The Metabolomics Core provides expertise to quantitate metabolites in biological samples using liquid chromatography/mass spectrometry. The Core serves the needs of SBP and external investigators by analyzing samples and interpretation of data.
We are partners with the University of Florida in the Southeast Center for Integrated Metabolomics (SECIM), one of six national centers funded by the National Institutes of Health to spur metabolomics research. The SBP core provides targeted metabolomics assays while the University of Florida provides global metabolomics and lipidomics assays using liquid chromatography/mass spectrometry in addition to metabolomics analysis by NMR.
Targeted metabolomics assays are available for the quantitative measurement of a wide variety of metabolites in cellular extracts, plasma, or tissues using liquid chromatography/mass spectrometry. These assays provide crucial information about metabolic pathways involving the three major classes of nutrients: lipids, carbohydrates, and protein. Our mission is to perform the assays and assist in the interpretation of the results. Our assays include the quantitation of:
• Acylcarnitines (products of fatty acid oxidation)
• Amino Acids (products of fatty acid and amino acid oxidation)
• Organic acids (TCA cycle intermediates; products of glucose and amino acid oxidation)
• Malonyl and Acetyl CoA
• NAD and NADH metabolites
A panel of 57 acylcarnitines is quantitated by liquid chromatography/mass spectrometry (Agilent 1290 HPLC/Agilent 6490 triple quadrupole mass spectrometer) with electrospray ionization. Calibration standards of the acylcarnitines are prepared in solvent that is spiked with stable isotope-labeled internal standards. Acylcarnitines are extracted from biological fluids and tissues using methanol and then derivatized with benzylhydroxylamine. Multiple reaction monitoring is used to detect acylcarnitine molecular ions that produce a characteristic acylcarnitine fragment ion that is formed by collision induced dissociation. Table 1 lists the acylcarnitines that are quantitated.
Measured acylcarnitines are grouped according to A) acetylcarnitine B) aliphatic saturated acylcarnitines C) aliphatic unsaturated acylcarnitines D) miscellaneous hydroxylated and dicarboxylic acylcarnitines.
Amino Acids and Urea Cycle Intermediates
Quantitation of 23 amino acids includes Gly, Ala, Pro, Val, Arg, Thr, Lys, Gln, Ser, Leu, Ile, Met, His, Phe, Tyr, Asn, Asp, Glu,Trp, Orn, Cit, 1-MeHis, and 3-MeHis is achieved using LC/MS/MS with electrospray ionization. Amino acids are extracted from biological fluids and tissues using methanol and derivatized with a quinoline functional group. Amino acids derivatives are separated on an Agilent 1290 HPLC interfaced to an Agilent 6490 triple quadrupole mass spectrometer. Multiple reaction monitoring is used to quantitate a fragment ion of the parent ion amino acid.
Quantitation of lactate, pyruvate, succinate, fumarate, malate, α-ketoglutarate, citrate, and 3-hydroxybutyrate is accomplished by liquid chromatography/mass spectrometry (Agilent 1290 HPLC/Agilent 6490 triple quadrupole mass spectrometer). Calibration standards are prepared in solvent and spiked with stable isotope-labeled internal standards. Organic acids are extracted from fluids and tissues using extraction and derivatization steps to form derivatives with benzylhydroxylamine. Detection of the derivatized amino acids is achieved by multiple reaction monitoring to quantitate a fragment ion of the parent ion organic acid.
Malonyl-CoA is quantitated by LC/MS/MS with electrospray ionization (Agilent 1290 HPLC/6490 triple quadrupole mass spectrometer). Calibration standards and tissues that are homogenized in 5% cold trichloroacetic acid are spiked with a stable isotope-labeled internal standard and extracted via solid phase extraction. Malonyl-CoA is quantitated by multiple reaction monitoring of a fragment ion of the malonyl-CoA parent ion.
NAD and NADH Metabolites
NMN, NAD, NADH, NADP, and NADPH are quantitated by LC/MS/MS with electrospray ionization (Agilent 1290 HPLC/6490 triple quadrupole mass spectrometer). Calibration standards and tissues that are homogenized in either acid or base are spiked with stable isotope-labeled internal standards. Metabolites are quantitated by multiple reaction monitoring of a fragment ion of each parent ion.
• Thermo Scientific UltiMate 3000 HPLC/TSQ Quantiva triple quadrupole mass spectrometer
• Agilent 1290 HPLC/6490 triple quadrupole mass spectrometer
For a Price List, please call (407)745-2129 or email us.