High-performance liquid chromatography-electrospray mass spectrometry (HPLC-ES/MS) was applied (HPLC-MS), using commercial capsaicin (Cayman Chemical, Ann Arbor, MI, USA) as the standard, as described earlier [51 (link)]. Three replicates from five pepper fruit samples (pericarp and placenta from the four varieties at the two ripening stages, green and red) were assayed. Basically, samples (0.5 g) were ground into a powder under liquid N2 and suspended into 2 mL AcN containing 100 ppm N-[(3,4-dimethoxyphenyl)methyl]-4-methyl-octanamide (DMBMO), as an internal standard. In our experimental conditions, an XBridge 2.1 × 10 mm pre-column and an XBridge 2.1 × 100 mm C18 3.5 µm column (Waters Corporation, Milford, MA, USA) were connected to an Alliance 2695 HPLC system coupled to a Micromass Quattro micro API triple quadrupole mass spectrometer (Waters). The retention time for capsaicin in our experimental conditions was 1.88 min, and the concentration of capsaicin was expressed as μg g−1 of fresh weight [51 (link)].
Micromass quattro micro api triple quadrupole mass spectrometer
Manufactured by Waters Corporation
Sourced in United States
The Micromass Quattro micro API triple quadrupole mass spectrometer is a high-performance analytical instrument used for precise and sensitive detection and quantification of chemical compounds. It utilizes tandem mass spectrometry technology to provide accurate mass measurements and structural information about molecules.
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4 protocols using micromass quattro micro api triple quadrupole mass spectrometer
1
Quantifying Capsaicin in Pepper Varieties
2
Quantifying PFCs in Breast Milk
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PFCs in breast milk were identified and quantified using high-performance liquid chromatography (2695 separation module) coupled with a Micromass Quattro micro API triple quadrupole mass spectrometer from (Waters, Milford, MA, USA). Then, 10 μL of the extract was injected onto a 2.1 mm × 100 mm, 3.5 μm Xterra MS C18 column (Waters, Milford, MA, USA) at a flow rate of 0.3 ml/min. The mobile phase was 2 mM ammonium acetate/methanol at a flow rate of 300 μL/min. The gradient begun at 10% methanol, amplified to 99% methanol at 12 min, and was kept for 3 min before switching back to 10% methanol and holding for 3 min for a total run time of 18 min. The API was executed in electrospray negative ionization mode with a source temperature of 100 °C, desolvation temperature of 250 °C, desolvation gas flow of 500 L/h, and cone gas flow of 200 L/h. The target compounds were identified using multiple reaction monitoring. Table 1 presents each analyte’s compound-specific MS/MS parameters and mass transitions.
3
Quantification of GSNO and GSH in Arabidopsis
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Arabidopsis samples (300 mg) were ground using a mortar and pestle in the presence of 1 ml of 0.1 M HCl. Homogenates were centrifuged at 15,000 g for 20 min at 4°C. The supernatants were collected and filtered through 0.22-mm polyvinylidene fluoride filters and immediately analyzed. All procedures were carried out at 4°C and were protected from light to avoid potential degradation of the analytes (GSNO and GSH). The LC–ES/MS system consisted of a Waters Allience 2695 HPLC system connected to a Micromass Quattro micro API triple quadrupole mass spectrometer, both obtained from the Waters Corporation. HPLC was carried out using an Atlantis® T3 3 μm 2.1 mm × 100 mm Column obtained from the Waters Corporation. The Micromass Quattro Micro API mass spectrometer was used in positive electrospray ionization mode for detection and quantification of GSNO and GSH (Airaki et al., 2011 (link); Leterrier et al., 2012 (link)).
4
Quantification of Choline and Betaine
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Endogenous (unlabeled) choline and its derivative betaine as well as [methyl-D 9 ]choline, [methyl-D 9 ]betaine and the internal standard [1,1,2,2-D 4 ]choline were analyzed with electrospray ionization tandem mass spectrometry (ESI-MS/MS) [24] , using an Atlantis® HILIC Silica 3µm column (2.1x30mm; 50°C) using a HP1100 LC system (Agilent Technologies, Heilbronn, Germany) on a Waters Micromass Quattro Micro API triple quadrupole mass spectrometer (Waters, Dreieich, Germany), supplied with an electrospray ionization interface. Components were analyzed by specific reaction monitoring (SRM) in the positive ionization mode, using mass by charge (m/z) transitions of 104 60 (choline), 108→61 (D 4 -choline), 113→69 ([D 9 -methyl]-choline), 118→59 (betaine), 127→68 ([D 9methyl]-betaine), 184→86 (phosphocholine) and 193→95 (D 9 -phosphocholine). Samples were quantified by comparing the areas of each SRM with external calibration curves for the respective metabolite using D 4 -Choline as internal standard. Comparison of EDTA plasma with serum of control samples (N=7) showed no difference for choline (means±SE) (13.07±1.05 and 12.68±0.69µmol/L, respectively; p=0.4505) whereas for betaine serum values were 27.8% increased in serum by paired t-test comparison (32.82±5.41 and 41.93±6.5µmol/L, respectively; p<0.0004). Consequently, serum betaine was corrected by multiplying values by 0.7827.
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