LC-MS analysis was performed using 6490 triple quadrupole mass spectrometer coupled to an Agilent 1290 series HPLC system (Agilent Technologies, Santa Clara, CA) with single reaction monitoring (SRM). This LC system is equipped with a degasser, binary pump, thermostatted auto sampler, and column oven. This SRM-based measurement of relative metabolite levels used normal phase chromatographic separation. 10 µl of resuspended samples were injected and analyzed using source parameters as follows: Gas temperature- 250 °C; Gas flow- 14 l/min; Nebulizer - 20psi; Sheath gas temperature - 350 °C; Sheath gas flow- 12 l/min; Capillary - 3000 V positive and 3000 V negative; Nozzle voltage- 1500 V positive and 1500 V negative. Approximately 8–11 data points were acquired per each detected metabolite.
Agilent 1290 series
Manufactured by Agilent Technologies
Sourced in United States, Germany
The Agilent 1290 series is a line of high-performance liquid chromatography (HPLC) systems designed for analytical applications. The 1290 series offers advanced technology and components to deliver reliable and efficient chromatographic separation and detection.
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Lab products found in correlation
Xbridge c18 column, by Waters Corporation (3 mentions)
Agilent 6460, by Agilent Technologies (2 mentions)
Agilent 6470, by Agilent Technologies (2 mentions)
Acquity uplc beh c18, by Waters Corporation (1 mentions)
Xevo tq s triple quadrupole mass spectrometer, by Waters Corporation (1 mentions)
Poroshell 300sb c8, by Agilent Technologies (1 mentions)
Eclipse plus c18 rrhd column, by Agilent Technologies (1 mentions)
Agilent 6530 q tof mass spectrometer, by Agilent Technologies (1 mentions)
Uv 1700, by Shimadzu (1 mentions)
Uplc beh amide column, by Waters Corporation (1 mentions)
Agilent 6460 series, by Agilent Technologies (1 mentions)
Eclipse plus c18, by Agilent Technologies (1 mentions)
Masshunter workstation software, by Agilent Technologies (1 mentions)
Acquity uplc hss t3 column, by Waters Corporation (1 mentions)
Agilent 6460 triple quadruple mass spectrometer, by Agilent Technologies (1 mentions)
3200 qtrap, by AB Sciex (1 mentions)
Zorbax eclipse c18 column, by Agilent Technologies (1 mentions)
16 protocols using agilent 1290 series
1
Targeted LC-MS Metabolite Profiling
2
HLJDD Herbal Compound Analysis by HPLC-MS
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Chromatographic analysis was performed on an Agilent 1290 series equipped with an Agilent photodiode array detector (Agilent Technologies, Waldbronn, Germany). Mobile phase was composed of two parts: (A) 0.1% formic acid in water; (B) methanol, in a gradient program: 0–4 min, 10% B; 4–15 min, 10–26% B; 15–27 min, 26–28% B; 27–35 min, 28–70% B; 35–55 min, 70–90% B; 55–60 min, 90% B. The flow rate was set at 1 mL·min−1 and the injection volume was 8 μL. The HLJDD and its herbs were detected in Fig. S1.
Quadrupole‐Time‐of‐Flight mass spectrometry was performed in the positive and negative mode. The optimal parameters were: gas temperature, 300 °C; drying gas flow rate, 8 L·min−1; nebulizer, 35 psig; capillary voltage, 4000 V; capillary current, 6.195 μA; fragmentor, 140 V; skimmer, 65 V; OCT 1 RF Vpp, 750 V. The HLJDD and its herbs were detected in Fig. S2 and compounds are listed in Table S1–S5.
Quadrupole‐Time‐of‐Flight mass spectrometry was performed in the positive and negative mode. The optimal parameters were: gas temperature, 300 °C; drying gas flow rate, 8 L·min−1; nebulizer, 35 psig; capillary voltage, 4000 V; capillary current, 6.195 μA; fragmentor, 140 V; skimmer, 65 V; OCT 1 RF Vpp, 750 V. The HLJDD and its herbs were detected in Fig. S2 and compounds are listed in Table S1–S5.
