The determination of AEA, PEA, OEA, and 2-arachidonoyl glycerol (2-AG) was done as previously described (Pastor et al., 2014 (link)). Briefly, aliquots of 175 μL of plasma were spiked with AEA-d4, PEA-d4, OEA-d4, and 2-AG-d5 (Cayman Chemical, Ann Harbor, MI), diluted up to 1 mL with 0.1M ammonium acetate pH 4.0 (Merck, Darmstadt, Germany), extracted with tert-butyl-methyl-ether (Merck) and analyzed by LC/MS-MS on a triple quadrupole mass spectrometer (Agilent 6410, Wilmington, DE) that operated with an electrospray ionization source (ESI) on the positive mode. Chromatographic separation was done with a Waters C18-CSH column (3.1 × 100 mm × 1.8 μm particle size), detection was done by selected reaction monitoring (SRM) and quantification was done by isotope dilution. 2-MGs were reported as the sum of isomer 1 and isomer 2 due to the instability of isomer 2 to isomerization.
Csh c18 column
Manufactured by Waters Corporation
Sourced in United States, Japan
The CSH C18 column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of compounds. It features a C18 stationary phase, which provides excellent retention and selectivity for a variety of organic molecules. The column is intended for use in analytical and preparative HPLC applications.
Automatically generated - may contain errors
Lab products found in correlation
Q exactive plus, by Thermo Fisher Scientific (4 mentions)
Lipidsearch software, by Thermo Fisher Scientific (3 mentions)
Nexera lc 30a, by Shimadzu (3 mentions)
Xevo tq s, by Waters Corporation (2 mentions)
I class uplc, by Waters Corporation (2 mentions)
Acquity uplc system, by Waters Corporation (2 mentions)
Progenesis qi, by Waters Corporation (2 mentions)
Facsaria 2, by BD (2 mentions)
Acquity uplc h class, by Waters Corporation (2 mentions)
Ultimate 3000 uhplc system, by Thermo Fisher Scientific (2 mentions)
Q exactive mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Acquity uplc 1 class, by Waters Corporation (2 mentions)
Vanquish uhplc system, by Thermo Fisher Scientific (2 mentions)
Aea d4, by Cayman Chemical (1 mentions)
2 ag d5, by Cayman Chemical (1 mentions)
Oea d4, by Cayman Chemical (1 mentions)
Pea d4, by Cayman Chemical (1 mentions)
Agilent 6410, by Agilent Technologies (1 mentions)
Tert butyl methyl ether, by Merck Group (1 mentions)
1290 infinity 2, by Agilent Technologies (1 mentions)
67 protocols using csh c18 column
1
Quantification of Endocannabinoids in Plasma
2
Quantitative UHPLC-MS/MS Analysis of Milk
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Samples were analyzed using UHPLC and tandem mass spectrometry (MS/MS), specifically by SRM. The UHPLC system (1290 Infinity II) and triple quadrupole mass analyzer (model 6495) were utilized (Agilent Technologies, CA, USA). Reversed-phase UHPLC separation utilized analytical column C18 CSH column (1.7 µm, 2.1 × 100 mm, Waters). MS/MS conditions were optimized using Optimizer software (Agilent Technologies). UHPLC separation was performed with water (A) and acetonitrile (B), both with an addition of 0.1% FA at 0.3 mL/min using 35 min analytical gradient (0.00 min 5% B, 25.00 min 30% B, 25.30 min 95% B, 30.00 min 95% B, 31.00 min 5% B, 35.00 min 5% B). Milk samples were analyzed in positive ion and dynamic SRM mode with a 3.5 min retention time window. The assay library is specified in supplementary materials (Supplemental Table 1 ). Five SRM transitions were used as qualifiers, and the most intense signal was used as a quantifier. The quantifiers are marked in bold in Supplemental Table 1 .
3
Rapid Quantification of Analytes by UPLC
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The Waters ACQUITY UPLC I-Class system was used for chromatographic analysis. By using a 20 min linear gradient, samples were transferred onto a C18 CSH column (100 mm × 2.1 mm, 1.7 μm; Waters) at 45°C at a flow rate of 0.4 mL/min and then chromatographic gradient elution procedure was performed. Mobile phase buffer A consisted of acetonitrile/water (1/4), 0.1% formic acid, and 10 mM ammonium format, and buffer B consisted of acetonitrile/isopropanol (1/9), 0.1% formic acid, and10 mM ammonium format.
