LC-MS/MS methods were based on EPA Standards 537 and 533 for PFAS [63 ,64 ], and EPA Method 544 for microcystins [65 ]. PFAS samples were first extracted and concentrated using weak anion exchange (WAX) solid phase extraction cartridges (Waters, Mississauga, ON, Canada), while microcystin samples were analyzed using direct injection. The LC-MS/MS system included an Agilent Poroshell EC-C18 column (Agilent, Santa Clara, CA, USA). Mobile phases consisted of water and acetonitrile, which both contained 0.1% acetic acid (for PFAS) or 0.1% formic acid (for microcystins), were applied under gradient conditions. Injection volumes were 100 μL for PFAS and 40 μL for microcystins. Sample run times were 8 min for PFAS and 9 min for microcystins at a flowrate of 0.3 mL/min. The LC system was coupled to an Agilent 6460 Triple Quadrupole Mass Spectrometer system operating in electrospray ionization negative (ESI−) mode for PFAS or electrospray ionization positive (ESI+) mode for microcystins, incorporating multiple reaction monitoring (MRM). For PFAS, 13C4-PFOA, 13C4-PFOS, 13C4-PFBA, and 13C4-PFBS were used as internal standards to monitor the relative response and quantify analytes.
Agilent poroshell ec c18 column
Manufactured by Agilent Technologies
Sourced in United States
The Agilent Poroshell EC-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 core-shell particle technology that provides efficient and rapid separations. The column is suitable for use in various HPLC applications.
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Lab products found in correlation
6420 triple quadrupole mass spectrometer, by Agilent Technologies (1 mentions)
Whatman, by Cytiva (1 mentions)
Agilent 1260 series hplc system, by Agilent Technologies (1 mentions)
Agilent 6460 triple quad lc ms system, by Agilent Technologies (1 mentions)
Agilent 1200 hplc system, by Agilent Technologies (1 mentions)
Freezing grinder, by Jingxin (1 mentions)
5 protocols using agilent poroshell ec c18 column
1
Sensitive LC-MS/MS Analysis of PFAS and Microcystins
2
Amino Acid Profile Analysis Protocol
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Approximately 25 mg of samples were suspended in 5 mL of 6 M HCl with boiling chips. Samples were then purged with nitrogen for 30 s and hydrolysed at 110 • C for 20 h. After hydrolysis, samples were cooled down to room temperature and filtered using No. 1 Whatman paper. Then, 10 µL of the hydrolysate was taken and suspended in 500 µL of 0.2 M sodium citrate buffer (pH 3), followed by derivatisation using the AccQ.Tag reagent, as previously described by Salazar et al. [25] (link) with some modifications. The derivatised solution was injected into an LC-MS, which consists of a 1260 Infinity Liquid Chromatography coupled with a 6420 Triple Quadrupole Mass Spectrometer (Agilent Technologies, Santa Clara, CA, USA). The chromatographic separation was performed using an Agilent Poroshell EC C-18 column (2.1 × 150 mm, 2.7 µm, Agilent Technologies, Santa Clara, CA, USA). The calibration curve ranged from 0.78-200 µM of amino acid standard mix solution spiked with glutamine and asparagine. In this method, cysteine, methionine, and tryptophan content may be lower than expected due to degradation via acid hydrolysis. Amino acid contents were expressed as g per 100 g protein.
3
Quantitative Analysis of Berberine and Metabolites by LC-MS
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Agilent 6460 Triple Quad LC-MS system (Agilent, Palo Alto, America) coupled with an Agilent 1260 series HPLC system was used for the analysis of berberine and its metabolites using the modified method of Feng et al. (2020) (link).
The separation was performed on an Agilent poroshell EC-C18 column (3.0 mm × 50 mm, 2.7 μm; Agilent, United States) using a gradient elution of the mobile phase that consisted of methanol (A) and water with 0.1% formic acid (B) at a flow rate of 0.5 ml/min. The composition of the mobile phase was as follows: 70% B for 0–0.5 min, 30% B for 1.2–2.5 min, and 70% B for 2.6–5 min. Mass spectra were recorded by electrospray ionization with a positive mode. Quantification was carried out using MRM at m/z 336.0 → 320.3/292.3 for berberine, m/z 322.1 → 307.1/279.1 for berberrubine (BBB), and m/z 356.2 → 192.2/165.2 for tetrahydropalmatine. Calibration was applied on a standard curve in the range of 0.05–100 ng/ml of berberine and BBB, respectively.
The separation was performed on an Agilent poroshell EC-C18 column (3.0 mm × 50 mm, 2.7 μm; Agilent, United States) using a gradient elution of the mobile phase that consisted of methanol (A) and water with 0.1% formic acid (B) at a flow rate of 0.5 ml/min. The composition of the mobile phase was as follows: 70% B for 0–0.5 min, 30% B for 1.2–2.5 min, and 70% B for 2.6–5 min. Mass spectra were recorded by electrospray ionization with a positive mode. Quantification was carried out using MRM at m/z 336.0 → 320.3/292.3 for berberine, m/z 322.1 → 307.1/279.1 for berberrubine (BBB), and m/z 356.2 → 192.2/165.2 for tetrahydropalmatine. Calibration was applied on a standard curve in the range of 0.05–100 ng/ml of berberine and BBB, respectively.
4
Metabolite Profiling of Frozen Orange Leaves
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Frozen orange leaf samples and small steel balls were placed in microfuge tubes and homogenized in a freezing grinder (Shanghai Jingxin, Shanghai, China) at 60 Hz for 2 min. Then, 50 mg of leaf powder from each sample was extracted with 1 mL of ice-cold 10% methanol/water (v/v) containing 0.125% acetic acid with constant shaking at 200 rpm (30 min, on ice), then centrifuged at 12,000 rpm for 10 min (4 °C). The supernatants from each sample were filtered through 0.22-μm microfilters, and aliquots were transferred to LC vials. The samples were analyzed using an Agilent 1200 HPLC system with an Agilent Poroshell EC-C18 column (150 mm × 3.0 mm, 2.7 μm, Agilent Technologies, Santa Clara, CA, USA) coupled to an AB Sciex Triple Quad 4000 mass spectrometer (AB Sciex, Framingham, MA, USA), as described previously [14 (link)]. Five independent plant replicates were used for each treatment.
5
Quantitative HPLC-MS Analysis of Compounds
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The identification of the extract was performed using the HPLC-MS technique with an Agilent 1260 Infiniti Series HPLC System (Agilent Technologies, Inc., Santa Clara, CA, USA). An Agilent poroshell EC-C18 column was used (Agilent Technologies, Inc., Santa Clara, CA, USA). The injection volume of the sample was 10 µL, acetonitrile, and 0.6% acidified water (80:20) was used as the mobile phase at a flow of 0.50 mL/min for 30 min. The results were analyzed with OpenLab ChemStation Edition software (Agilent Technologies, Inc., Santa Clara, CA, USA).
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