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Journal article
Diisopropylethylamine/hexafluoroisopropanol-mediated ion-pairing ultra-high-performance liquid chromatography/mass spectrometry for phosphate and carboxylate metabolite analysis: utility for studying cellular metabolism
Rapid communications in mass spectrometry, v 30(16), pp 1835-1845
30 Aug 2016
PMID: 27476658
Featured in Collection : UN Sustainable Development Goals @ Drexel
Abstract
RationaleMass spectrometric (MS) analysis of low molecular weight polar metabolites can be challenging because of poor chromatographic resolution of isomers and insufficient ionization efficiency. These metabolites include intermediates in key metabolic pathways, such as glycolysis, the pentose phosphate pathway, and the Krebs cycle. Therefore, sensitive, specific, and comprehensive quantitative analysis of these metabolites in biological fluids or cell culture models can provide insight into multiple disease states where perturbed metabolism plays a role.
MethodsAn ion-pairing reversed-phase ultra-high-performance liquid chromatography (IP-RP-UHPLC)/MS approach to separate and analyze biochemically relevant phosphate- and carboxylic acid-containing metabolites was developed. Diisopropylethylamine (DIPEA) was used as an IP reagent in combination with reversed-phase liquid chromatography (RP-LC) and a triple quadrupole mass spectrometer using selected reaction monitoring (SRM) and negative electrospray ionization (NESI). An additional reagent, hexafluoroisopropanol (HFIP), which has been previously used to improve sensitivity of nucleotide analysis by UHPLC/MS, was used to enhance sensitivity.
ResultsHFIP versus acetic acid, when added with the IP base, increased the sensitivity of IP-RP-UHPLC/NESI-MS up to 10-fold for certain analytes including fructose-1,6-bisphosphate, phosphoenolpyruvate, and 6-phosphogluconate. It also improved the retention of the metabolites on a C-18 reversed-phase column, and allowed the chromatographic separation of important isomeric metabolites. This methodology was amenable to quantification of key metabolites in cell culture experiments. The applicability of the method was demonstrated by monitoring the metabolic adaptations resulting from rapamycin treatment of DB-1 human melanoma cells.
ConclusionsA rapid, sensitive, and specific IP-RP-UHPLC/NESI-MS method was used to quantify metabolites from several biochemical pathways. IP with DIPEA and HFIP increased the sensitivity and improved chromatographic separation when used with reversed-phase UHPLC.
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Details
- Title
- Diisopropylethylamine/hexafluoroisopropanol-mediated ion-pairing ultra-high-performance liquid chromatography/mass spectrometry for phosphate and carboxylate metabolite analysis: utility for studying cellular metabolism
- Creators
- Lili Guo - Penn SRP and Center for Excellence in Environmental Toxicology, Department of Systems Pharmacology and Translational Therapeutics University of Pennsylvania Philadelphia PA 19104 USAAndrew J. Worth - Penn SRP and Center for Excellence in Environmental Toxicology, Department of Systems Pharmacology and Translational Therapeutics University of Pennsylvania Philadelphia PA 19104 USAClementina Mesaros - Penn SRP and Center for Excellence in Environmental Toxicology, Department of Systems Pharmacology and Translational Therapeutics University of Pennsylvania Philadelphia PA 19104 USANathaniel W. Snyder - Drexel UniversityJerry D. Glickson - University of PennsylvaniaIan A. Blair - Penn SRP and Center for Excellence in Environmental Toxicology, Department of Systems Pharmacology and Translational Therapeutics University of Pennsylvania Philadelphia PA 19104 USA
- Publication Details
- Rapid communications in mass spectrometry, v 30(16), pp 1835-1845
- Publisher
- Wiley
- Number of pages
- 11
- Grant note
- U54HL117798; P42ES023720; P30ES013508; P30CA016520; K22ES26235; T32ES019851; R01CA129544; R01CA172820 / NIH; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA U54HL117798 / NATIONAL HEART, LUNG, AND BLOOD INSTITUTE; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA; NIH National Heart Lung & Blood Institute (NHLBI) Pennsylvania Department of Health CURE grant R01CA129544 / NATIONAL CANCER INSTITUTE; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA; NIH National Cancer Institute (NCI) T32ES019851 / NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA; NIH National Institute of Environmental Health Sciences (NIEHS)
- Resource Type
- Journal article
- Language
- English
- Academic Unit
- A.J. Drexel Autism Institute
- Web of Science ID
- WOS:000381081600001
- Scopus ID
- 2-s2.0-84979942233
- Other Identifier
- 991019167801404721
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- Collaboration types
- Domestic collaboration
- Web of Science research areas
- Biochemical Research Methods
- Chemistry, Analytical
- Spectroscopy