Serum metformin concentrations were quantified using high-performance liquid chromatography (HPLC). For sample preparation, 50 μl of internal standard (phenformin 500 mg/l in methanol) and 250 μl of acetonitrile were added to 100 μl of the sample. After vortex mixing, this mixed sample was centrifuged at 4000g for 10 min. Ten microliter of the supernatant was injected into the HPLC column (Zorbax SB-CN 5 μm 4.6 × 150 mm [Agilent]), protected by a precolumn (H3-10C5 [Hichrom]), which was kept at 30 °C and detected by a photodiode array detector (234 nm). Isocratic elution was performed with a mobile phase consisting of sodium sulphate (pH of 2.3) and acetonitrile in a 770:230 ratio at a flow rate of 1.2 ml/min. The elution times for metformin and the internal standard were 2.1 and 3.0 min, respectively. The assay was linear over 0.2–5.0, and 0.2 mg/l was the limit of quantification (LOQ). Intra- and inter-assay coefficients of variation were within 0.8–3.6 and 4–8.5%, respectively. The concentrations of the quality controls were 0.5, 2.5, and 4.5 mg/l (low, middle and high, concentration, respectively).
Zorbax sb cn
Manufactured by Agilent Technologies
Sourced in Canada, United States
The Zorbax SB-CN is a reversed-phase liquid chromatography column. It is designed for the separation and analysis of a variety of organic compounds.
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5 protocols using zorbax sb cn
1
Quantifying Serum Metformin via HPLC
2
Quantitative LC-UV Analysis of Ascorbic Acid
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We used a liquid chromatographic separation with ultraviolet light detection (LC–UV) method adapted and re-validated from established LC–UV protocols [10 (link),11 ,12 (link)]. The LC system consisted of a solvent delivery pump (Model P4000; Thermo Separation Products; San Jose, CA, USA), which pumped a mixture of 5% methanol and 95% 0.05 M phosphoric acid (HPLC grade, cat # P3786; Sigma Aldrich, Oakville, ON, Canada) through a 15 cm × 4.6 mm reversed-phase 5 µm column (Zorbax SB-CN; Agilent Technologies Canada Inc., Mississauga, ON, Canada) at 1.0 mL/min. Three microliters of each prepared sample, quality control or standard was injected directly onto the LC column using an autoinjector (Ultra WISP 715; Waters Scientific, Toronto, ON, Canada) in duplicate.
The column effluent was monitored with a variable wavelength UV detector (UV 6000 Thermo Separation Products; San Jose, CA, USA). The signal from the detector at 246 nm was integrated and recorded with a chromatography data system (Chrom Quest, version 5.0, ThermoFisher Scientific Inc., Nepean, ON, Canada). The area under the AA peak at 246 nm was subjected to least squares linear regression and the actual AA concentration in each sample determined by interpolation from the standard curve.
The column effluent was monitored with a variable wavelength UV detector (UV 6000 Thermo Separation Products; San Jose, CA, USA). The signal from the detector at 246 nm was integrated and recorded with a chromatography data system (Chrom Quest, version 5.0, ThermoFisher Scientific Inc., Nepean, ON, Canada). The area under the AA peak at 246 nm was subjected to least squares linear regression and the actual AA concentration in each sample determined by interpolation from the standard curve.
3
Purification of Thorectandrin A from Crude Extract
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An aliquot (60 mL) of the EtOH crude extract was decanted, concentrated in vacuo, and partitioned between n-BuOH (20 mL) and H2O (20 mL) (Figure S3 ). MS analysis of the partitions indicated localization of the target GNPS cluster in the n-BuOH partition. The n-BuOH soluble material was dissolved in MeOH and subjected to HPLC fractionation (Agilent Zorbax SB-CN 9.4 × 250 mm, 5 μm, 3 mL/min gradient elution over 25 min from 10% MeCN/H2O to 60% MeCN/H2O, with constant 0.01% TFA modifier), to yield thorectandrin A (1 ) (tR = 15.2 min, 1.2 mg, 0.6%) (Figure S4 ).
4
Famotidine Quantification via HPLC
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Agilent Zorbax SB-CN (50 mm × 2.1 mm I.D., 5 micron) was selected as the analytical column. The mobile phase was composed of methanol: 20 mM ammonium acetate (55 : 45, v/v). The flow rate of the mobile phase was set at 0.6 mL/min and the injection volume was 10 μL. The column temperature was set at 20°C [8 (link)]. The retention time of famotidine was found to be approximately 1.37 min as shown in Table 1 .
5
HPLC Analysis with Zorbax SB-CN Column
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The liquid chromatography system was a Lachrom Elite–Hitachi (Merck) composed of an organizer, an autosampler L‐2200, two pumps L‐2130, a column oven L‐2350 and a diode array detector (DAD) L‐2455. The system was controlled by EZchrom Elite version 3.18 HPLC System Manager Software (Merck–Hitachi Japan ). The analysis was performed on a Zorbax SB‐CN 150 × 4.6 mm, 5 μm equipped with 5 μm guard cartridges Zorbax SB‐CN 12.5 × 4.6 mm (Agilent Technologies, USA ). The SPE was performed on Isolute‐96‐CBA 100 mg/2 mL 96 fixed well plates (Biotage AB, Uppsala , Sweden ).
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