Yeast conditioned medium was passed through 0.2 μm PVDF syringe filters and 3 kDa MWCO centrifugal filters (Millipore), diluted 1:10, then 1 μl was used for amino acid analysis on a liquid chromatography (Agilent 1290 Infinity) and tandem mass spectrometry (Agilent 6460) system, as described elsewhere (Mülleder et al., 2016a ). Method covered all proteinogenic amino acids (except cysteine), ornithine, citrulline, and α /γ-aminobutyric acid. In short, amino acids were separated by hydrophilic interaction chromatography with gradient elution on a Waters ACQUITY UPLC BEH Amide column (2.1 x 100 mm, 1.7 μm) using a binary solvent system of 50:50 acetonitrile:water and 95:5:5 acetonitrile:methanol:water, both containing 0.176% formic acid, 10 mM ammonium formate. The compounds were identified by comparing retention time and fragmentation pattern with analytical standards (Sigma-Aldrich). The obtained signals, operating the instrument in selected reaction monitoring mode, were processed and quantified by external calibration with Agilent MassHunter software.
Analytical standards
Manufactured by Merck Group
Sourced in United States, Germany
Analytical standards are reference materials used to validate and calibrate analytical instruments and methods. They provide a consistent and reliable way to measure and quantify the performance of these systems.
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Lab products found in correlation
Masshunter software, by Agilent Technologies (3 mentions)
Acquity uplc system, by Waters Corporation (2 mentions)
Methyl tricosanoate, by Merck Group (2 mentions)
Bf3 methanol solution, by Merck Group (2 mentions)
Gc 7820a 5977e msd, by Agilent Technologies (2 mentions)
Slb il60 column, by Merck Group (2 mentions)
Acquity uplc h class, by Waters Corporation (2 mentions)
Qda mass detector, by Waters Corporation (2 mentions)
Cortecs uplc c18 column, by Waters Corporation (2 mentions)
6 aminoquinoyl n hydroxysuccimidly carbamate aqc reagent, by Waters Corporation (2 mentions)
Empower tm 3, by Waters Corporation (2 mentions)
Acquity uplc beh amide column, by Waters Corporation (1 mentions)
Mwco centrifugal filter, by Merck Group (1 mentions)
1290 infinity, by Agilent Technologies (1 mentions)
Dionex integrion hpic system, by Thermo Fisher Scientific (1 mentions)
Aers 500 suppressor, by Thermo Fisher Scientific (1 mentions)
C18 reversed phase column, by Phenomenex (1 mentions)
Spd m20a, by Shimadzu (1 mentions)
Analytical grade, by Merck Group (1 mentions)
6890n gc ms system, by Agilent Technologies (1 mentions)
17 protocols using analytical standards
1
Yeast Conditioned Medium Amino Acid Analysis
2
Ion Chromatographic Analysis of Pot Ale
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Concentrations of lactate, acetate, phosphate, succinate, nitrate, nitrite, chloride and sulphate were determined in pot ale by Ion Chromatography (IC) using a Dionex Integrion HPIC system (Thermo Fisher Scientific, UK). Analytes were separated on a Dionex IonPac AS11-HC-4 µm column (4 × 250 mm) equipped with a Dionex IonPac AG11-HC-4 µm guard column (4 × 50 mm) and Dionex AERS500 suppressor. A diluted KOH / ultrapure water mobile phase was used with a gradient elution program of the following: 0–8 min (1.5 mM), 8–18 min (15 mM), 18–23 min (15 mM), 23–24 min (24 mM) and 24–30 min (60 mM). A flow rate of 0.38 mL min–1 was used. A sample volume of 2 µL was injected once the background conductivity was < 1 µS. Data acquisition and analysis was performed using Chromeleon 7.1 software. Calibration curves were generated using analytical standards (Sigma-Aldrich, UK), at a range of 5–100 mg/L. Pot ale samples were diluted 1/100 prior to analysis. Pot ale samples (triplicate) spiked at a concentration of 10 mg L−1 using analytical standards of acetate, lactate, chloride, nitrite, succinate, carbonate, sulphate, phosphate and nitrate (Sigma-Aldrich, UK) gave recoveries ranging between 95–103%.
