What suggestion can you offer for Analysis of Metabolites such as Glucose, Pyruvate, Taurocholic Acid, Gamma Glutamylcysteine, Glycochenodeoxycholate, Glycerol-3-phosphate, Spermidine, Taurine, and S-Adenosylmethionine?
Suggestion: We would recommend starting by using the Cogent Diamond Hydride™ HPLC Column for this Analysis. Many of these compounds are quite Polar and would be very difficult to Separate by Conventional Reversed Phase Methods and The Diamond Hydride™ Column can retain compounds based on Aqueous Normal Phase (ANP) Chromatography. There are a variety of Functional Groups present in these Analytes so it is difficult to say which Mobile Phase Additive would be most effective overall.
Start with 10mM Ammonium Acetate in Solvent A and Solvent B and next try 0.1% Formic Acid. You may even want to consider Ammonium Acetate in one Solvent and Formic Acid in the other if you find that you need a pH Gradient.
Additives to consider are Ammonium Formate and Acetic Acid. Use of either Isopropanol or Methanol as 50% of the A Solvent for Complex Matrices is recommended. This is because these Solvents help to Wash Strongly Adsorbed Matrix Components from the Column and extend Column life.
An example Gradient might be:
—A: 50:50 DI Water / Methanol / 0.1% Formic Acid
—B: Acetonitrile / 0.1% Formic Acid
You would start at high Organic and end at High water (in this case 50% water due to the methanol in the A Solvent) and keep a hold time to help wash some strongly retained matrix components off the Column. You can adjust the Gradient steepness if you need more Separation.