Standard compound

Standard compounds were purchased from Sigma-Aldrich (Steinheim, Germany) in analytical purity. α-Ribazole was synthesized according to Wienhausen et al. [25 (link)]. Ultrapure water was obtained by filtration of deionized water in an arium® pro ultrapure water system (Sartorius, Göttingen, Germany). Acetonitrile and methanol were purchased from Biosolve (Dieuze, France) in ULC/MS grade.

Gas chromatography/mass spectrometry (GC/MS) analysis of volatile compounds was carried out using the headspace solid-phase microextraction (SPME) sampling technique as described previously by Plessas et al. [33 (link)]. Volatile compounds were identified by comparison with standard compounds (Sigma-Aldrich, St. Louis, MO, USA) and MS data with those in NIST107, NIST21, and SZTERP libraries. For semi-quantitative analysis of volatiles, 4-methyl-2-pentanol (Sigma-Aldrich) diluted in pure ethanol was used as the internal standard (IS) at various concentrations (4, 40, and 400 μg/g of sample). The volatile compounds were quantified by dividing the peak areas of the compounds of interest by the peak area of the IS and multiplying this ratio by the initial concentration of the IS (expressed as μg/g). All assays were carried out in triplicate.

Analyses of total solids, protein, fat, lactose and urea-N were conducted using infrared method [24 ] while the flow cytometry method was used to determine SCC [25 ]. For carotenoids and vitamin A, the methodology described by Hulshof et al. [26 (link)] was used. Extraction was performed using ammonium hydroxide and ethanol (2.5:6 v/v), followed by the addition of ethyl ether (0.0025% BHT) and petroleum ether (1:1 v/v). The samples were then centrifuged (4,000 rpm, 4 min), and the supernatant evaporated under N₂ flow at 40 °C. The residue was saponified (5% KOH for 3 h at 37 °C, 200 rpm) and re-extracted with hexane. Aliquots of the concentrated sample were injected into a liquid chromatograph (Shimzadu LC-10A), equipped with a C18 reverse-phase column (Vydac218TP54, 250 mm × 4.6 mm × 5 µm), protected by a 5 µm C18 reverse-phase guard column (Vydac218GK54), and a UV-visible detector operating at 450 and 392 for carotenoids and vitamin A, respectively. The eluent was MeOH:CH₃CN (90:10, v/v, 1 mL/min) and the identification of the compounds of interest was performed using retention times of standard compounds (Sigma-Aldrich, St. Louis, MO, USA).

HPLC-grade solvents including methanol, water, and acetonitrile were purchased from Fisher Scientific (USA). Standard compounds and analytical grade reagents used in this study were purchased from Sigma Chemical Co.(USA).

Methanol was purchased from Fisher Scientific (Pittsburgh, PA, USA). Pyridine, methoxyamine-hydrochloride, N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA), and standard compounds were obtained from Sigma Chemical Co. (St. Louis, MO, USA).