3
Quantification of Aflatoxins in Maize
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Each extract (from ∼200-mg ground, lyophilized maize seed) was redissolved in methanol (1.5 ml) and centrifuged to remove particulate. The supernatant was analyzed using a Waters ACQUITY UPLC system (40% methanol in water, BEH C18 1.7 μm, 2.1 mm × 50 mm column) using fluorescence detection (Ex = 365 nm, Em = 440 nm). Samples were diluted 10-fold if the aflatoxin signal saturated the detector. Analytical standards (Sigma-Aldrich, St. Louis, MO, United States) were used to identify and quantify aflatoxins: aflatoxin B1 (AFB1) and aflatoxin B2 (AFB2). Aflatoxin content was expressed in ng/g (ppb) fresh weight of homogenized kernels.
4
Quantification of Capsaicinoids in Samples
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To determine the content of capsaicin (CAP) and dihydrocapsaicin (DHC), an extraction procedure was carried out according to Zheng et al. [23 ]. Quantification was carried out by HPLC as described by Nag et al. [24 (link)]. A reversed-phase C18 column (250 cm × 4.6 mm, 5 μm, 100 Å, Phenomenex, Torrance, USA) was used. The mobile phase was water (A) : acetonitrile (B) (35 : 65) v/v in isocratic mode. The pH of the water was adjusted to 3.8 with 1% glacial acetic acid (v/v). The flow rate was 1.0 mL/min. The column temperature was kept at 25°C, and the injection volume was 20 μL. A PDA detector was used (SPD-M20A, Shimadzu, Kyoto, Japan), with monitoring at 230 nm. Calibration curves for both CAP and DHC were made from analytical standards (Sigma-Aldrich, Saint Louis, USA), whose concentrations ranged from 1 to 80 μg/mL. Figure S1 shows the chromatogram of the standards. On the other hand, the pungency level of the samples was determined by calculations according to AOAC method No. 995.03 [21 ].
5
Quantifying Aflatoxins in Maize Kernels
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Homogenized maize kernel tissue (∼20–70 mg) was extracted with ethyl acetate/acetone (1:1)/0.1 % formic acid (1 ml) for 24 h at room temperature. The extracts were filtered through cotton plugs and the filtrates were concentrated under nitrogen to dryness. Each extract was re-dissolved in acetonitrile (1 mg/ml), filtered through a 0.22 μm Spin-X centrifuge tube filter, and analyzed on a Waters Acquity UPLC system (40% MeOH in water, BEH C18 1.7μm, 2.1 × 50 mm column) using fluorescence detection (Ex = 365 nm, Em = 440 nm). Samples were diluted 10-fold if the aflatoxin signal saturated the detector. Analytical standards (Sigma–Aldrich, St. Louis, MO, United States) were used to identify and quantify aflatoxins: aflatoxin B1 (AFB1, retention time = 4.60 min.); aflatoxin B2 (AFB2, retention time = 3.55 min.). Aflatoxin contents were expressed in ng/mg fresh weight of homogenized maize kernels.
6
GC-MS Analysis of Ajowan Essential Oils
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A total of 6 µL of the ajowan EOs and 594 µL of analytical-grade n-hexane (Sigma-Aldrich, Milan, Italy) were injected in split-mode (split ratio 1:50) into an Agilent 6890 N GC-MS system, endowed with a 5973 N single quadrupole detector and an autosampler 7863. As a stationary phase, a 5% phenyl-methylpolysiloxane coated capillary column (Agilent HP-5MS, 30 m length, 0.25 mm internal diameter, 0.1 μm film thickness) was used. The temperature program was set as follows: 5 min isothermal at 60 °C, then ramp at 4 °C/min to 22 °C and, finally, ramp at 11 °C/min until 280 °C, maintained for 15 min. The flow rate of the carrier gas (helium 99.5%) was 1 mL/min and both the injector and detector were at 280 °C. The mass spectra were achieved between 29.0 uma and 400.0 uma in a full scan through the electron impact mode (EI, 70 eV). The major chemical constituents of the T. ammi EOs, namely p-cymene, γ-terpinene, and thymol, as well as carvacrol, were identified using analytical standards (Sigma-Aldrich, Milan, Italy), whereas the other components were identified by combining the temperature-programmed retention indices (RIs) and the mass spectra (MS), as detailed in our previous study [59 (link)].