4
Lipid Profiling by LC-MS/MS
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The charged surface hybrid (CSH) C18 column (1.7 µm, 2.1 mm, 100 mm; Waters, Milford, Massachusetts, USA) was chosen for the LC separation utilizing reverse phase chromatography. Then, 3 µL of the sample was injected after the lipid extracts were redissolved in 200 L of 90% isopropanol/acetonitrile and centrifuged at 14,000 g for 15 min. Acetonitrile-water (6:4 v/v), which also contained 0.1 mM ammonium formate and 0.1% formic acid, served as solvent A, while acetonitrile-isopropanol (1:9 v/v) with 0.1% formic acid and 0.1 mM ammonium formate served as solvent B. The initial mobile phase was 30% solvent B at a flow rate of 300 µL/min. After equilibrating at 5% solvent B for 10 min, it was linearly raised to 100% solvent B in 23 min.
Q-Exactive Plus (Thermo Fisher Scientific, USA) was used to acquire mass spectra in both positive and negative modes. The electron spray ionization (ESI) parameters were optimized and preset for all measurements as follows: source temperature, 300 ℃; capillary temperature, 350 ℃; ion spray voltage, 3,000 V; S-Lens radio frequency level, 50%; scanning range, 200–1,800 m/z.
Q-Exactive Plus (Thermo Fisher Scientific, USA) was used to acquire mass spectra in both positive and negative modes. The electron spray ionization (ESI) parameters were optimized and preset for all measurements as follows: source temperature, 300 ℃; capillary temperature, 350 ℃; ion spray voltage, 3,000 V; S-Lens radio frequency level, 50%; scanning range, 200–1,800 m/z.
5
UPLC-QTOF-MS Analysis of Compound Mixtures
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The UPLC was equipped with an CSH C18 column (1.7 μm, 2.1 × 100 mm; Waters) and a PDA detector (Waters, Inc.) to analyze samples at a flow rate of 0.3 ml/min at 45°C. The sample injection volume was 2 µl. Elution A was acetonitrile, and elution B was H2O containing 1% (v/v) formic acid. The gradient elution was performed as follows: 0–4 min, 100%–95% B; 4–6 min, 95–90% B; 6–7 min, 90–60% B; 7–8 min, 60%−20% B; 8–10 min, 20%–0% B; 10–12 min, 0% B.
ESI-MS spectra were obtained in negative ionization mode using MALDI SYNAPT Q-TOF MS (Waters, Inc.). In this ESI-MS run program, the capillary voltage was 3.0 kV, the detection voltage was 2.0 kV, and the cone voltage was 20 V. The source block temperature and desolation temperature were 100 and 400°C, respectively. The desolation gas flow rate and the cone gas flow rate were set to 700 L/h and 500 L/h, respectively. The ESI-MS spectra spanned the mass range of 50–2000 m/z at a collision energy (eV) of 6/25 V. Besides, the mass was corrected by flow rate of 30 μl/min with 200 pg/μl of leucine enkephalin, and the corrected mass was 554.2615 Da in negative ion mode.
ESI-MS spectra were obtained in negative ionization mode using MALDI SYNAPT Q-TOF MS (Waters, Inc.). In this ESI-MS run program, the capillary voltage was 3.0 kV, the detection voltage was 2.0 kV, and the cone voltage was 20 V. The source block temperature and desolation temperature were 100 and 400°C, respectively. The desolation gas flow rate and the cone gas flow rate were set to 700 L/h and 500 L/h, respectively. The ESI-MS spectra spanned the mass range of 50–2000 m/z at a collision energy (eV) of 6/25 V. Besides, the mass was corrected by flow rate of 30 μl/min with 200 pg/μl of leucine enkephalin, and the corrected mass was 554.2615 Da in negative ion mode.
6
Quantitative Lipidomic Analysis by UPLC-MS
7
Quantitative Analysis of Protein Cleavage
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This analysis was performed on an Aquity UPLC-I-Class (SM-FTN) coupled to a Xevo TQ-S Triple Quadrupole mass spectrometer (Waters) equipped with an electrospray ionization (ESI) source in multiple reaction monitoring (MRM) mode. The cleavage products were chromatographically separated on a 150 × 2.1 mm i.d., 1.7 μm, 130 Å CSH-C18 column (Waters). The mobile phase consisted of 1% formic acid in H2O (eluent A) and 1% formic acid in acetonitrile:H2O (8:2 v/v; eluent B) in a linear gradient. Identification criteria included retention time and at least two MRM transitions for each cleavage product with the expected transition intensity ratios. The cleaved N- and C-terminal products were qualitatively evaluated in comparison to positive and negative control samples (normal human serum, NHS, with or without spiked toxin).
