The LC system consisted of an Agilent (Palo Alto, CA, USA) 1100 autosampler and binary pump, a Phenomenex (Torrance, CA, USA) Synergi Polar-RP (4 μm, 2 mm × 100 mm) column kept at ambient temperature, and a gradient mobile phase. Mobile phase solvent A was 0.1% (v/v) formic acid in acetonitrile, and mobile phase solvent B was 0.1% (v/v) formic acid in water. The initial mobile phase was 40% solvent A and 60% solvent B pumped at a flow rate of 0.3 mL/min. Between 0 and 12 min, the percentage of solvent A was increased linearly to 80%. At 12.1 min, the flow rate was increased to 0.5 mL/min, which was maintained until 17 min. Between 17 and 17.1 min, the percentage of solvent A was decreased linearly to 40% and held until 24 min, at which time the next sample was injected into the LC system. For WW751, the initial mobile phase was 35% solvent A and 65% solvent B pumped at a flow rate of 0.3 mL/min. Between 5.6 and 9 min, Between 9.1 min and 15min, the percentage of solvent A was decreased linearly to 35% and then held until 24 min, at which time the next sample was injected into the LC system.
Synergi polar rp
Manufactured by Phenomenex
Sourced in United States, Germany
Synergi Polar-RP is a reversed-phase high-performance liquid chromatography (HPLC) column developed by Phenomenex. It features a proprietary polar-embedded stationary phase designed to provide enhanced selectivity and retention for polar and hydrophilic analytes.
Automatically generated - may contain errors
Lab products found in correlation
Analyst 1, by AB Sciex (2 mentions)
Ultimate 3000 hplc, by Thermo Fisher Scientific (1 mentions)
Q exactive mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Pursuit pfp, by Agilent Technologies (1 mentions)
Polymerx rp 1, by Phenomenex (1 mentions)
Zorbax c8, by Agilent Technologies (1 mentions)
Guard column, by Phenomenex (1 mentions)
Api 5500, by AB Sciex (1 mentions)
Omni bead ruptor homogenizer, by Omni International (1 mentions)
Agilent 1260 lc, by Agilent Technologies (1 mentions)
Api 4000 triple quadrupole mass spectrometer, by AB Sciex (1 mentions)
Multiquant software version 3, by AB Sciex (1 mentions)
Lc 20ab, by Shimadzu (1 mentions)
Cto 20ac, by AB Sciex (1 mentions)
Cbm 20a, by AB Sciex (1 mentions)
Analyst version 1, by AB Sciex (1 mentions)
Sil 20ac ht, by AB Sciex (1 mentions)
Agilent 6470 triple quad lc ms ms system, by Agilent Technologies (1 mentions)
Agilent 1260 hplc system, by Agilent Technologies (1 mentions)
Ultimate 3000 uplc system, by Thermo Fisher Scientific (1 mentions)
15 protocols using synergi polar rp
1
Reversed-Phase HPLC Gradient Separation
2
Untargeted Metabolomic Profiling of Placental Tissue
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Frozen placental tissue (n = 84 subjects, early pregnancy BMI 18.5–45.0) was homogenized to a fine powder in liquid nitrogen and homogenates (~50 mg) were extracted with ice-cold 80% aqueous methanol containing isotope labeled standard [1,2,3,4-13C4]α-ketoglutaric acid (Cambridge Isotope Laboratories) and maintained at -20°C for 1 h. Subsequently, the extracts were centrifuged at 13,800 × g for 10 minutes and the supernatants were transferred to glass autosampler vials for HPLC electrospray ionization-mass spectrometry (HPLC-ESI-MS) analysis on a Thermo Fisher Q Exactive mass spectrometer with on-line separation by a Thermo Fisher/Dionex Ultimate 3000 HPLC. HPLC conditions were: column, Synergi Polar-RP, 4 μm, 2 × 150 mm (Phenomenex); mobile phase A, 0.1% formic acid in water; mobile phase B, 0.1% formic acid in acetonitrile; flow rate, 250 μl/min; gradient, 1% B to 5% B over 5 minutes, 5% B to 95% B over 1 minutes and held at 95% B for 2 minutes. Metabolite identification was based on the metabolite accurate mass (± 5 ppm) and agreement with the HPLC retention time of authentic standards. Quantification was made by integration of extracted ion chromatograms of αKG followed by comparison with the corresponding standard curves.
