Analysis of adenosine phosphates in HepG-2 cell by a HPLC–ESI-MS system with porous graphitic carbon as stationary phase

Wang, Jun; Lin, Tao; Lai, Jia-Ping; Cai, Zongwei; Yang, M. S.

doi:10.1016/j.jchromb.2009.05.027

Cited by 40 publications

(47 citation statements)

References 30 publications

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“…16,19 The extraction was generally performed at a low temperature; however, there were no effects on the recovery rate using a precooled solvent to -30 C (data not shown). Based on our investigations, the addition of ACN:water (70:30) extraction was found to be the best extraction technique.…”

Section: Resultsmentioning

confidence: 96%

“…For protein precipitation, PCA was mainly employed when analysis was performed either with HPLC or LC-MS devices. 18,19,27 Highly variable recoveries were observed when PCA was used as the extraction solvent with the exception of NAD + . In addition, the peak shape of GTP and GDP highlighted weak retention and became worse (data not shown).…”

Section: Resultsmentioning

confidence: 99%

“…Organic solvents as extraction solvents 18,19,27 were widely used and known to have a good ability to precipitate proteins. Organic solvents such as IPA, ACN, MeOH, and EtOH were also evaluated as extraction solvents.…”

Section: Resultsmentioning

confidence: 99%

“…16 These biochemicals have now been measured in a variety of systems, including cultured cells, [17][18][19] isolated tissues, 15 food substances, [12][13][14][20][21][22][23][24][25] fungi, 26 and biological samples. 11 Previously, we also reported the use of the HPLC method for determining the purine content of food, 14 beer, and beer-like alcoholic beverages, 12 whereas LC-MS was suitable for the simultaneous analysis of 23 species of purine bases, pyrimidines, nucleosides, and nucleotides.…”

Section: Introductionmentioning

confidence: 99%

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Analysis of Intra- and Extracellular Levels of Purine Bases, Nucleosides, and Nucleotides in HepG2 Cells by High-performance Liquid Chromatography

2015

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To evaluate cellular uptake and purine transport, we developed a high-performance liquid chromatography method for intra-and extracellular purine quantification. Our aim was to develop an effective method for simultaneously quantifying the substrate and metabolites with high sensitivity. C18 columns from different manufacturers were tested for simultaneous quantification of 22 different purine bases, nucleosides, and nucleotides. We used a YMC-Triart C18 column. The analysis conditions, including extraction solutions for the cells and cell culture medium, were optimized to achieve good quantification. Linearity, accuracy, determination limits, and recovery were assessed and showed good performance. The developed HPLC method was successfully applied to the qualitative analysis of 22 different intra-and extracellular purines, demonstrating that it is useful for studying the overall pattern of purine metabolism. This method could also be useful for evaluating metabolic dynamics of purines under a variety of stimulatory conditions of culture cells.

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Section: Resultsmentioning

confidence: 96%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Analysis of Intra- and Extracellular Levels of Purine Bases, Nucleosides, and Nucleotides in HepG2 Cells by High-performance Liquid Chromatography

2015

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“…The commercial kits (ab65313, abcam; K255-200, BioVision; TB288, Promega; FL-AA, Sigma-Aldrich) and LC methods using fluorometric detection or mass spectrometry (Levitt et al 1984;Ramos-Salazar and Baines 1985;Fujimori et al 1990; Kawamoto et al 1998;Katayama et al 2001;Tuytten et al 2002;Xing et al 2004;Klawitter et al 2007;Wang et al 2009;Birkler et al 2010;Pabst et al 2010;Jiang et al 2012) can only detect some of these compounds simultaneously.…”

Section: Introductionmentioning

confidence: 99%

A Novel Method for Measuring the ATP-Related Compounds in Human Erythrocytes

Aragón-Martínez

Galicia-Castillo

Isiordia-Espinoza

et al. 2014

Tohoku J. Exp. Med.

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The ATP-related compounds in whole blood or red blood cells have been used to evaluate the energy status of erythrocytes and the degradation level of the phosphorylated compounds under various conditions, such as chronic renal failure, drug monitoring, cancer, exposure to environmental toxics, and organ preservation. The complete interpretation of the energetic homeostasis of erythrocytes is only performed using the compounds involved in the degradation pathway for adenine nucleotides alongside the uric acid value. For the first time, we report a liquid chromatographic method using a diode array detector that measures all of these compounds in a small human whole blood sample (125 μL) within an acceptable time of 20 min. The stability was evaluated for all of the compounds and ranged from 96.3 to 105.1% versus the day zero values. The measurement had an adequate sensitivity for the ATP-related compounds (detection limits from 0.001 to 0.097 μmol/L and quantification limits from 0.004 to 0.294 μmol/L). This method is particularly useful for measuring inosine monophosphate, inosine, hypoxanthine, and uric acid. Moreover, this assay had acceptable linearity (r > 0.990), precision (coefficients of variation ranged from 0.1 to 2.0%), specificity (similar retention times and spectra in all samples) and recoveries (ranged from 89.2 to 104.9%). The newly developed method is invaluable for assessing the energetic homeostasis of red blood cells under diverse conditions, such as in vitro experiments and clinical settings.

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