Separation Technique for the Determination of Highly Polar Metabolites in Biological Samples

Iwasaki, Yusuke; Sawada, Takahiro; Hatayama, Kentaro; Ohyagi, Akihito; Tsukuda, Yuri; Namekawa, Kyohei; Ito, Rie; Saito, Koichi; Nakazawa, Hiroyuki

doi:10.3390/metabo2030496

Cited by 43 publications

(21 citation statements)

References 109 publications

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“…The CE-TOFMS technique has shown to be an accurate method for the analysis of highly polar and ionic metabolites. 48) Therefore, in this study we used CE-TOFMS to examine the dynamic changes of various hydrophilic metabolites in the 786-O Res cells. However, hydrophobic metabolites, such as those involved in lipid metabolism, would need to be examined using LC-MS or GC-MS to obtain a comprehensive picture of the metabolic changes associated with the acquisition of sunitinib resistance.…”

Section: Discussionmentioning

confidence: 99%

Investigation of Metabolomic Changes in Sunitinib-Resistant Human Renal Carcinoma 786-O Cells by Capillary Electrophoresis-Time of Flight Mass Spectrometry

Hatakeyama

Fujiwara

Sato

et al. 2018

Biological & Pharmaceutical Bulletin

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Acquired resistance to sunitinib is a challenge in the treatment of renal cell carcinoma (RCC). The dysregulation of cellular metabolism is prevalent during resistance acquisition. It is known that in sunitinib-resistant RCC 786-O (786-O Res) cells sunitinib is mainly sequestered in the intracellular lysosomes. However, the relevance between sunitinib resistance and cellular metabolism has not been examined. In this study, we examined the metabolic changes in 786-O Res by using capillary electrophoresis-time of flight mass spectrometry. The cell line 786-O Res was established via persistent treatment with sunitinib, where increase in intracellular sunitinib, and sizes of lysosomes and nuclei were enhanced as compared with those in the parental 786-O (786-O Par) cells. Metabolic analyses revealed that out of the 110 metabolites examined, 13 were up-regulated and 4 were down-regulated in the 786-O Res cells. The glycolysis, tricarboxylic acid cycle and pentose phosphate pathway (PPP) were identified as being altered in the sunitinib-resistant cells, which resulted in the enhanced metabolisms of energy, nucleic acids, and glutathione redox cycle. As sunitinib was sequestered in the enlarged lysosomes in 786-O Res, the enriched energy metabolism might contribute to the maintenance of luminal pH in lysosomes via the H+ ATPase. The changes in the PPP could contribute to nuclei enlargement through up-regulation of nucleic acid biosynthesis and protect 786-O Res from cytotoxicity induced by sunitinib through up-regulation of reduced glutathione. Though the direct link between sunitinib resistance and metabolic alternation remains to be elucidated, this metabolomics study provides fundamental insights into acquisition of sunitinib resistance.

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Section: Discussionmentioning

confidence: 99%

Investigation of Metabolomic Changes in Sunitinib-Resistant Human Renal Carcinoma 786-O Cells by Capillary Electrophoresis-Time of Flight Mass Spectrometry

Hatakeyama

Fujiwara

Sato

et al. 2018

Biological & Pharmaceutical Bulletin

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“…1). Additionally, improvements in ionization methods and separation techniques have helped reduce matrix effects due to co-eluting compounds common in complex mixtures, but ion suppression remains a primary issue for MS metabolomics [11, 53]. Electrospray ionization mass spectrometry (ESI-MS) sensitivity is directly dependent on a compound's pKa and hydrophobicity, which can be negatively impacted by the heterogeneous composition of a metabolomics sample [54].…”

Section: Introductionmentioning

confidence: 99%

Beyond the paradigm: Combining mass spectrometry and nuclear magnetic resonance for metabolomics

Marshall

Powers

2017

Progress in Nuclear Magnetic Resonance Spectroscopy

182

136

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Metabolomics is undergoing tremendous growth and is being employed to solve a diversity of biological problems from environmental issues to the identification of biomarkers for human diseases. Nuclear magnetic resonance (NMR) and mass spectrometry (MS) are the analytical tools that are routinely, but separately, used to obtain metabolomics data sets due to their versatility, accessibility, and unique strengths. NMR requires minimal sample handling without the need for chromatography, is easily quantitative, and provides multiple means of metabolite identification, but is limited to detecting the most abundant metabolites (≥ 1 μM). Conversely, mass spectrometry has the ability to measure metabolites at very low concentrations (femtomolar to attomolar) and has a higher resolution (∼103-104) and dynamic range (∼103-104), but quantitation is a challenge and sample complexity may limit metabolite detection because of ion suppression. Consequently, liquid chromatography (LC) or gas chromatography (GC) is commonly employed in conjunction with MS, but this may lead to other sources of error. As a result, NMR and mass spectrometry are highly complementary, and combining the two techniques is likely to improve the overall quality of a study and enhance the coverage of the metabolome. While the majority of metabolomic studies use a single analytical source, there is a growing appreciation of the inherent value of combining NMR and MS for metabolomics. An overview of the current state of utilizing both NMR and MS for metabolomics will be presented.

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“…The mobile phase was optimized to maximize peak responses of the combination of acetonitrile phosphate buffer (40:60; pH 7.0). Mixing of the mobile phase was also optimized because it can affect the pH of the phosphate buffer and influence reactions between metformin HCl and SDS ion pair [7][8][9][10][11]. The preferred mixing method involved manual mixing by the analyst.…”

Section: Optimization Of Chromatography Conditionsmentioning

confidence: 99%

Development and Validation of a High-Performance Liquid Chromatography Method for Determining Metformin Hydrochloride in Dried Blood Spots

2018

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Objective: This study aimed to develop an analytical method for determining metformin concentrations in human dried blood spots (DBS) using optimal chromatographic conditions and DBS preparation methods and validate the analytical methods.Methods: Analysis metformin HCl performed using C-18 columns (250 mm × 4.6 mm) at a column temperature of 40°C with a mobile phase acetonitrile phosphate buffer (40:60% v/v) at pH 7.0 using a flow rate of 0.8 mL/min, a photodiode array detector at a wavelength of 234 nm, and atorvastatin calcium as an internal standard. Samples were prepared by protein precipitation with 60% methanol and then drying using nitrogen gas at 60°C for 15 min before reconstituting in 200 µL of the mobile phase.Results: Invalidation procedures, the present methods met the acceptance criteria stated in the EMEA Bioanalytical Guideline 2011. The method was also linear at the concentration range of 25.0-5000.0 ng/mL (r=0.9997). Conclusion:The developed method for determination of metformin HCl in DBS sample was valid with a concentration range of 25.0-5000.0 ng/mL and can be applied to in vivo studies.

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Separation Technique for the Determination of Highly Polar Metabolites in Biological Samples

Cited by 43 publications

References 109 publications

Investigation of Metabolomic Changes in Sunitinib-Resistant Human Renal Carcinoma 786-O Cells by Capillary Electrophoresis-Time of Flight Mass Spectrometry

Investigation of Metabolomic Changes in Sunitinib-Resistant Human Renal Carcinoma 786-O Cells by Capillary Electrophoresis-Time of Flight Mass Spectrometry

Beyond the paradigm: Combining mass spectrometry and nuclear magnetic resonance for metabolomics

Development and Validation of a High-Performance Liquid Chromatography Method for Determining Metformin Hydrochloride in Dried Blood Spots

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