The extraction protocol of anthocyanins was as follows: 2.5 g of pericarps were incubated with of 25 mL of MeOH-HCl (pH3) in dark for 24 h and then centrifuged at 12,000 g (RT). Supernatant was collected and dried in a vacuum (40°C) and then dissolved in 5 mL of 0.01% (v/v) HCl. All extracts were filtered through a 0.22- μm membrane before injection. Waters Acquity UPLC chromatograph with a C18 column (Luna, 5 µm, 4.6 mm × 250 mm) was used to determine the content of anthocyanins. The solvent and gradient method was as follows: solvent A, 10% aqueous formic acid; solvent B, methanol; constant gradient from 5% to 60% B within 20 min, from 60% to 100% B for 5 min, and then maintain 100% B for 5 min. The injection sling was 10 μL. The flow rate was 1 mL min−1. The detection was at 520 nm, and the column oven temperature was maintained at 35°C. The mass spectrometry conditions were as follows: electrospray ion source; positive ion mode; capillary voltage, 3, 500 V; nebulizer, 45 psi; dry gas: nitrogen; cone gas flow, 12 L min−1; dry temperature, 300°C; ion trap, scan from Mass to charge ratio (m/z) 200 to 1,300. The anthocyanins were quantified by external calibration using Cyanin-3-O-glucoside (Cy3G) and Peonidin-3-O-glucoside (Pn3G) standard (Extrasynthese, France).
Acquity uplc chromatograph
Manufactured by Waters Corporation
Sourced in United States
The Acquity UPLC chromatograph is a high-performance liquid chromatography (HPLC) system developed by Waters Corporation. It is designed to separate, identify, and quantify components in a mixture with high resolution and sensitivity. The Acquity UPLC utilizes ultra-high-pressure liquid chromatography (UPLC) technology to achieve faster analysis times and improved separation efficiency compared to traditional HPLC systems.
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Lab products found in correlation
Acquity beh c18 column, by Waters Corporation (3 mentions)
Tqd quadrupole mass spectrometer, by Waters Corporation (2 mentions)
C18 column, by Waters Corporation (1 mentions)
Xevo qtof hybrid quadrupole time of flight mass spectrometer, by Waters Corporation (1 mentions)
Acquity uplc beh c18, by Waters Corporation (1 mentions)
Synapt hdms qtof mass spectrometer, by Waters Corporation (1 mentions)
Xevo tq s triple quadrupole ms, by Waters Corporation (1 mentions)
Fatty acid free bsa, by Merck Group (1 mentions)
Xevo tq s mass spectrometer, by Waters Corporation (1 mentions)
Lithium heparin, by Sarstedt (1 mentions)
Microvette tube, by Sarstedt (1 mentions)
Acquity uplc hss c18 column, by Waters Corporation (1 mentions)
Thermo accela uplc system, by Thermo Fisher Scientific (1 mentions)
Gamma 2 16 lsc freeze dryer, by Martin Christ (1 mentions)
Avance 3 hd ascend 500 mhz, by Bruker (1 mentions)
Ph meter, by Hanna Instruments (1 mentions)
Pda eλ detector, by Waters Corporation (1 mentions)
Empower 3, by Waters Corporation (1 mentions)
Waters acquity uplc beh c18, by Waters Corporation (1 mentions)
Triple quadrupole mass spectrometer tqd, by Waters Corporation (1 mentions)
14 protocols using acquity uplc chromatograph
1
Extraction and Quantification of Anthocyanins
2
UPLC-ESI-QTOF Metabolite Profiling
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Ultra-performance liquid chromatography coupled with electrospray ionization mass spectrometry using a Waters Acquity UPLC chromatograph (Waters, Milford, MA, USA) equipped with a XEVO QTOF hybrid quadrupole time-of-flight mass spectrometer (Waters, USA) was used for sample profiling. The analysis was performed in negative ion mode in the m/z range of 550–1,500. The injection volume was 5 µL. The ionization source parameters were as follows: ionization source temperature was 150°C, desolvation temperature was 600°C, capillary voltage was 3 kV, sample injection cone voltage was 40 V, and nitrogen (desolvation gas) flow rate was 1,000 L h–1. Chromatographic separation conditions were as follows: Acquity UPLC BEH C18 (50 × 2.1 mm, 1.7 μm, Waters, USA) was used with column temperature 40°C, and mobile phase flow rate was 0.4 mL min–1. A 0.1% (v/v) solution of formic acid in water (solvent A) and a 0.1% (v/v) solution of formic acid in acetonitrile (solvent B) were used as the mobile phase. Chromatographic separation was performed using a gradient elution mode. The composition of the mobile phase changed according to the following program (B, % by volume): 0–2 min, 20%; 2–20 min, 20%; 20–20.10 min, 20%; 20.10–25.00 min, 20%–37%; 25.00–26.00 min, 37%–46%; and 26.00–26.10 min, 46%–95%.
