title: Sensitive determination of glucose in Dulbecco's modified Eagle medium by high-performance liquid chromatography with 1-phenyl-3-methyl-5-pyrazolone derivatization: application to gluconeogenesis studies.
abstract: A new pre-column derivative high-performance liquid chromatography (HPLC) method for determination of d-glucose with 3-O-methyl-d-glucose (3-OMG) as the internal standard was developed and validated in order to study the gluconeogenesis in HepG2 cells. Samples were derivatized with 1-phenyl-3-methy-5-pyrazolone at 70°C for 50 min. Glucose and 3-OMG were extracted by liquid-liquid extraction and separated on a YMC-Triart C18 column, with a gradient mobile phase composed of acetonitrile and 20 mm ammonium acetate solution containing 0.09% tri-ethylamine at a flow rate of 1.0 mL/min. The eluate were detected using a UV detector at 250 nm. The assay was linear over the range 0.39-25 μm (R(2) = 0.9997, n = 5) and the lower limit of quantitation was 0.39 μm (0.070 mg/mL). Intra- and inter-day precision and accuracy were <15% and within ±3%, respectively. After validation, the HPLC method was applied to investigate the gluconeogenesis in Dulbecco's modified Eagle medium (DMEM) cultured HepG2 cells. Glucose concentration was determined to be about 1-2.5 μm in this gluconeogenesis assay. In conclusion, this method has been shown to determine small amounts of glucose in DMEM successfully, with lower limit of quantitation and better sensitivity when compared with common commercial glucose assay kits.
authors: Center of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing, 210009, China.
authors: Zhaoli
authors: Z
authors: Ling
authors: Center of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing, 210009, China.
authors: Ping
authors: P
authors: Xu
authors: Center of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing, 210009, China.
authors: Zeyu
authors: Z
authors: Zhong
authors: Center of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing, 210009, China.
authors: Fan
authors: F
authors: Wang
authors: Center of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing, 210009, China.
authors: Nan
authors: N
authors: Shu
authors: Center of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing, 210009, China.
authors: Ji
authors: J
authors: Zhang
authors: Center of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing, 210009, China.
authors: Xiange
authors: X
authors: Tang
authors: Center of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing, 210009, China.
authors: Li
authors: L
authors: Liu
authors: Center of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing, 210009, China.
authors: Xiaodong
authors: X
authors: Liu
mesh_terms: D000983:Antipyrine / Q000031:analogs & derivatives / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003470:Culture Media / Q000737:chemistry*; D000077553:Edaravone; D005943:Gluconeogenesis; D005947:Glucose / Q000032:analysis*; D056945:Hep G2 Cells; D006801:Humans; D057230:Limit of Detection; D059625:Liquid-Liquid Extraction
chemical_list: D003470:Culture Media; D005947:Glucose; D000077553:Edaravone; D000983:Antipyrine
keywords: 1-phenyl-3-methyl-5-pyrazolone; HepG2 cells; derivatization; gluconeogenesis; glucose; high-performance liquid chromatography