GEN-MKT-18-7897-A
Aug 10, 2017 | Blogs, Life Science Research, Metabolomics | 0 comments
I recently had the opportunity to catch up with Baljit Ubhi to discuss the top questions you’re asking in regards to using Microflow HILIIC Chromatography for Targeted Metabolomics. Here’s what Bal said:
Many of the metabolites of interest in the study of metabolomics are extremely polar and therefore often unable to be analyzed through traditional coupling of reversed phase (RP) chromatography and mass spectrometry. Also to detect and quantify key metabolites from pathways of biochemical importance, samples must be run on both reversed phase and normal phase, in negative and positive ion modes requiring a total of four injections.
We have implemented hydrophilic chromatography (HILIC) with microflow and mass spectrometry to develop a method for screening over 300 polar metabolites. HILIC allows separation by partitioning of analytes between an aqueous enriched layer of a polar stationary phase. HILIC conditions typically use high organic, with a moderate amount of a salt (i.e. 20-100mM ammonium formate or acetate, pH 4.4 or 5.5. respectively). This method deviates by having 20 mM ammonium hydroxide in both mobile phases to provide constant pH of 9.0 during the chromatographic separation. The high pH deprotonates the stationary phase and allows for better selectivity of the polar metabolites. The method is multiple reaction monitoring (MRM) with positive/negative polarity switching allowing the collection of these key metabolites in a single injection!
While microflow has become increasingly popular for many applications, microflow for metabolomics has not been readily employed because the typically used (aqueous) sample solvent does not allow for injecting larger volumes of samples without sacrificing chromatographic resolution. However, by simply reconstituting the sample in an organic solvent (95% acetonitrile, pH 9), we were able to inject up to 5 µL onto the microLC column, while maintaining excellent peak shape.
The microflow Luna-NH2 HILIC chromatography provides excellent chromatographic separation of polar, hydrophilic metabolites. The cross-linked aminopropyl phase gives a slightly different selectivity than the traditional amide phase; it gives higher coverage of the metabolome when compared to the amide functionality. This allows for improved sensitivity with signal-to-noise (S/N) improvement of up to 60X and up to 50% higher coverage of the metabolome than traditional analytical approaches (see table above).
The Echo® MS+ system is a novel platform for Acoustic Ejection Mass Spectrometry (AEMS) and combines the speed of acoustic sampling with the selectivity of mass spectrometry. This platform has been designed for high throughput analysis of small and large molecules. The technology combines Acoustic Droplet Ejection (ADE), an Open Port Interface (OPI) and could be coupled with the SCIEX Triple Quad 6500+ system or the ZenoTOF 7600 system.
The Echo® MS+ system comprises of an open-port interface (OPI) and acoustic droplet ejection (ADE) module which could be coupled with a mass spectrometer. The mass spectrometer could either be a SCIEX Triple Quad 6500+ system or the ZenoTOF 7600 system. This non-liquid chromatography based; high-throughput screening platform enables rapid analysis of compounds at speeds of up to 1 sample/second.
The ability to consistently achieve reproducible results on many complex samples across multiple days is critical to a routine clinical laboratory. Laboratories relying on analytical instrumentation require stability and robustness to perform a variety of screening and confirmatory assays with confidence. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become the preferred analytical method in the clinical laboratory to reliably perform clinical testing as it provides best-in-class performance and reliability for the most challenging assays. LC-MS/MS offers the required levels of sensitivity and specificity for the detection and quantitation of molecules from complex biological samples, helping laboratories deliver highly accurate data for a variety of clinically relevant analytes across a wide range of assays.
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