The concentrations of 20 proteinogenic amino acids and one non-proteinogenic amino acid (γ-aminobutyric acid, GABA) in the processing waters and acidic bean extracts were determined by HPLC coupled to tandem MS (HPLC-MS/MS) by means of an Alliance 2695 chromatograph and Micromass Quattro MicroTM mass spectrometer (Waters), as described previously (Zhang et al., 2019 (link)). Quantification was performed through internal standardization in triplicate. L -2-amino butyric acid (1.2 ng/mL; Merck) was used as IS. All samples were microcentrifuged (19,400 × g for 15 min at 10°C) and filtered (Chromafil 0.2 μm PTFE filters) before injection (10 μL) into the column.
Alliance 2695 chromatograph
Manufactured by Waters Corporation
Sourced in United States
The Alliance 2695 chromatograph is a high-performance liquid chromatography (HPLC) instrument designed for analytical and preparative applications. It features an integrated solvent delivery system, autosampler, and detector, enabling precise and automated sample analysis. The core function of the Alliance 2695 is to separate, identify, and quantify components within a sample mixture.
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Lab products found in correlation
Hplc grade acetonitrile, by Merck Group (1 mentions)
Waters 2996 photodiode array detector, by Waters Corporation (1 mentions)
Atlantis c18 column, by Waters Corporation (1 mentions)
Caffeic acid, by Merck Group (1 mentions)
Chlorogenic acid, by Merck Group (1 mentions)
Empower software, by Waters Corporation (1 mentions)
Hypersil bds c18 column, by Thermo Fisher Scientific (1 mentions)
4 protocols using alliance 2695 chromatograph
1
Quantification of Amino Acids in Processing Waters and Extracts
2
HPLC Quantification of Hydroxytyrosol
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Hydroxytyrosol was measured with-High Performance Liquid Chromatography (HPLC) on a Waters Alliance 2695 chromatograph. Separation was carried out in a reverse-phase C-18 column (Phenomenex Prodigy, 5 μm, ODS-3 100 Å, 100 mm × 4.6 mm). The mobile phase was 0.1% trifluoroacetic acid (TFA, Sigma Aldrich, St. Louis, MO, USA) in HPLC–MS-grade water (A) by Sigma Aldrich, and HPLC-grade acetonitrile (B) by Sigma Aldrich. The hydroxytyrosol standard was purchased from Sigma Aldrich and its purity was >98%. The chromatographic method was a gradient elution starting at 10% acetonitrile and staying for 10 min and then gradually increasing the organic phase up to 90% acetonitrile over the course of 30 min. The focus was given to hydroxytyrosol quantification, which was eluted at approximately 8 min and therefore the other peaks after 10 min were deemed not important. An example of a chromatogram obtained through HPLC is presented in Appendix A , Figure A1 . Error bars correspond to the standard deviation of measurements carried out in triplicate.
3
RPLC Quantification of Organic Acids in B. badius
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RPLC was used for the separation and quantification of ten organic acids (acetic, citric, formic, fumaric, lactic, maleic, malic, malonic, oxalic, and succinic) in B. badius samples. RPLC analyses were conducted with a Waters Alliance 2695 Chromatograph coupled with a Waters 2996 Photodiode Array Detector (Waters Corp., Milford, MA, USA). Separation was performed on a Waters Atlantis C18 column (250 × 4.6 mm) with 5 μm particle size. RPLC conditions were as follows: mobile phase, 25 mM KH2PO4 adjusted to pH 2.5 with concentrated phosphoric acid and methanol (95:5, v/v); flow rate 0.8 mL min−1; UV detection wavelength 220 nm; at an ambient temperature of 25 ± 2 °C; and injection volume 10 μL. For the analysis of mushroom samples, gradient elution was used in every fifth sample to obtain 95 % methanol in 15 min to fully flush the column of hydrophobic compounds from previous injections (Cawthray 2003 (link)).
4
HPLC Analysis of Chlorogenic and Caffeic Acids
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The levels of chlorogenic acids and caffeic acid were determined by HPLC, as adapted by Trugo and Macrae [65 (link)], using a Waters Alliance 2695 chromatograph, 2996 PDA and Empower® software (Waters, Milford, MA, USA). A Hypersil C18 BDS column (5 cm × 4.6 mm and 2.6 μm; Thermo Scientific, Milford, MA, USA) was used. The mobile phase gradient consisted of the initial composition of 5% methanol (phase A) and 95% formic acid (phase B), maintained for 6 min. After 8 min, the composition of the mobile phase reached 80% of phase A and remained there for up to 10 min. After 11 min, the composition reached the plateau of 100% of phase A, and from 12 min to 15 min, the composition returned to the initial conditions. Detection was performed at 320–325 nm, the flow rate of the mobile phase was 1 mL/min and the injection volume was 3 μL. Samples were extracted in an ultrasonic bath for 20 min with 20% acetonitrile in ultrapure water (v/v). The samples were then centrifuged, microfiltered in disposable hydrophilic Teflon filter units with a porosity of 0.22 μm. A pre-calibrated system with chlorogenic acid and caffeic acid external standards (Sigma-Aldrich, New York, NY, USA) was prepared by weighing about 30 mg of the standard into a solubilized 25 mL of water and pelleted with 0.5% formic acid.
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