3
Quantification of Berberine and Epicatechin
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The collected samples were centrifuged at 3,000 rpm and 4°C for 10 minutes, and the obtained plasma samples were frozen at below −70°C until analysis. The plasma concentration was determined by a liquid chromatography (Berberine, Agilent 1290 series, Agilent Technologies, Santa Clara, CA, USA; Epictechin, Waters Acquity UPLC 1-class, Waters Corporation, Milford, MA, USA) coupled with a mass spectrometer (Berberine, TQ5500 system, ABSCIEX, Framingham, MA, USA; Epicatechin, Xevo TQ-s triple quadrupole mass spectrometer, Waters Corporation). The blood samples of berberine and epicatechin were treated with methanol for precipitation of proteins. In addition, berberine-d6 and scopoletin was used as an internal standard for quantitation of berberine and epicatechin, respectively. Chromatographic separation was performed under gradient conditions using a Kinetex 1.7 μ C18 100A (2.0 × 50 mm, 1.7 μm, Phenomenex, Torrance, CA, USA) for berberine and using a ACQUITY UPLC BEH C18 (2.1 × 50 mm, 1.7 μm, Waters Technologies, Drinagh North, Ireland) fore epicatechin. The calibration curves were linear over the range of 1–50 pg/mL for berberine and 0.5–20 ng/mL for epicatechin (r2 ≥ 0.9969 and r2 ≥ 0.9964, respectively). The in-study imprecision of berberine and epicatechin for the quality control samples was less than 15%, and the accuracy range for both was within 85–115%.
4
Purification of CyRPA Proteins
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FreeStyle 293-F cells were transfected with pcDNA3.1_BVM_CyRPA(26–362)_6xHis and pcDNA3.1_BMV_CyRPA(26–362/N145Q-N322Q-N338Q)_6xHis plasmids using a riDOM-based transfection system [43 (link)]. Prior to transfection at 1.2 × 106 cells/ml, cells were diluted 1:2 with fresh culture medium and transfected with 0.4 mg/l expression plasmids and transfection reagents. Cell supernatants containing secreted proteins were typically harvested 72–96 h post-transfection. Histidine-tagged proteins were purified by immobilized metal ion affinity chromatography (IMAC). The purity and integrity of the purified proteins were analysed by RP-HPLC on an Agilent 1290 Series with a Poroshell 300SB-C8, 1 × 75 mm column (Agilent). Chromatography was performed with a non-linear (H2O + 0.01 % TFA/Acetonitrile + 0.08 % TFA) gradient system. The protein concentration was determined by measuring the OD280 (1 Abs = 1 mg/ml). The purified recombinant proteins were identified as the expected G-CyRPA and N-CyRPA proteins by western blot analysis with PfCyRPA-specific mAbs [32 (link)].
5
UPLC-MS/MS Quantitation of Spermidine
6
Berberine Quantification by LC-MS/MS
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The analysis was performed on an Agilent 1290 series liquid chromatography system (Agilent Technologies, Palo Alto, CA), and an Agilent 6460 triple-quadruple mass spectrometer (Agilent Technologies, CA) with Turbo Ion spray. The chromatographic separation of berberine was performed on Waters X-Bridge C18 column (3.0 × 100 mm, i.d.; 3.5 μm) at room temperature. The mobile phase was water (containing 0.1% formic acid) and acetonitrile (40:60, v: v) at a flow rate of 0.4 mL/min.
The mass scan mode was positive MRM mode. The precursor ion and product ion are m/z 336.2 → 320.2 for berberine and m/z 363.5 → 121.0 for IS, respectively. The collision energy for berberine and IS were 30 and 25 ev, respectively. The MS/MS conditions were optimized as follows: fragmentor, 140 V; capillary voltage, 4 kV; Nozzle voltage, 500 V; nebulizer gas pressure (N2), 40 psig; drying gas flow (N2), 10 L/min; gas temperature, 350 °C; sheath gas temperature, 400 °C; sheath gas flow, 11 L/min.
The mass scan mode was positive MRM mode. The precursor ion and product ion are m/z 336.2 → 320.2 for berberine and m/z 363.5 → 121.0 for IS, respectively. The collision energy for berberine and IS were 30 and 25 ev, respectively. The MS/MS conditions were optimized as follows: fragmentor, 140 V; capillary voltage, 4 kV; Nozzle voltage, 500 V; nebulizer gas pressure (N2), 40 psig; drying gas flow (N2), 10 L/min; gas temperature, 350 °C; sheath gas temperature, 400 °C; sheath gas flow, 11 L/min.
7
LCMS-MS Quantification of Chlorogenic Acid
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The analysis was performed on an Agilent 1290 series liquid chromatography system (Agilent Technologies, Palo Alto, CA) and an Agilent 6470 triple-quadruple mass spectrometer (Agilent Technologies, Santa Clara, CA). Chromatographic separation of CPA and rutin was performed on Waters X-Bridge C18 column (3.0 × 100 mm, i.d.; 3.5 μm, Milford, MA) at room temperature (25 °C). The mobile phase was water (containing 1 mM ammonium formate and 0.05% formic acid) and methanol (35:65, v:v) with isocratic elution at a flow rate of 0.3 mL/min, and the analysis time was 1.5 min. The injection volume was 2 μL and the auto-sampler temperature was maintained at 25 °C.
