Stabilwax ms column

The extraction of lipids for fatty acids (FA) analysis was performed with chloroform/methanol (2:1 v/v), and FA methyl esters (FAME) were prepared according to Pérez-Palacios et al. [74 (link)]. The fatty acid composition of the Spirulina samples was identified using a gas chromatograph GC-2010 Plus (Shimadzu Europa GmbH, Duisburg, Germany) equipped with Mass Spectrometer GCMS-QP2010 (Shimadzu Europa GmbH, Duisburg, Germany). Separation was carried out on a Stabilwax-MS column (30 m length, 0.25 mmID, and 0.25 μm df) (Restek Corporation, Bellefonte, PA, USA). Oven temperature program started at 50 °C, then increased at a rate of 8 °C/min to 220 °C, held for 1 min at 220 °C, increased again at a rate of 20 °C/min to 240 °C and, finally, held throughout 10 min. The injector temperature was 240 °C, interface −240 °C, and ion source 240 °C. The carrier gas was helium at a flow-rate of 0.91 mL/min. The individual FAME peaks were identified by comparing their retention times with FAME standards (Merck & Co., Inc., Kenilworth, NJ, USA).

A non-targeted screening through headspace/solid-phase microextraction coupled to gas chromatography with time-of-flight mass spectrometry (HS/SPME-GC-TOF-MS) of the ground cocoa pulp, cocoa beans, and chocolate-making samples was conducted, in triplicate, using a Trace 1300 gas chromatograph (Thermo Fisher Scientific) equipped with a Stabilwax-MS column (Restek), as described previously (Díaz-Muñoz et al., 2021 (link)). This metabolite fingerprinting technique served as a selection of VOCs to be quantified.

The extraction of lipids for fatty acid (FA) quantification was undertaken with chloroform/methanol (2:1, v/v), and fatty acid methyl esters (FAME) were prepared according to the protocol described by Pérez-Palacios et al. [33 (link)]. The FA composition in BCOL was identified using a gas chromatograph GC-2010 Plus (Shimadzu Europa GmbH, Duisburg, Germany) equipped with a mass spectrometer GC/MS-QP2010 (Shimadzu Europa GmbH, Duisburg, Germany). Separation was carried out on a Stabilwax-MS column (30 m length × 0.25 mm diameter and 0.25 μm diameter particle size) (Restek Corporation, Bellefonte, PA, USA). The mass spectrometer operated at full scan mode, and the analyte was injected in split mode at a 1:60 split ratio. The following parameters were used: MS ion source temperature: 240 °C; MS interface temperature 240 °C; helium (carrier gas) flow-rate: 0.90 mL/min; injector temperature: 240 °C; and oven temperature program: 50 °C (4 min), 10 °C/min to 110 °C (1 min), 15 °C/min to 160 °C (2 min), 2.5 °C/min to 195 °C (1 min), 2 °C/min to 230 °C (1 min), and 2 °C/min to 240 °C (12 min). The individual FAME peaks were identified by comparing their retention times with FAME standards (Merck & Co., Inc., Kenilworth, NJ, USA). The quantification was determined using the corrected area normalization.

Semi-quantitative volatile fingerprinting of the GB samples was conducted by headspace/solid-phase microextraction coupled with GC and time-of-flight MS (HS/SPME-GC-TOF-MS) in triplicate, as described previously (Zhang et al., 2019 (link)). A Trace 1300 gas chromatograph (Thermo Fisher, Waltham, MA, United States) equipped with a Stabilwax^®-MS column (Restek) and a BenchTOF-HD mass spectrometer (Markes International, Llantrisant, Wales, United Kingdom) was used. GB powder (1.0 g) was incubated in a 10 mL screw-top headspace vial at 50°C for 10 min, followed by extraction using a SPME fiber (DVB/CAR/PDMS, 50/30 μm; Supelco, Merck) for 45 min. The raw data were deconvoluted with TOF-DS software (Markes), followed by identification of each peak via the NIST library (National Institute of Standards and Technology, Gaithersburg, MD, United States) and the Kovats Index (Afeefy et al., 2017 (link)). The peak area of each compound identified, normalized to the peak area of the IS and adjusted for the moisture content, was a measure of the aroma intensity (Zhang et al., 2019 (link)).

The extraction of lipids for FA quantification was undertaken with chloroform/methanol (2:1, v/v) and fatty acid methyl esters (FAME) were prepared according to the protocol described by Pérez-Palacios et al. (25 (link)). The FA composition of the lentil samples was identified using a gas chromatograph GC-2010 Plus (Shimadzu Europa GmbH, Duisburg, Germany) equipped with a mass spectrometer GCMS-QP2010 (Shimadzu Europa GmbH, Duisburg, Germany). Separation was carried out on a Stabilwax-MS column (30 m length, 0.25 mmID and 0.25 μm df) (Restek Corporation, Bellefonte, US). The mass spectrometer operated at full scan mode and the analyte was injected in split mode at 1:60 split ratio. The following parameters were used: MS ion source temperature: 240°C; MS interface temperature 240°C; helium (carrier gas) flow-rate: 0.90 mL/min; injector: 240°C and oven temperature programme was: 50°C (4 min), 10°C/min to 110°C (1 min), 15°C/min to 160°C (2 min), 2.5°C/min to 195°C (1 min), 2°C/min to 230°C (1 min) and 2°C/min to 240°C (12 min). The individual FAME peaks were identified by comparing their retention times with FAME standards (Merck & Co., Inc., Kenilworth, NJ, USA). The quantification was determined by using the corrected area normalization method.