The SCIEXSCIEX QTRAP 6500+ mass spectrometer and a Shimadzu LC30AD liquid chromatography system was used to analyze the supernatant. The Waters XBridge Amide (100 mm × 4.6 mm i.d., 3.5 μm) was used for LC separation. A 5μL sample was needed. The electrospray ionization mass spectra were acquired in positive ion mode (4850 V ion spray voltage) and negative ion mode (4500 V ion spray voltage), respectively. The multiple reaction monitoring (MRM) acquisition methods were used to collect MS information simultaneously. The heated capillary temperature was maintained at 475 °C. The curtain gas flow, nebulizer, and heater gas were set to 25, 33, and 33 arbitrary units, respectively.
Qtrap 6500 mass spectrometer
Manufactured by Shimadzu
Sourced in United States
The QTRAP 6500 mass spectrometer is a high-performance analytical instrument designed for a wide range of applications. It features a triple quadrupole configuration that enables sensitive and selective detection of target analytes in complex samples. The instrument provides accurate mass measurement and advanced data acquisition modes to support comprehensive qualitative and quantitative analyses.
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4 protocols using qtrap 6500 mass spectrometer
1
Metabolite Profiling via QTRAP LC-MS
2
Quantifying Plant Metabolite Profiles
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Soluble sugars, sugar phosphates, organic acids, and free amino acids were extracted with methanol/chloroform/water using a protocol modified from Ma et al. (2017) (link) (Supplemental Methods ), taking care to keep samples chilled throughout. The extracted filtered samples were run on an AB Sciex QTRAP 6500 mass spectrometer linked to a Shimadzu HPLC, with ions being detected and monitored using a targeted multiple-reaction monitoring (MRM) approach. Metabolites were eluted and run using parameters as previously described (Czajka et al., 2020 (link)), using positive ionization for amino acids and negative ionization for all other metabolites. Protein quantification was performed by directly hydrolyzing the protein from leaf samples and quantifying the resulting individual amino acids as previously described (Kambhampati et al., 2019 (link)). Metabolite concentrations and recoveries were calculated based on external calibration curves and internal standards (PIPES for sugar phosphates and organic acids, ribitol for sugars, and norvaline for amino acids). A 1/x2 weighting factor was applied for external calibration curves as suggested by Gu et al. (2014) (link).
3
Quantification of Endocannabinoids in Serum
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AEA, 2-AG, AA, OEA, and PEA were extracted, purified, and quantified in serum by stable isotope dilution liquid chromatography/tandem mass spectrometry (LC-MS/MS) as previously described [22 (link),23 (link),24 (link)]. LC-MS/MS analyses were conducted on a Sciex (Framingham, MA, USA) QTRAP® 6500+ mass spectrometer coupled with a Shimadzu (Kyoto, Japan) UHPLC system. Liquid chromatographic separation was obtained using 5 μL injections of samples onto a Kinetex 2.6 μm C18 (100 × 2.1 mm) column from Phenomenex (Torrance, CA, USA). The autosampler was set at 4 °C, and the column was maintained at 40 °C during the entire analysis. Endocannabinoids were detected in positive ion mode using electron spray ionization (ESI) and a multiple reaction monitoring (MRM) mode of acquisition, using d4-AEA as internal standard (IS). The IonDriveTM Turbo V source temperature was set at 450 °C with an ion spray voltage of 4000 V. The curtain gas was set at 30.0 psi. The nebulizer gas (Gas 1) was set to 40 psi, and the turbo heater gas (Gas 2) was set to 40 psi. The dwell time was set to 30 ms. The collision energy (CE), declustering potential (DP), and collision cell exit potential (CXP) for the monitored transitions are listed in Table 1 . The LC-MS/MS chromatogram is presented in Figure 1 . The serum levels of AEA, 2-AG, OEA, PEA, and AA were measured in duplicate against standard curves.
4
Quantifying Plant Metabolite Profiles
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