3
Quantification of PQ and C-PQ by LC-MS
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PQ and C-PQ levels were determined by liquid chromatography-mass spectrometry (LC-MS) as previously described (39 (link)). The system consisted of a Shimadzu LCMS-2010A mass spectrometer operated using electrospray ionization (ESI) in positive ion detection mode. Data were collected in the selected ion monitoring mode at 325.35 m/z for quinine (internal standard; retention time, 3.7 min), 260.30 m/z for PQ (retention time, 5 min), and 275.25 m/z for C-PQ (retention time, 8 min). The analytical column was a Phenomenex Synergi Polar RP (150 by 2 mm, 4 μm), preceded by a Polar RP security guard column (2 by 4 mm; Phenomenex, Torrance, CA). The standard curve ranged from 4 to 1,000 ng/ml, with a lower limit of quantitation of 4 ng/ml and a lower level of detection of 1 ng/ml. All control values were within 15% of their nominal value.
4
LC-MS/MS Based Analytical Protocol
5
Quantitative LC-MS/MS Metabolite Analysis
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The LC–MS/MS system connected to Agilent 6470 triple-quadrupole mass spectrometer (Agilent, Wilmington, DE, USA) via electrospray ionization (ESI) interface. The mobile phase consisted of methanol: water (65:35, v:v) with 0.1% formic acid. The analytical column of a Synergi Polar-RP (4 µm particle size, 150 × 2 mm, Phenomenex, Torrance, CA, USA) equipped with an guard column (4 × 2 mm, Phenomenex) was used. The sample injection volume, dwell time, and flow rate were 10 µL, 10 min, and 0.3 mL/min, respectively.
6
Simultaneous Quantification of NaHA and CisPt
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The simultaneous quantification of NaHA and CisPt in the film-forming mixture and in samples from the release experiments, was performed in different reverse phase chromatographic conditions. The column used was a Synergi Polar-RP (4 µm, 4.6 × 150 mm; Phenomenex, Torrance, CA, USA). Isocratic elution at room temperature was carried out with 25 mM KH2PO4, adjusted to pH 5.8 ± 0.1 with 1 M KOH. The detection wavelength was 210 nm. The flow rate was 0.6 mL/min and the injection volume 10 µL. In these conditions the retention times of NaHA and CisPt were 1.5 and 3.4 min, respectively. The method linearity was confirmed in the 1.2–46.4 µg/mL cisplatin concentration range (y = 16.065x + 0.4436; R2 = 1.0000) and in the 50–250 µg/mL NaHA concentration range (y = 1.616x − 3.1234; R2 = 0.9996).
7
Quantification of Isoniazid in Human Hair
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We used an Agilent LC 1260 (Sta. Cruz, CA) equipped with a binary pump and an AB Sciex API 5500 (Foster City, CA) as the analytical platform for our method. We pulverized human hair samples (2 mg, about 10–25 one centimeter strands) using an Omni Bead Ruptor® homogenizer (Kennesaw, GA) and extracted the pulverized hair samples in methanol. The extracts (10 microliter (uL)) were then separated on a Phenomenex Synergi Polar-RP (2.1 x 100 mm, 2.5 μm particle size) column (Torrance, CA) using water with 0.2% (v/v) formic acid as mobile phase at a flow rate of 0.4 mL/min. We monitored INH at multiple reaction mode using an electrospray ion source operated in positive polarity. We used two transitions to monitor INH: 138.1 > 79.0 m/z (quantifier) and 138.1 >120.9 m/z (qualifier).