3
Metabolomic Analysis by UPLC-QTOF-MS
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Metabolomic analyses were performed in a Waters Acquity UPLC chromatograph hyphenated to a Waters Synapt HDMS Q-ToF mass spectrometer (Waters, UK). Cells were processed and analyzed by following a previously optimized analytical strategy20 21 (link). In summary, different metabolome extractions were combined to obtain polar and nonpolar fractions, which were then analyzed separately by hydrophilic interaction liquid chromatography (HILIC) and reversed-phase (RP) liquid chromatographic techniques (see Supplementary Information ).
4
Measurement of Free Plasma PUFAs
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For the measurement of free PUFA (μg/ml), 100 μl of the blood sample from each piglet was precipitated using 100 μl of internal standard (deuterated ARA, DHA, and EPA obtained from Sigma-Aldrich) solution in IPA (POCh). After that, it was acidified by addition of 100 μl of 2% HCOOH. Samples were subsequently extracted using 600 μl of hexane (Avantor Baker) and the organic layer was evaporated under nitrogen. Samples were solubilized in 100 μl of 5% NH4OH in 65% MeOH and 10 μl was injected into LC/MS. Samples were then analysed using Waters Xevo TQ-S triple quadrupole MS coupled with Waters Acquity UPLC chromatograph. The column used was an ACQUITY UPLC BEH C18 Column, 1.7 μm, 2.1 mm × 50 mm thermostated in 60°C. Mobile phase A was 0.1% NH4OH in MQ water; mobile phase B was pure LC/MS grade ACN (Avantor Baker). A linear gradient was used, starting from 40% to 95% B over 1.7 min. MS was operating in negative MRM mode. Parameters were as follows: capillary voltage 3.2, desolvation temperature 500°C, desolvation gas (nitrogen) flow 900 l /h, and cone gas flow 150 l /h. Calibration and control samples were prepared by spiking PUFA standards into 45 mg/ml fatty acid-free BSA (Sigma-Aldrich, St. Louis, MO, USA) solution in 0.9% NaCl.
5
Plasma L-arginine and L-ornithine quantification
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Blood from the facial veins puncture was collected into Microvette® tubes with lithium heparin (Sarstedt). Samples were centrifuged at 1000 × g for 10 min, plasma was separated and kept frozen at − 20 °C until analysis. Measurements of the plasma concentration of ʟ-arginine and ʟ-ornithine were performed by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method on Waters Xevo TQ-S mass spectrometer equipped with Waters Acquity UPLC chromatograph (Waters) in the Mass Spectrometry Lab at the Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.
6
UPLC-MS Analysis of Phenolic Acids
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The phenolic acid content was analyzed utilizing a Waters ACQUITY UPLC Chromatograph (Waters Corp., Milford, MA, USA), equipped with a PDA and a triple-quadrupole mass detector (Waters Corp., Milford, MA, USA). Samples (50 mg/mL) were first separated on a Waters ACQUITY UPLC® HSS C18 column (100 × 2.1 mm, 1.8 μm) at 30 °C. The mobile phase consisted of solvent A (0.1% formic acid in water) and solvent B (acetonitrile with 0.1% formic acid). The elution step (0.50 mL/min) was carried out with a gradient of solvent B: 0–0.5 min, 8% B; 0.5–8 min, 8–20% B; 8–8.10 min, 20–95% B; 8.10–10 min, 95% B; 10–10.10%, 95–8% B; 10.10–12 min, 8% B. The sample injection volume was 2.5 μL (full loop mode). The detection of phenolic acids was performed in the negative ionization mode by applying a selected reaction monitoring method. The condition of the MS analysis was published by Czaban et al. [42 ]. The concentration of phenolic acids in snack pellet extracts was calculated on the basis of calibration curves (Table 10 ).
