Uhplc ms ms

Collected urine samples were centrifuged at 3000 rpm for 5 min, and 1.5 mL supernatant was filtered through a 0.45 μm pore (13 mm filter), vacuum dried and stored at −80 °C until analysis. Dried samples were reconstituted in 0.2 mL ammonium acetate 13 Mm Ph 4/0.1% formic acid in acetonitrile (50:50, v/v), vortexed for 2 min and sonicated for an additional 10 min. After sonication, samples were vortexed again (2 min) and centrifuged at 10,000× g rpm for 5 min at 4 °C. Supernatants were collected and filtered through a Millex-HV13 0.22 μm pore membrane (Millipore Corp., Bedford, MA, USA).
Identification and quantification of glucosinolates and their metabolic derivatives in the urine of participants was performed by ultra-high pressure liquid chromatography coupled to electrospray ionization and a 6460 tandem mass spectrometer with triple quadrupole technology (UHPLC/MS/MS, Agilent Technologies, Waldron, Germany). Chromatographic separation was carried out using a ZORBAX Eclipse Plus C18 column (2.1 × 50 mm², 1.8 μm) with a chromatographic gradient created with the solvents (A) 13 mM ammonium acetate pH 7 and (B) acetonitrile/formic acid (99.99:0.01, v/v) according to the method specified in [39 (link)], which separates intact glucosinolates and indoles, updated for the analysis of the compounds specifically present in the administered matrix.

The samples were extracted as described for phytohormones (Hura, Dziurka et al., 2017 (link)) and cleaned up in the same manner as for aromatic acids (Hura et al., 2016 (link)). D‐BeA was added as an internal standard prior to extraction. The compounds of interest (p‐coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol) were measured employing UHPLC‐MS/MS (Agilent Technologies) in positive ion mode MRM. An Ascentis Express RP‐Amide analytical column (2.7 μm, 2.1 × 150 mm; Supelco, Bellefonte) in a linear gradient of 3% ACN in H₂O versus ACN with 0.01% (vol/vol) HCOOH was used. Further technical details are given in Table S1.

The frozen apple flesh (−80 °C storage) was ground under cryogenic conditions (−196 °C) using AK11 mill (IKA), rapidly weighted into ice cold MeOH with sample weight to solvent volume (w/v) ratios of 1:4, shaken at room temperature for 15 min, and centrifuged (10,000× g, 4 °C) for 10 min. The volume was adjusted, samples were filtered through a 0.2 µm PVDF filter (Agilent technologies) into dark vials and directly injected onto UHPLC-MS/MS (Agilent technologies), and analyzed as described [39 (link)].

The identification and quantification of glucosinolates was conducted using ultra high-pressure liquid chromatography coupled to electrospray ionisation and a 6460 tandem mass spectrometer with triple quadrupole technology (UHPLC/MS/MS, Agilent Technologies, Waldron, Germany). The chromatographic separation was achieved by using a ZORBAX Eclipse Plus C18 (2.1 × 50 mm, 1.8 µm) (Agilent Technologies, Waldron, Germany) through a chromatographic gradient developed by applying different percentages of the solvents (A) 13 mM ammonium acetate pH 7 and (B) acetonitrile/formic acid (99.99:0.01, v/v), according to the multipurpose method optimised by Domínguez-Perles et al. [46 (link)], which separates intact glucosinolates, isothiocyanates, and their metabolites (Supplementary Table S1). The intact glucosinolates and isothiocyanates were identified following their MS2 fragmentation pattern [mass/charge (m/z) ratio] by applying positive or negative ionisation mode depending on the compound considered at the optima ESI conditions for the maximal detection of the analytes, and their retention time in comparison with authentic standards (Supplementary Table S2). The glucosinolate contents of the urine and plasma samples analysed were expressed as nanograms per mL (ng/mL).