The LC/MS/MS method was conducted as described in Reference [12 ]. The method was carried out using a Waters Acquity UPLC Sample Manager and a Waters Acquity UPLC Binary Solvent Manager connected to a Waters Quattro Premier XE triple-quadruple mass spectrometer and Mass Lynx 1.5.2 software (Waters, Milford, MA, USA). An Agilent Zorbax XDB-C18 (3.5 mm, 2.1 mm × 50 mm) was also used. The gradient mode was used to achieve triptolide and internal standard using mixtures of 40 mM ammonium formate (mobile phase A) and methanol with 0.1% (v/v) formic acid (mobile phase B) at a flow rate of 0.5 mL/min. Mobile phase A was 98% at 0.1–0.6 min, then a linearly programmed gradient from 98% to 5% at 3–4.5 min, at last, mobile phase A ramp to 98% at 4.5–6 min toward the end of the analysis. The system was auto-injected with 10 μL of each analyte. Multiple reaction monitoring (MRM) mode was applied to achieve quantification with monitoring precursor-product ion transitions of m/z 361.0→105.0 for triptolide using electrospray ionization mass spectrometry (ESI-MS) on an API-4000, with Turbo Ionspray. The system was in positive ESI-mode during the run. The desolvation temperature was set at 550 °C and the nebulizer gas pressure was 60 psi.
Acquity uplc binary solvent manager
Manufactured by Waters Corporation
Sourced in United States
The ACQUITY UPLC Binary Solvent Manager is a high-performance liquid chromatography (HPLC) module designed to precisely deliver and combine two different solvents for use in ultra-performance liquid chromatography (UPLC) systems. It provides accurate and reproducible solvent flow and composition control to support reliable and efficient UPLC separations.
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Lab products found in correlation
Acquity uplc sample manager, by Waters Corporation (2 mentions)
Xevo g2 xs qtof, by Waters Corporation (2 mentions)
Zorbax xdb c18, by Agilent Technologies (1 mentions)
Acquity uplc system, by Waters Corporation (1 mentions)
Masslynx 4, by Waters Corporation (1 mentions)
Hd imaging 1, by Waters Corporation (1 mentions)
Desi source, by Waters Corporation (1 mentions)
Xevo tq xs ms, by Waters Corporation (1 mentions)
Quattro premier xe triple quadrupole mass spectrometer, by Waters Corporation (1 mentions)
8 protocols using acquity uplc binary solvent manager
1
Sensitive LC-MS/MS Quantification of Triptolide
2
Quantitative Analysis of Methylxanthines in Tea
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The chromatographic analyses of the standards caffeine, theobromine, and theophylline as well as all real samples were performed on an Acquity UPLC system (Waters Corp., Milford, MA, USA), including an Acquity UPLC binary solvent manager, a sample manager, and a column heater.
The liquid chromatographic separations of all analyzed samples were achieved under isocratic conditions using Nucleodur C18 polar column Tec. (50 × 2 mm i.d.; particle size: 1.8 μm) from Macherey-Nagel (Düren, Germany). The column temperature was kept at 70°C, and the sample manager was maintained at 10°C. A binary mixture composed of acetonitrile/water (10 : 90, v/v) with 0.1% formic acid at a flow rate of 0.5 mL/min was employed as the mobile phase. Peak area was used for quantitative evaluations. All standard and real samples were injected five times. Standard deviations and statistical parameters were calculated using Microsoft Office Excel software 2013 package. The sample injection volume was fixed at 5.0 μL. Table 2 summarizes the optimum conditions of UHPLC. Due to the presence of many different ingredients in the tea such as catechins, polyphenols, flavonoids, and amino acids, the late-eluted compounds from the sample matrix (which are undetectable under the stated MS conditions) were washed with acetonitrile and water after analysis of each tea sample.
The liquid chromatographic separations of all analyzed samples were achieved under isocratic conditions using Nucleodur C18 polar column Tec. (50 × 2 mm i.d.; particle size: 1.8 μm) from Macherey-Nagel (Düren, Germany). The column temperature was kept at 70°C, and the sample manager was maintained at 10°C. A binary mixture composed of acetonitrile/water (10 : 90, v/v) with 0.1% formic acid at a flow rate of 0.5 mL/min was employed as the mobile phase. Peak area was used for quantitative evaluations. All standard and real samples were injected five times. Standard deviations and statistical parameters were calculated using Microsoft Office Excel software 2013 package. The sample injection volume was fixed at 5.0 μL.
3
UPLC-MRM Analysis of Compounds 1, 10, 13, 16, 83, and 84
4
Direct Flow Injection Analysis of Spent Hemodialysates
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The direct flow injection analysis of spent hemodialysates was performed using the online desalination tube connected with an ESI source equipped with a Q-TOF MS (Synapt G2 Q-TOF MS, Waters, Milford, MA, USA) in positive ion mode. The spray solvent (20 mM ammonium acetate in 50% methanol) was maintained at a flow rate of 0.2 mL/min using a solvent pump (ACQUITY UPLC Binary Solvent Manager, Waters, Milford, MA, USA). The ESI source conditions in positive ion mode were optimized using a capillary voltage of 4.0 kV, a cone voltage of 30 V, a source temperature of 150°C, a desolvation temperature of 450°C, a cone gas flow of 50 L/h, and a desolvation gas flow of 800 L/h. Ions from spent hemodialysates were obtained in a range of m/z 100 to 1000. The mass spectra were calibrated using sodium formate solution (500 μM) in 2-propanol: water (90:10, v/v) prior to measurements. We used lock spray (leucine-enkephalin solution; m/z 556.28) to obtain high mass accuracy.
