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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
Author(s) -
Ling Zhaoli,
Xu Ping,
Zhong Zeyu,
Wang Fan,
Shu Nan,
Zhang Ji,
Tang Xiange,
Liu Li,
Liu Xiaodong
Publication year - 2016
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.3589
Subject(s) - chemistry , chromatography , derivatization , high performance liquid chromatography , ammonium acetate , detection limit , gluconeogenesis , extraction (chemistry) , acetonitrile , biochemistry , enzyme
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 C 18 column, with a gradient mobile phase composed of acetonitrile and 20 m m 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 μ m ( R 2 = 0.9997, n = 5) and the lower limit of quantitation was 0.39 μ m (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 μ m 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. Copyright © 2015 John Wiley & Sons, Ltd.