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 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.
The Diamond Hydride™ Column can retain compounds based on Aqueous Normal Phase (ANP) chromatography and would be the best suited. 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 A and B Solvents 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. Other
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% DI Water/ 50% Methanol/ 0.1% Formic Acid
B: Acetonitrile/ 0.1% Formic Acid
| time/(min) | %B |
| 0 | 95 |
| 2 | 95 |
| 10 | 0 |
| 14 | 0 |
| 15 | 95 |
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.