5
DESI-MSI for Epinastine Mapping
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DESI-MSI data of the sections on glass slides were acquired using a DESI source (Waters) equipped with a quadrupole time-of-flight mass spectrometer (Xevo G2-XS QTof; Waters) in the positive ion mode. Before the analysis, the DESI-MSI instrument was calibrated using a 10 mM sodium formate solution prepared in 50% methanol. An ACQUITY UPLC binary solvent manager (Waters) was used to deliver 98% methanol as DESI spray solvent at a speed of 3 µL/ min. The capillary and cone voltages were set to 3.5 kV and 50 V, respectively. The source temperature was set at 120 °C. Mass spectra were acquired in the m/z 100-1,000 range. The pixel size was 100 μm. The mass resolution was set at 20,000. The Mass Lynx 4.1 and HD Imaging 1.4 software (Waters) were used to obtain the DESI-MSI data. A distribution image of the upper eyelid sections was created using protonated epinastine ions at m/z 250.13 without normalization.
6
Mass Spectrometry Analysis of Spent Hemodialysates
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The direct flow injection of spent hemodialysates was performed using the online desalination tube connected with an ESI source equipped with a TQ-MS (Xevo TQ-XS MS, Waters, Milford, MA, USA) in positive ion mode. The spray solvent (0.1% formic acid in 50% methanol) was maintained at a flow rate of 0.2 mL/min using a solvent pump (ACQUITY UPLC Binary Solvent Manager, Waters, Milford, MA, USA). We optimized the TQ-MS parameters for maximum sensitivity as follows, scan mode: multiple reaction monitoring (MRM), ionization mode: positive, capillary voltage: 3.0 kV, cone voltage: 30 V, source temperature of 150°C, desolvation temperature: 500°C, desolvation gas flow: 600 L/h, cone gas flow: 150 L/h, N2 gas pressure: 7.0 bar.
7
UPLC/MS/MS Analysis of Metabolites
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The UPLC/MS/MS method was conducted using a Waters Acquity UPLC Sample Manager and a Waters Acquity UPLC Binary Solvent Manager connected to a Waters Quattro Premier XE triple-quadrupole mass spectrometer equipped with a combined ESI probe and Mass Lynx 1.4 software (Waters, MA, USA). An Acquity UPLC BEH C18 column (1.7 µm, 2.1 × 100 mm2) was used. Instrument settings were as follows: ESI+; source temperature, 110 °C; desolvation temperature, 350 °C; capillary voltage, 3.2 kV; desolvation N2, 600 L/h; cone N2, 50 L/h; LM resolution 1, 13.5; and HM resolution 1, 13.5. The data acquisition was carried out at m/z 50–500 Da in the total ion scan mode. The MS/MS spectra were acquired at m/z 50–300 Da in the daughter ion scan mode. The solvent system consisted of solvent A (0.1% CH3COOH) and solvent B (100% acetonitrile). The flow rate was 0.2 mL/min. The use of different HPLC gradients of 10–80% B for 16 min for imperatorin and isoimperatorin after exposure to human liver microsomes was necessary to separate the metabolites from the parent compound.
8
DESI-MS Analysis of NAD+ Standards
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The MSI analysis was performed using a DESI source attached to a quadrupole time-of-flight mass spectrometer (Xevo G2-XS Q-TOF, Waters, Milford, MA). The mass spectra were obtained in negative ion mode. Before measurement, calibration was done externally using 500 µmol/L sodium formate solution in 2-propanol: water (90:10, v/v). The spray solvent (98:2; methanol: water, v/v) was delivered continuously at a flow rate of 2 µL/min using a solvent pump (ACQUITY UPLC Binary Solvent Manager, Waters, Milford, MA). Prosolia's Omni Spray-controlled 2-dimensional moving stage was used to scan the defined areas. Parameters were set as follows: spray impact angle of 68°, emitter tip to the surface distance of ≈1.0 mm, emitter to ion transfer capillary orifice of ≈5 mm, ion transfer capillary orifice to the surface of ≈0.5 mm, emitter exposed from sprayer tip of ≈0.5 mm, a capillary voltage of 3.6 kV, a solvent flow rate of 2 µL/min, source temperature of 150 °C, sampling cone of 50 V, and a nitrogen pressure of 0.4 kPa. For NAD + standard, liquid droplets (0.3 µL) of solutions were placed on a glass slide and allowed to dry at room temperature. The samples were scanned at a velocity of 100 µm/s. The pixel size was set as 100 µm×100 µm.
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