The mass scan mode was positive MRM mode, and the mass parameters were optimized using Optimizer software. The precursor ion and product ion are m/z 607.3 → 365.3 for CPA and m/z 610.9 → 355.9 for rutin (internal standard), respectively. The collision energy for CPA and rutin was 30 and 25 eV, respectively. The MS/MS conditions were optimized as follows: fragmentor, 160 V; capillary voltage, 4 kV; Nozzle voltage, 500 V; nebulizer gas pressure (N2), 40 psig; drying gas flow (N2), 10 L/min; gas temperature, 350 °C; sheath gas temperature, 400 °C; sheath gas flow, 11 L/min.
The mass scan mode was positive MRM mode, and the mass parameters were optimized using Optimizer software. The precursor ion and product ion are m/z 607.3 → 365.3 for CPA and m/z 610.9 → 355.9 for rutin (internal standard), respectively. The collision energy for CPA and rutin was 30 and 25 eV, respectively. The MS/MS conditions were optimized as follows: fragmentor, 160 V; capillary voltage, 4 kV; Nozzle voltage, 500 V; nebulizer gas pressure (N2), 40 psig; drying gas flow (N2), 10 L/min; gas temperature, 350 °C; sheath gas temperature, 400 °C; sheath gas flow, 11 L/min.
8
Chromatographic Analysis of Formulations
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Chromatographic analysis was performed on an Agilent 1290 Series (Agilent Corp., Santa Clara, CA, USA,) UPLC system equipped with a binary pump, micro degasser, an auto sampler and a thermostatically controlled column compartment. Chromatographic separation was carried out at 25 oC on a Zorbax RRHD Eclipse Plus C18 column (2.1 × 50 mm, 1.8 μm). The mobile phase consisted of 0.1% formic acid solution (A) and ACN (B) using a gradient elution of 0–5% B at 0–6 min, 5–8% B at 6–15 min, 8–15% B at 15–20 min, 15–20% B at 20–30 min, 20–30% at 30–35 min, 30–35% at 35–45 min, 35–40% at 45–60 min. The flow rate was kept at 0.2 mL/min, and the sample volume injected was set at 5 μL. Detections were carried out by Agilent 6530 Q/TOF mass spectrometer (Agilent Corp., Santa Clara, CA, USA) equipped with an ESI interface. The parameters of operation were as follows: drying gas N2 flow rate, 10.0 L/min; temperature, 330 oC; nebulizer, 35 psig; capillary, 3000 V; skimmer, 60 V; OCT RFV, 250 V. Each sample was analyzed in both the positive and negative modes due to the selective sensitivities to different components of the formulation-providing better information for molecular formulae and structural identification. Mass spectra were recorded across the range m/z 100–1000 with accurate mass measurements.
9
HPLC Analysis of SPS Bioactive Compounds
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The levels of astilbin (1), hesperidin (2), acacetin (3), senkyunolide A (4), imperatorin (5), magnolol (6), glycyrrhizic acid (7), and pulegone (8) were evaluated by HPLC (Agilent 1290 series, Santa Clara, CA). The HPLC of the SPS extract and standard compounds was conducted at the Korea Basic Science Institute (KBSI) (Seoul, Korea). The samples (10 μl) were injected into a Kinetex C18 column (4.6 × 250 mm, 5 μm, Phenomenex) with a guard column (UHPLC C18, AJ0-8768, Phenomenex). The mobile phases included (A) 0.1% phosphoric acid and (B) acetonitrile. The flow rate was 0.9 ml/min. The solvent gradient was set to the following: 10 to 90% (B) for 25 min and equilibration for 5 min. Astilbin, hesperidin, acacetin, senkyunolide A, imperatorin, and magnolol were detected at 210 nm. Glycyrrhizic acid and pulegone were detected at 254 nm. The column temperature was 35°C. The components of SPS were quantified from standard curves.
10
Standardization of Peanut Extract
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The qualitative tests such as Biuret test, Xanthoproteic test, Ninhydrin test and Millon’s test were performed to standardize the crude peanut extract. The UV absorption was evaluated using a UV-double beam spectrophotometer, UV-1700, Shimadzu (Kyoto, Japan). The samples were uniformly mixed at 0.1 mg/mL in the corresponding extraction buffer and then scanned from 240 nm to 600 nm at 25 °C. The FT-IR analysis of the obtained crude peanut extract and all the excipients was done by Shimadzu (Kyoto, Japan), to identify the specific functional groups of the drug. LC (Agilent 1290 series, Agilent Technologies, Palo Alto, CA) was performed by injecting 5.0 µL of the obtained peanut extract onto a 0.32 × 150 mm Symmetry300 C18 5 µm particle size column (Waters, Bedford, MA) at a flow rate of 0.3 ml/min. A 0–50% acetonitrile with 0.5% acetic acid gradient was used for the separation. Mass peaks of the allergens were detected using Mass spectrometer (Agilent 6460 QQQ) in positive ionization mode at mass range scanned from 200 to 2000amu. The run time is about 40 min.
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