The Duke Proteomics and Metabolomics Shared Resource performs multiplexed quantitative analysis of amino acids, biogenic amines, glycerophospholipids, sphingolipids, and acylcarnitines with the Biocrates AbsoluteIDQ p180 kit. The Biocrates p180 kit allows for a wide range of biological and therapeutic studies with samples originating from cell cultures to animal models and human biological fluids although it has been developed specifically for plasma samples. Pricing is performed on a per sample basis, plus the cost of consumables from Biocrates. For more details see our pricing page or contact Lisa St. John-Williams or Will Thompson.
The Biocrates AbsoluteIDQ p180 kit uses a 96-well plate layout as shown below, which can accommodate up to 80 study samples in addition to necessary blanks and standards. Each well requires 10 uL of sample matrix for analysis. More specific information on the exact metabolites which can be detected by this kit can be found here on the Biocrates AbsoluteIDQ p180 list of metabolites.
Although up to 188 metabolites can be detected with the p180 kit, actual coverage within any particular matrix varies widely depending on the metabolite abundance in those particular matrices. Additionally, only some of the potentially identifiable metabolites will be relevant to your experiment. Biocrates provides an introductory illustration to the main experimental application areas, which can provide a starting point for tailoring your own metabolite investigations. We have performed experiments in the Proteomics and Metabolomics Shared Resource to further define biochemical coverage across a variety of matrices to assist in aligning this platform with your study of interest. The table below shows the expected performance of the platform with respect to the approximate number of metabolites in each class you might expect to measure from a variety of biological matrices using this platform.
With this kit, analysis of most amino acids and biogenic amines is facilitated by derivatization with phenylisothiocyanate. The derivatization reaction yields phenylthiocarbamyl derivatives, which allows for improved chromatographic separation of the analytes of interest. Glycerophospholipids, sphingolipids and acylcarnitines undergo the same sample preparation steps but remain underivatized and subsequently analyzed directly by flow-injection analysis tandem mass spectrometry (FIA-MS/MS). Metabolite quantitation is achieved with the use of stable-isotope labeled internal standards pre-pipetted onto the kit plate. Most biogenic amines and amino acids are fully quantified in each experiment with a seven-point calibration curve. All other analytes are semi-quantitated with a single point standard and concentration linearity is assumed. The concentration range detected by a Biocrates p180 kit varies with the analyte of interest and the sample matrix.
The figures above illustrate the differences between FIA-MS/MS and LC-MS/MS. Figure A shows a smaller time window compared to figure B, and there is no chromatographic separation in flow-injection analysis. The mass spectrometric analysis is performed on a Waters Xevo TQ-S tandem mass spectrometer and an Acquity UPLC is used for the liquid chromatography separation. Biocrates provides additional information about the p180 kit in their application notes. An example of a report provided by the Duke Proteomics and Metabolomics Shared Resource as a final product of our analysis is available for download below. If you have further questions about metabolite identification and quantification in our laboratory, please contact us.