7
UPLC-MS/MS Analysis of Compounds
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Liquid chromatography-mass spectrometry was performed using a UPLC/MS/MS system consisting of an ACQUITY UPLC chromatograph from Waters (Milford, MA, USA) and a TQD quadrupole mass-spectrometer (Waters) with registration of positive ions using the ESI MS method with an ACQUITY BEH column C18 (1.7 microns, 50 × 2.1 mm, Waters), flow rate 0.5 mL/min, 35 °C, and elution with a gradient of 5–100% CH3CN in 20 mM of HCOOH for 4 min.
8
Quantitative UPLC-MS/MS Analysis of Onc112
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Liquid chromatography–mass spectrometry was performed using a UPLC/MS/MS system consisting of an Acquity UPLC chromatograph (Waters Corporation, Milford, MA, USA) and a TQD quadrupole mass spectrometer (Waters Corporation, Milford, MA, USA) with the registration of positive ions carried out using the ESI-MS method with an Acquity BEH column C18 (1.7 microns, 50 × 2.1 mm; Waters Corporation, Milford, MA, USA), a flow rate of 0.5 mL/min, a temperature of 35 °C, and elution with a gradient of 5–100% CH3CN in 20 mM of HCOOH for 4 min. UPLC-MS analysis of Onc112 was performed using a Thermo Finnigan LCQ Deca XP ion trap spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) with the Thermo Accela UPLC system (Thermo Fisher Scientific, Waltham, MA, USA) equipped with an Atlantis T3 C18 (150 × 2 mm) column (Waters Corporation, Milford, MA, USA).
9
Analytical Instrumentation for Bioassays
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Gamma 2–16 LSC freeze dryer, Martin Christ Gefriertrocknungsanlagen GmbH, Germany. Waters ACQUITY UPLC chromatograph (Waters Corp., Milford, MA, USA) equipped with a binary pump and MS and DAD detectors. Prep- (Laborota 20 control automatic) and medium-scale (Hei-VAP) rotary evaporators (Heidolph, Germany). Nuclear magnetic resonance (NMR) spectrometer—an Avance III HD Ascend 500 MHz (Bruker BioSpin, Rheinstetten, Germany). The pH-meter (Hanna Instruments, Luton, UK), UV-Vis ABL&E Jasco V550 spectrophotometer (Cluj-Napoca, Romania), and Tetratec® air pumps (Tetra, Melle, Germany) have been used in in vitro and in vivo bioassays.
10
Quantification of Bioactive Compounds in Lamium album
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The concentrations of bioactive compounds in the methanolic extracts were determined using a UPLC-PDA-TQD system, which consisted of the Acquity UPLC chromatograph (Waters, Manchester, MA, USA) coupled with a photodiode detector (PDA eλ Detector) (Waters, Manchester, MA, USA) and an electrospray ionization (ESI) triple quadrupole mass spectrometer (TQD) (Waters, Manchester, MA, USA). Mobile phase consisted of two solutions: line A-water containing 0.1% HCOOH and line B methanol containing 0.1% HCOOH. The phenolics were separated at 30 • C on the analytical columna Waters ACQUITY UPLC BEH C18 (150 × 2.1 mm/ID, with 1.7 µm particle size) (Waters, Manchester, MA, USA) with flow rate 0.35 mL/min and using gradient elution: from 10 to 60% of B line during 15 min followed by the return to the initial conditions. Temperature of samples in the autosampler was 10 • C. UV spectral data for all peaks were recorded in the range of 190-450 nm. Data processing was done using Empower 3 (Waters, Manchester, MA, USA). All samples of Lamium album extracts were filtered through a 0.20 µm syringe filter (Chromafil, Macherey-Nagel, Duren, Germany) before analyses and were injected to the chromagraphic system in triplicate. Identification of bioactive compounds was done using matching retention times, UV and mass spectra (MRM) data of standards.
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