 10% (w/v) aqueous trichloroacetic acid in ultrapure water and 50 μL of 7.2% (w/v) BHT in ethanol for 45 s with a high-performance homogenizer (Miccra D-8 Homogenizer, Falc Instruments, Treviglio, Italy) into a 50 mL polypropylene tube. The homogenizer was cleaned with 10 mL of 10% (w/v) aqueous trichloroacetic acid in ultrapure water, collecting the solution in the tube. Subsequently, the mixture was centrifuged for 15 min at 4000 rpm and 4 °C. Then, it was filtered through a paper filter, collecting the extract. A total of 1 mL of the vortexed extract was mixed with 10 mM 2-thiobarbituric (TBA). The mixture was homogenized and heated through agitation (45 min, 100 rpm at 100 °C) to form MDA-TBA₂. After cooling, 150 μL was pipetted into a 2 mL amber screw-cap vial with 850 μL of a mixture of ACN:ultrapure water at a ratio of 30:70 (v:v). Finally, the extracts were injected into an ACQUITY UPLC H-Class liquid chromatograph (Waters, Milford, MA, USA).

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.