The Missing Link in Your Metabolomics Analysis

The normal tendency for any analyst is to stay in their comfort zone with HPLC techniques. Sometimes this works, but often not! I see reverse-phase columns forced into application areas which they are not designed for or suited.

Often hydrophilic interaction liquid chromatography (HILIC), or mixed-mode columns are the next reluctant steps taken when dealing with hydrophobic, polar, ionizable or charged ionic compounds. There is not usually the third step! However, there are times when HILIC chromatography simply does not have the required resolution, selectivity, or effectively runs the analysis with the desired peak shape. A good example is metabolomic analysis of the compounds in central metabolism and the energy cycles. The compounds in the central metabolomic pathways are all negatively charged with either single or multiple carboxylic acid functionalities or phosphorylated. Also, several of these compounds are isobaric isomers such as glucose 1 and 6 phosphate. These cannot be distinguished with MS only (even by HRAM-MS) and require good chromatographic separation. Such separations are not easy to accomplish with HILIC and more difficult with HPLC. Usually, the multiply phosphorylated nucleotide’s, such as adenosine triphosphate (ATP), peak shape is very poor. On the other hand, Ion exchange chromatography (IC) produces great separations for all these compounds with great peak shapes for quantitation.

I hear “IC is not compatible with mass spectrometry (MS)” often. Well, let me tell you about a growing trend. IC-MS is becoming increasingly popular as people try it and realize this is very easy to do, and the results are simply astounding. There is no need to make up eluent. The system only pumps pure water into an eluent generator (EG), which electrolytically produces precise concentrations of the potassium hydroxide (KOH) pushing-ion from 0 to 200mM at the head of the ion exchange column. Interestingly, as the KOH gradient concentration increases, there is also a pH gradient produced. It goes right through the pK_a range of the individual hydroxyl (OH) groups on these analytes, around pH 12. As the OH groups dissociate and form a negative charge, it will give tighter binding to the anion exchange column, allowing the separation of almost identical sugar molecules with the smallest differences in the pK_a values of the individual OH groups. I have not seen this level of selectivity with any other chromatography technique for these compounds.

The IC system then uses a continuously electrolytically regenerated charged membrane suppressor to convert the KOH back to H20 before entering the MS. The system is easy to use and extremely precise. The peak shape and the separation are the best I have ever seen for charged metabolites. However, it is outside the comfort zone of the standard HPLC user. Or is it? The ion chromatography system is an HPLC pump that pumps the sample from an HPLC autosampler through a column and then a detector. Sounds familiar? It is like standard HPLC to me but with an anion exchange column instead of a C18 column.

Wait, it is easier than standard HPLC as you do not have to make up an eluent because the system does this for you. The MS system also sees the purest background eluent it has ever seen, as the starting eluent was just pure water. It has gone through another ion exchange purification system, the suppressor, before it reaches the MS with a continuously monitored background conductivity of below 1µs. The sensitive conductivity detection can be used as a preference for some analytes for quantification instead of MS signal.

Make your life easier give it a try for these difficult and challenging analytes.