Proteinogenic amino acids (i.e., alanine, isoleucine, leucine, phenylalanine, tyrosine, and valine) were quantified in the aqueous extracts of the cocoa beans, in duplicate, by UPLC-MS/MS, using an Acquity UPLC system, equipped with a HSS T3 column and coupled to a Quattro Micro tandem mass spectrometer with a ZSpray electrospray ionization source in the positive ionization mode (Waters). The flow rate of the mobile phase, composed of 5% (v/v) acetonitrile (Thermo Fischer Scientific) with 1 mM formic acid (Merck) and 1 mM pentadecafluorooctanoic acid (PDFOA, Sigma-Aldrich; eluent A) and 90% (v/v) acetonitrile with 1 mM formic acid and 0.5 mM PDFOA (eluent B; Sigma-Aldrich), was 0.23 ml/min. For elution, the following gradient was applied: 0 to 1 min, 99% eluent A and 1% eluent B; 1 to 8 min, 30% eluent A and 70% eluent B; 8 to 10 min, 0% eluent A and 100% eluent B; and 10 to 25 min, 99% eluent A and 1% eluent B. Quantification was performed by external calibration, including an IS solution [8 mg of 2-amino butyric acid (IS) dissolved in 900 ml of ultrapure water (MilliQ) and 1 mM formic acid (Sigma-Aldrich)]. All samples were microcentrifuged (19,400 × g for 15 min at 10°C) and filtered (0.2-μm H-PTFE Millex filters, Merck) before injection (10 μl) into the column.
Formic acid
Manufactured by Merck Group
Sourced in Germany, United States, Italy, United Kingdom, France, Spain, China, Poland, India, Switzerland, Sao Tome and Principe, Belgium, Australia, Canada, Ireland, Macao, Hungary, Czechia, Netherlands, Portugal, Brazil, Singapore, Austria, Mexico, Chile, Sweden, Bulgaria, Denmark, Malaysia, Norway, New Zealand, Japan, Romania, Finland, Indonesia
Formic acid is a colorless, pungent-smelling liquid chemical compound. It is the simplest carboxylic acid, with the chemical formula HCOOH. Formic acid is widely used in various industrial and laboratory applications.
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Lab products found in correlation
Acetonitrile, by Merck Group (1365 mentions)
Methanol, by Merck Group (1060 mentions)
Ammonium formate, by Merck Group (404 mentions)
Acetic acid, by Merck Group (389 mentions)
Gallic acid, by Merck Group (295 mentions)
Ammonium acetate, by Merck Group (287 mentions)
Milli q system, by Merck Group (275 mentions)
Acetonitrile, by Thermo Fisher Scientific (256 mentions)
Hydrochloric acid (hcl), by Merck Group (239 mentions)
Ethanol, by Merck Group (239 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (226 mentions)
Sodium hydroxide (naoh), by Merck Group (217 mentions)
Methanol, by Thermo Fisher Scientific (205 mentions)
Hplc grade acetonitrile, by Merck Group (195 mentions)
Quercetin, by Merck Group (193 mentions)
Milli q water purification system, by Merck Group (178 mentions)
Ammonium bicarbonate, by Merck Group (169 mentions)
Iodoacetamide, by Merck Group (167 mentions)
Trifluoroacetic acid, by Merck Group (153 mentions)
Ethyl acetate, by Merck Group (152 mentions)
4 933 protocols using formic acid
1
Quantification of Amino Acids in Cocoa Beans
2
HPLC Quantification of Polyphenols
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Reagents
The reagents were as follows: a; H2O-deionized high pressure liquid chromatography (HPLC) water, b; acetonitrile (Merck, for liquid chromatography), c; methanol (Merck, for liquid chromatography), d; formic acid (Merck), e; chemical reference standards containing catechin (≥98% HPLC), caffeic acid (≥99.0% HPLC), ferulic acid (≥99.0% HPLC), and taxifolin (≥98% HPLC) all from Sigma Aldrich, f; HPLC mobile phase which mobile phase A was 0.1 % formic acid in H2O, and mobile phase B was 0.1 % formic acid in acetonitrile.
The reagents were as follows: a; H2O-deionized high pressure liquid chromatography (HPLC) water, b; acetonitrile (Merck, for liquid chromatography), c; methanol (Merck, for liquid chromatography), d; formic acid (Merck), e; chemical reference standards containing catechin (≥98% HPLC), caffeic acid (≥99.0% HPLC), ferulic acid (≥99.0% HPLC), and taxifolin (≥98% HPLC) all from Sigma Aldrich, f; HPLC mobile phase which mobile phase A was 0.1 % formic acid in H2O, and mobile phase B was 0.1 % formic acid in acetonitrile.