We analyzed the data obtained from our LC-MS/MS runs using AB Sciex Analyst 1.6 and AB Sciex MultiQuant 2.1 software packages (Foster City, CA). We identified and confirmed INH based on its retention time and the peak area ratio between its two transitions. We quantified INH in hair samples by isotope dilution method using INH-d4 as internal standard. We monitored INH-d4 using the transition 142.1 > 82.9 m/z.
We analyzed the data obtained from our LC-MS/MS runs using AB Sciex Analyst 1.6 and AB Sciex MultiQuant 2.1 software packages (Foster City, CA). We identified and confirmed INH based on its retention time and the peak area ratio between its two transitions. We quantified INH in hair samples by isotope dilution method using INH-d4 as internal standard. We monitored INH-d4 using the transition 142.1 > 82.9 m/z.
8
LC-MS/MS Analysis of Organic Compounds
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The LC-MS/MS system consisted of an HPLC apparatus (Shimadzu LC-20AB, SIL-20AC HT, CTO-20AC, CBM-20A, Duisburg, Germany) and an API 4000 triple quadrupole mass spectrometer (Sciex, Darmstadt, Germany). The substance-specific parameters used for multiple-reaction-monitoring (MRM) measurement are listed in Online Resource 2. The following source parameters were applied: ion spray voltage (ESI+), 5500 V; temperature, 550 °C; nebulizer gas, 50 psi; heating gas, 60 psi; curtain gas, 30 psi; and collision gas (nitrogen), level 9. A Synergi™ Polar-RP (150 × 2 mm, 4 μm; Phenomenex, Aschaffenburg, Germany) was used as an analytical column protected by a guard column. The binary linear gradient consisted of eluent A (deionized water containing 5 mmol/L ammonium formate, 0.1% formic acid) and eluent B (LC-MS-grade methanol, 5 mmol/L ammonium formate, 0.1% formic acid) with a flow rate of 0.4 mL/min: 0 min 10% B, 10 min 40% B, and 26 min 100% B. The column was equilibrated at starting conditions for 6 min prior to each run. Analyst (Version 1.6.2) and MultiQuant software (Version 3.0.1), both provided by Sciex (Darmstadt, Germany), were used for data acquisition and processing.
9
Quantification of OP-A Adducts
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OP-A was adjusted to 100 µM in 1 mL methanol and then mixed with 1 mL of either 50 mM GSH or 50 mM NAC. After 3 h incubation at 40°C, the reaction was quenched by the addition of 20 fold ice-cold methanol. For monitoring of OP-A alone and its formation of adducts with NAC or GSH, 1 μL aliquot of the mixture was directly injected into an Agilent 6470 Triple Quad LC-MS/MS system (Agilent, Wilmington, DE, USA) coupled to an Agilent 1260 HPLC system. Chromatographic separation was achieved using a Synergi Polar RP (4 µm, 2.0 mm i.d. × 150 mm, Phenomenex, Torrance, CA, USA).
10
LC-MS Identification of YCF5 Compounds
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To identify the YCF5 compounds in extracts, YCF5 samples were subjected to liquid chromatography (LC)-mass spectrometry (MS) using a Dionex Ultimate 3000 UPLC system coupled to a Thermo Scientific Q Exactive Orbitrap mass spectrometer. Samples were loaded onto the Phenomenex Synergi Polar-RP (2 × 150 mm, 4 μm) at 40 ℃. The flow rate was 0.3 mL/min. Mobile phase A was composed of water and 0.1% formic acid, and mobile phase B was composed of acetonitrile and 0.1% formic acid. The optimum gradient elution conditions were as follows: 0% B (0–5 min), linear gradient from 0% B to 5% B (5−7 min), 5% B to 20% B (7−10 min), 20% B to 25% B (10−20 min), 25% B to 50% B (20−23 min), and 50% B to 100% B (23−40 min), 100% B for a further 3 min (40−43 min), then back to 0% B (43−45 min), 0% B for 5 min, and stop at 50 min. The injection volume was 5.00 μL.
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