Agilent 5977a

The samples of A. officinarum volatile oil were analyzed on an Agilent 7890B gas chromatography system coupled to an Agilent 5977A MSD system (Agilent Technologies Inc., Santa Clara, CA, USA). GC conditions: Capillary column HP-5MS (30 m × 0.25 mm × 0.25 µm) was utilized to separate the volatile oil components. Helium was used as the carrier gas at a constant flow rate of 1 mL/min through the column. The injector temperature was maintained at 250 °C. Injection volume was 1 µL by split mode (split ratio is 50:1). The oven temperature was programmed as follows: The initial oven temperature was 70 °C, held for 3 min, ramped to 100 °C at a rate of 3 °C/min, held for 3 min, and then ramped to 120 °C at 10 °C/min, to 140 °C at a rate of 2 °C/min, held for 3 min, finally ramped to 220 °C at 10 °C/min. MS conditions: EI source, electron energy of 70 eV, ionization temperature 230 °C, interface temperature at 280 °C, The temperature of MS quadrupole 150 °C, quantity scanning range was from 20 amu to 500 amu.

The muscle myofibrillar fraction was isolated as previously described (Atherton et al., 2010; Greenhaff et al., 2008), and L‐[ring‐¹³C₆]‐phenylalanine incorporation into myofibrillar protein was determined by gas chromatography combustion isotope ratio mass spectrometry (GC–C‐IRMS, Delta‐plus XP, Thermo, Hemel Hampstead, UK). Separation was achieved on a 25 m · 0.25 mm · 1.0 μ‐film DB 1701 capillary column (Agilent Technologies, West Lothian, United Kingdom). Gas chromatography mass spectrometry (GC‐MS, Agilent‐5977a, California, USA) was used to determine muscle intracellular L‐[ring‐¹³C₆]‐phenylalanine enrichment. The sarcoplasmic fraction containing the intramuscular free amino acid pool was precipitated, and the supernatant was purified by cation‐exchange chromatography, using Dowex H⁺ resin and derivatised as their t‐BDMS derivatives before measurement of phenylalanine enrichment by GC‐MS (Atherton et al., 2010; Greenhaff et al., 2008; Mitchell et al., 2015).

The cabbage volatile profile was determined by gas chromatography with mass spectrometry (GC-MS) by a solid-phase micro-extraction (SPME) method. The extraction method was based on modified procedure that was described by [16 (link)]. Briefly, the samples were prepared by stirring 5 mL of sample and 1 g of sodium chloride in a sealed vial. After preheating (5 min. at 40 °C) volatiles were collected by SPME for 20 min. at 40 °C using 65 μm PDMS/DVB fibre (Supelco Inc., Bellefonte, PA, USA). An analysis was carried out with gas chromatography Agilent 5890 B with a mass detector Agilent 5977 A. The capillary column used in this experiment was CP-WAX52CB (Agilent, 60 m × 250 μm × 0.25 μm). Helium (He) 5.0 (purity 99.999%; Messer, Austria) was used as a carrier gas. Working conditions were as follows: injector temperature at 250 °C; MSD interface temperature 250 °C; oven temperature programmed from 40 °C (2 min. hold) to 230 °C (5 min, hold) at 6 °C/min; carrier gas (He) at a flow rate of 1 mL/min. (average velocity 25.502 cm/sec); injection port operated in split less mode. The compounds were identified by comparing their mass spectra with the spectral library (Wiley 9, NIST 0.8) and expressed as peak area. Three replicate measurements were performed for each sample.

The VOCs of turbot samples were determined using the method portrayed by Li et al. [38 (link)]. Thereby, 3 g minced muscle samples and 6 mL saturated NaCl solution were transferred into a 20-mL sample vial. A 65 μm PDMS/DVB fiber (Supelco, PA, USA) was exposed to the headspace of the vial at 50 °C for 25 min. After extraction, the fiber was directly desorbed into the injection port of the GC at 250 °C. The analytes were determined by GC/MS (GC, Agilent 7890B; MS, Agilent 5977A, Agilent, CA, USA) equipped with a methyl polysiloxane capillary column (HP-5MS, Agilent; 30 m × 0.25 mm × 0.25 μm film thickness). The carrier gas was helium at 1.0 mL/min. The GC column temperature procedure was set as follows: kept at 40 °C for 10 min, increased to 240 °C at 5 °C/min, increased to 280 °C at 20 °C/min, and held for 8 min. The MS operated in electron impact (EI) mode with EI energy of 70 eV; and collected data at a rate of 0.7 scans/s over a range of m/z 40–650. The VOCs were tentatively identified by the comparison of actual mass spectra with the published authentic spectra database in the GC/MS libraries (NIST2011), and the compounds with MS match index over 800 were reported.

Thirteen commercially available EOs from seven plant families were used in the assay. According to the information provided by the producers, the EOs were obtained by hydro-distillation. The semi-quantitative composition of the EO samples was determined by gas chromatography coupled with mass spectrometry (GC-MS) using an Agilent 7890B oven coupled with Agilent 5977A mass detector (Agilent Technologies Inc., Palo Alto, CA, USA) and CombiPal autosampler 120 (CTC Analytics AG, Zwingen, Switzerland). The methodology for the determination of EOs components was detailed in our previous study [43 (link)]. Details about the composition of the EOs are listed in Table 1.

Samples (1 g) were placed in a glass vial (10 mL) and heated at 40 °C for 5 min. Released volatiles were adsorbed on SPME fiber polydimethylsiloxane/carboxen/divinylbenzene (PDMS/Carboxen/DVB, Sigma-Aldrich Corp., St. Louis, MO, USA) at 40 °C for 20 min. The volatiles adsorbed on SPME were separated using an Agilent 7890B gas chromatograph (Agilent Technologies Inc., Santa Clara, CA, USA) with a fused silica capillary column (DB-WAX, 30 m × 0.25 mm × 0.25 μm; Agilent Technologies Inc.). Volatiles isolated were analyzed using an Agilent 5977A mass spectrometer (Agilent Technologies Inc.). Operating conditions were as follows: injector temperature, 250 °C; helium flow rate, 1 mL/min; oven temperature, 35 °C for 5 min and then programmed to increase at 5 °C/min to 120 °C, and increased at 15 °C/min to 220 °C and held for 6 min. Mass spectra were obtained by EI ionization at 70 eV over 29 to 290 mass units, with an ion source temperature of 230 °C. Volatile compounds were identified by comparison of their mass spectra similarities with standard compounds and the NIST 02 spectral library of the gas chromatography-mass spectrometry (GC-MS).

This was performed using a targeted gas chromatography-mass spectrometry protocol, as previously described [31 (link)]. Sample analysis was performed on an Agilent 7890B GC system coupled to an Agilent 5977A mass selective detector (Agilent, Santa Clara, CA, USA). Patient samples were run alongside negative controls and quality control samples (pooled aliquots of all patient samples; one run after every ten patient samples) to ensure no source contamination and to assess for signal drift. Three injections were undertaken for each sample. Analysis of data was performed using MassHunter software (Agilent), with SCFA levels calculated via integration of spectra from patient samples and comparison with freshly prepared calibration curves using SCFA standards (Merck, Darmstadt, Germany).