3
Quantification of All-trans Retinoic Acid
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ATRA was quantified in serum as previously described (43 (link)). Briefly, 100 μl of serum and 1 μl of ATRA-d5 [50 μg/ml; internal standard, Toronto Research Chemicals (TRC)] were transferred to a 15-ml glass culture tube and extracted in 200 μl of acetonitrile (Sigma-Aldrich) and 2-μl formic acid (Sigma-Aldrich) and 10 ml of hexanes (Sigma-Aldrich). The hexane extracts was separated by centrifugation, dried under nitrogen flow, and reconstituted in 100 μl of acetonitrile for LC-MS/MS analysis. The whole process was performed on ice and under yellow light to avoid the degradation.
The concentration of ATRA from serum was measured using the AB Sciex 4000 QTRAP LC-MS/MS System equipped with atmospheric-pressure chemical ionization (APCI) in positive ion mode. Briefly, ATRA was separated with the 2.1 mm by 100 mm Supelcosil ABZ + PLUS column (3 μm; Sigma-Aldrich) with the following running solvents: A, H2O with 0.1% formic acid; B, acetonitrile with 0.1% formic acid. Quantification is based on peak area ratio of ATRA to the ATRA-d5 and the ATRA-d5 concentration.
The concentration of ATRA from serum was measured using the AB Sciex 4000 QTRAP LC-MS/MS System equipped with atmospheric-pressure chemical ionization (APCI) in positive ion mode. Briefly, ATRA was separated with the 2.1 mm by 100 mm Supelcosil ABZ + PLUS column (3 μm; Sigma-Aldrich) with the following running solvents: A, H2O with 0.1% formic acid; B, acetonitrile with 0.1% formic acid. Quantification is based on peak area ratio of ATRA to the ATRA-d5 and the ATRA-d5 concentration.
4
Quantitative Analysis of Organic Compounds
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Acetonitrile, methanol, and acetone (HPLC grade, Riedel-de-Haën, Seelze, Germany) were used as solvents as well as 0.1% formic acid (Merck, Darmstadt, Germany) in ultrapure water. The ultrapure water with resistance 18.2 MΩ cm and TOC < 5 ppb was prepared by Milli-Q IQ 7000 device from Merck (Darmstadt, Germany).
The aqueous mobile phase components were 0.1% formic acid solution (pH 2.7), 5.0 mM ammonium bicarbonate buffer (pH 8.0), and 0.1% ammonium solution (pH 10.0). formic acid, ammonium bicarbonate were purchased from and ammonium (25%, MS grade) was purchased from Merck (Darmstadt, Germany). The organic phase used was acetonitrile (HPLC grade, Riedel-de-Haën, Seelze, Germany).
The aqueous mobile phase components were 0.1% formic acid solution (pH 2.7), 5.0 mM ammonium bicarbonate buffer (pH 8.0), and 0.1% ammonium solution (pH 10.0). formic acid, ammonium bicarbonate were purchased from and ammonium (25%, MS grade) was purchased from Merck (Darmstadt, Germany). The organic phase used was acetonitrile (HPLC grade, Riedel-de-Haën, Seelze, Germany).
5
Phosphopeptide Enrichment and Isobaric Labeling
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The major fraction of lyophilized peptides (95%) were re-dissolved in 28.5% lactic acid (Sigma), 57% acetonitrile (LC-MS grade, Sigma), 0.28% TFA (Sigma), and applied to TiO2 spin tips (88303, Thermo Pierce) for phosphopeptide enrichment according to manufacturer’s protocol. Eluted phosphopeptides were acidified with formic acid (pH 2-3), desalted using ZipTip C18 tips (100 μL, Millipore), and lyophilized. The two sets of peptides (phospho-enriched and unenriched) were separately labeled with 10-plex isobaric tandem mass tags (90406, Thermo Scientific) according to manufacturer’s protocol with slight modification. TMT reagents were reconstituted to 8 mg/mL in anhydrous acetonitrile (Sigma) and added to lyophilized peptides dissolved in 100 uL of 200 mM HEPES buffer, pH 8.0 (~8:1 reagent/peptide ratio). Labeling reaction was carried out in room temperature for 1 hr with gentle shaking, and quenched with 5 uL of 5% hydroxylamine (Thermo Scientific). Labeled peptides were combined into a single pool per experiment, acidified with formic acid (pH 2-3), desalted using ZipTip C18 tips (100 μL, Millipore), and lyophilized. The final processed peptides were dissolved in LC-MS/MS Buffer A (H2O with 0.1% formic acid, LC-MS grade, Sigma Aldrich) for LC-MS/MS analysis.