8
Lipid Extraction and Profiling by MTBE-LC-MS
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Lipids were extracted according to MTBE method. Briefly, samples were homogenized with 200 µl water and 240 µl methanol. Then 800 µl of MTBE was added and the mixture was exposed to ultrasound for 20 min at 4 °C followed by sitting still for 30 min at room temperature. The solution was centrifuged at 14,000 × g for 15 min at 10 °C and the upper organic solvent layer was obtained and dried under nitrogen. Reverse phase chromatography was selected for LC separation using CSH C18 column (1.7 µm, 2.1 mm × 100 mm, Waters). The lipid extracts were re-dissolved in 200 µl of 90% isopropanol/ acetonitrile, centrifuged at 14,000 × g for 15 min, and finally 3 µl of sample was injected. Solvent A was acetonitrile–water (6:4, v/v) with 0.1% formic acid and 0.1 mM ammonium formate, and solvent B was acetonitrile–isopropanol (1:9, v/v) with 0.1% formic acid and 0.1 mM ammonium formate. The initial mobile phase was consisted of 30% solvent B at a flow rate of 300 µl/min. It was held for 2 min, and then linearly increased to 100% solvent B in 23 min, followed by equilibrating at 5% solvent B for 10 min. Mass spectraspectrometry was acquired by performed using a Q-Exactive Plus spectrometer (Thermo Fisher Scientific) in positive and negative mode, respectively76 (link).
9
Targeted Metabolite Separation and Detection
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Equipments for separation and detection of metabolites were the same as that for untargeted metabolomics above.
Chromatographic separation was performed on CSH C18 column (Waters, USA). At positive ion mode with mobile phase A consisting 60% acetonitrile in water + 10 mM ammonium formate + 0.1% formic acid and mobile phase B consisting 90% isopropanol + 10% acetonitrile + 10 mM ammonium formate + 0.1% formic acid. At positive ion mode, with mobile phase A consisting 60% acetonitrile in water + 10 mM ammonium formate and mobile phase B consisting 90% isopropanol + 10% acetonitrile + 10 mM ammonium formate. The column temperature was maintained at 55°C. The gradient conditions were as follows: 40-43% B over 0~2 min, 43-50% B over 2-2.1 min, 50-54% B over 2.1-7 min, 54-70% B over 7-7.1 min, 70-99% B over 7.1-13 min, 99-40% B over 13-13.1 min, held constant at 99-40% B over 13.1~15 min and washed with 40% B over 13.1-15 min. The flow rate was 0.4 mL/min and the injection volume was 5 μL.
Q Exactive perform was used for primary and secondary mass spectrometry data acquisition. The stepped normalized collision energy was set to 15, 30 and 45 eV. All other parameters were set the same as that above.
Chromatographic separation was performed on CSH C18 column (Waters, USA). At positive ion mode with mobile phase A consisting 60% acetonitrile in water + 10 mM ammonium formate + 0.1% formic acid and mobile phase B consisting 90% isopropanol + 10% acetonitrile + 10 mM ammonium formate + 0.1% formic acid. At positive ion mode, with mobile phase A consisting 60% acetonitrile in water + 10 mM ammonium formate and mobile phase B consisting 90% isopropanol + 10% acetonitrile + 10 mM ammonium formate. The column temperature was maintained at 55°C. The gradient conditions were as follows: 40-43% B over 0~2 min, 43-50% B over 2-2.1 min, 50-54% B over 2.1-7 min, 54-70% B over 7-7.1 min, 70-99% B over 7.1-13 min, 99-40% B over 13-13.1 min, held constant at 99-40% B over 13.1~15 min and washed with 40% B over 13.1-15 min. The flow rate was 0.4 mL/min and the injection volume was 5 μL.
Q Exactive perform was used for primary and secondary mass spectrometry data acquisition. The stepped normalized collision energy was set to 15, 30 and 45 eV. All other parameters were set the same as that above.
10
Quantitative Analysis of Saponin Concentrations
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Dry matter content was measured using a precision electronic balance (AB204-N, Mettler Toledo Shanghai Co., Ltd). The samples were dried at 105 °C in an oven (DZF-6050, Shanghai Jing Hong Laboratory Instrument Co., Ltd.) for 3 h and then kept in a desiccator for 0.5 h. The quantitative analysis method for the determination of the saponin concentrations was reported in previous work [26 (link)]. The method was performed on a Waters Acquity UPLC system (Waters, Milford, MA, USA) using a Waters CSH C18 column (50 mm × 2.1 mm i.d., 1.7 μm) at 40 °C. The mobile phase consisted of 0.01% formic acid–water (A) and 0.01% formic acid–acetonitrile (B) using the following gradient program: 0–6 min, 18–20% B; 6–6.8 min, 20–30% B; 6.8–11 min, 30–35% B; 11–17 min, 35–90% B; and 17–25 min, 90% B. The flowrate was 0.35 mL/min, and the volume of the sample injection was 5 μL. The detector wavelength was set to 203 nm.
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