7
Enzymatic Tyrosinase Assay Protocol
8
Organic Acid Production Quantification
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To determine organic acid production, bacteria were grown in CDM [64 ] supplemented with 7.5 mM glucose as a carbon source. Cultures were centrifuged at 13,000 rpm for 5 min and supernatants were diluted 1:5 in water and filtered through 0.22-μm syringe filters (MSI, USA). Samples were analyzed by high-performance liquid chromatography (HPLC) (LC-20AT Prominence; Shimadzu Corp., Japan) equipped with a UV detector (SPD-20AV; Shimadzu Corp.) using an Aminex HTX-87H column (Bio-Rad Laboratories, USA) at 50 °C. The mobile phase consisted of 5 mM H2SO4 with a flow rate of 0.6 ml/min. Detection was performed at 210 nm and analytical standards (Sigma-Aldrich Co., USA) were used for quantification by external calibration curves.
9
HPLC method for tylosin A and D
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Plasma concentrations of tylosin A and D were measured by an in-house developed high-performance liquid chromatography (HPLC) method. Waters Alliance HPLC system (Waters, USA) equipped with a 2996 PDA detector and a Hypersil GOLD aQ (5 µm) 150 × 4.6 mm column (Thermo Fisher Scientific, USA) were used to separate and quantify both compounds. The mobile phase comprised 70% 0.02 M KH2PO4 (Sigma-Aldrich, Japan) at pH 2.4 and 30% acetonitrile (J.T. Baker, USA) and was set at a flow rate of 1 mL/min. Tylosin A and D were detected by UV absorption at 286 nm. Retention time for tylosin A was 10.5 min and for tylosin D it was 8.2 min. Plasma samples (0.5 mL) were extracted with 1.5 mL ethyl acetate for 15 min. To facilitate separation, samples were centrifuged at 37,000 ×g for 15 min and frozen in −70°C for 3 min. After that, supernatant was collected and dried in vacuum at 45°C. Dry residues were dissolved in 250 µL of water and transferred to autosampler vials. Plasma concentrations of tylosin A and D were calculated based on calibration curves prepared using blank plasma spiked with analytical standards (Sigma-Aldrich, Germany). Linearity, specificity, recovery, inter-assay and intra-assay coefficient of variation (CV) were assessed. The limit of quantification (LOQ) was determined as 10:1 signal-to-noise ratio, while limit of detection (LOD) was determined at a ratio of 3:1.
10
Hydrophobic Pesticides Extraction and Analysis
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The pesticides studied include three hydrophobic (log Kow > 4) compounds: CHL
Stock solutions of each pesticide in methanol were prepared using analytical standards purchased from Sigma-Aldrich (Steinheim, Germany) and stored in the dark at -20 °C until use.
Internal standards of d10-chlorpyrifos and phenoxy-d5-fenvalerate were also purchased from Sigma-Aldrich; d7-oxadiazon was purchased from LGC standards (Teddington, Middlesex, UK).
Ethyl acetate, acetone, water, dichloromethane, hexane, and methanol solvents were purchased form J.T. Baker (Waltham, Massachusetts, USA). Chloroform was purchased from Carlo Erba (Val De Reuil, Eure, France). Formic acid (98-100%) was purchased from Merck (Darmstadt, Germany). The quality of all solvents was according to organic trace analysis. Sulphuric acid was obtained from Scharlau (Spain), and mineral salts and other chemicals were purchased from Scharlab (Spain). GF/A and CG/C glass fibre filters were obtained from Whatman (GE Healthcare, USA). Table 1 (a) [32] , (b) [33] , (c) [34] (link), (d) [35] (link), (e) [36] (link)
Stock solutions of each pesticide in methanol were prepared using analytical standards purchased from Sigma-Aldrich (Steinheim, Germany) and stored in the dark at -20 °C until use.
Internal standards of d10-chlorpyrifos and phenoxy-d5-fenvalerate were also purchased from Sigma-Aldrich; d7-oxadiazon was purchased from LGC standards (Teddington, Middlesex, UK).
Ethyl acetate, acetone, water, dichloromethane, hexane, and methanol solvents were purchased form J.T. Baker (Waltham, Massachusetts, USA). Chloroform was purchased from Carlo Erba (Val De Reuil, Eure, France). Formic acid (98-100%) was purchased from Merck (Darmstadt, Germany). The quality of all solvents was according to organic trace analysis. Sulphuric acid was obtained from Scharlau (Spain), and mineral salts and other chemicals were purchased from Scharlab (Spain). GF/A and CG/C glass fibre filters were obtained from Whatman (GE Healthcare, USA). Table 1 (a) [32] , (b) [33] , (c) [34] (link), (d) [35] (link), (e) [36] (link)
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