6
Aqueous and Organic Fraction Analysis
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Aqueous fractions were collected as described above and transferred to an ultrafiltration tube and the solution filtered by centrifugation at 9,000 g for 2 hours at 4 °C. 350 μl were recovered after filtration and concentrated during 3 hours and further resuspended in 50 μl of Milli-Q H2O for the LC/MS and CE/MS analysis. Organic fractions were concentrated and resuspended in isopropanol 70% (Sigma, UK), and the mixture were analyzed by nanoflow LC/MS. LC/MS was performed by liquid chromatography coupled to a quadrupole time of-flight mass spectrometer (Agilent Chip/6250 QTOF-MS) using a Zorbax 80 SB-C18 Chip Agilent (5μm 150mm x 75μm 2.5mm, 500nl) using a gradient of buffers A (2% acetonitrile, Millipore, USA, 98% Milli-Q H2O and 0.1% Formic Acid Sigma, UK) and B (95% acetonitrile, 5% Milli-Q H2O and 0.1% Formic Acid Sigma) ; B: 0% to 40%, minute 0 to 11; 40% to 100%, minute 11 to 14; 100% minute 14 to 15 and 100% to 0%, minute 14 to 15. The CE/MS analysis was performed using an Agilent G1603A CE-TOFMS system as previously described [75 (link), 76 (link)].
7
Phosphopeptide Enrichment and Isobaric Labeling
8
Simultaneous Determination of Ractopamine and Salbutamol
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Reference standard ractopamine and salbutamol were purchased from Sigma–Aldrich (St. Louis, MO, USA). Internal standard ractopamine-d5 and salbutamol-d6 were purchased from RIVM (Bilthoven, Netherlands). Methanol, acetonitrile, ethyl acetate and dichloromethane, all of LC grade, were purchased from Merck (Darmstadt, Germany). Sodium hydroxide, hydrochloric acid (37%) and formic acid (98–100%), all of the reagent grade, were purchased from Merck (Darmstadt, Germany). Ammonium formate solution (10M) was purchased from Sigma–Aldrich (St. Louis, MO, USA). Alcohol (95%) was purchased from ECHO chemical (Miaoli, Taiwan). De-ionized water (18.2 MΩ) was purified by the Millipore Synergy 185 ultrapure water system (Billerica, MA).
Formate buffer (pH = 3.0) was prepared according to a standard protocol used in the lab by adding formic acid 97%–0.01% at 5 mM ammonium formate in a de-ionized solution.
Formate buffer (pH = 3.0) was prepared according to a standard protocol used in the lab by adding formic acid 97%–0.01% at 5 mM ammonium formate in a de-ionized solution.
9
Multi-Pesticide Quantification by LC-MS
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Fifty-three pesticide standards (high purity grade, >90%) were purchased from Dr. Ehrenstorfer GmbH. Acetonitrile (ACN, LC-MS grade), methanol (MeOH, LC-MS grade) for HPLC with 99.80% of purity, and formic acid (FA, ACS reagent) were from Merck (Merck, Singapore). ammonium formate (LC-MS grade, Sigma-Aldrich, Singapore) was also used for preparation of the mobile phase. Standard compounds were classified into 30 groups and used to prepare individual stock solutions around 1000 μg·mL−1 in appropriate solvents such as acetone (GC-MS grade), methanol, n-hexane (GC-MS grade), acetonitrile, and ethanol (ACS reagent) in amber vials. The mixed standard solutions of all target analytes (10 μg·mL−1) were prepared and diluted with acetonitrile. Stock standards were stored in the amber LC vial at 4°C. The working standard solutions were daily prepared by diluting the mixed standard solution in the mobile phase. The mobile phase was daily prepared by dissolving appropriate amount of ammonium formate in methanol/deionized water (Milli-Q Integral 3, Merck Millipore, France) containing 0.1% formic acid. The mobile phase was degassed in the ultrasonic bath (S 100H, Elma, Germany) to eliminate dissolved gas.
10
Quantitative Proteomic Sample Preparation
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N-Ethylmaleimide, 6-aminocaproic acid, benzamidine hydrochloride hydrate, dithiothreitol (DTT), iodoacetamide, ammonium bicarbonate (AMBIC), formic acid, and HPLC grade acetonitrile and Solvents A (0.1% formic acid) and B (0.1% formic acid in acetonitrile) for liquid chromatography-mass spectrometry (LC-MS) were from Sigma–Aldrich (St. Louis MO, USA). Guanidine hydrochloride (GdnHCl) and anhydrous sodium acetate (NaAc) were from Merck (Darmstadt, Germany). Trypsin Gold (MS grade) was purchased from Promega (Madison WI, USA). The Pierce Quantitative Colorimetric Peptide Assay and SOLAμ™ Solid Phase Extraction (SPE) HRP (horse radish peroxidase) 2mg/1 ml 96-well plates were from Thermo Fisher Scientific (Rockford IL, USA), and Nanosep® 30K Omega Centrifugal Devices were from Pall Life Sciences (Ann Arbor MI, USA).
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