I'm trying to set up an HPLC method for separation and relative quantification of lactate and pyruvate as substrate and product of a dehydrogenase enzyme. I'm using the attached article as starting point, using ion pairing with tetrabutylammonium (TBA) sulfate (the paper say phosphate but in principle I think it should not make a difference).
The column is a C18 Nucleodur 5um, 150 mm length at 1 mL per minute with 75% A (50 mM phosphate buffer, 6 mM TBA, pH 6,5) and 25% B (A+20% acetonitrile, final 5%).
In the chromatogram, purple is a 4 mM lactate standard, blue is a 1 mM pyruvate standard while black is the combination of both standards at the same concentration. As you can see, there is a shift of around 0,25 minutes between the standards when they are injected individually and when they are combined, which it would be what is expected in the assay. Both are prepared in LC grade water.
What could be the reason for this shift, I cannot think of any interaction that would lead to this.
Thanks for your input!
A reference standard can shift in retention time because the molecule is influenced by the micro-environment of the column and mobile phase. An example in Vitamin A reference standard dissolved in Methanol and a fat or oil sample analysed with a 'dilute and shoot' method. Thus, the sample should be matrixed matched with the reference standard by spiking the sample.
hi,
I experience this issue too.
- Retention time shifts may be related to variations in mobile phase composition, expecially in your case, where you have a mobile phase B containing ACN. I try to be clearer: according to van deemter equation, particle diffusion in coloumn is strictly related to mobile phase viscosity. Consequently, the retention time shift in standard elution, may be dependent on not properly prepared mobile phases. (ACN modifies solution viscosity). all this hypothesizing that these shifts are observed in different runs with different phases.
- moreover, another explanation may be room temperature fluctuations that also influence solvent viscosity (in the hypothesis that you are working in a not thermostatated environment);
- Finally the problem may be strictly instrumental (I have experienced this problem too). In my chromatograms I observed a perfect overlap between chromatograms in the first minutes, but I begun to observe dramatic shifts in peak retention times later, when the other pump of my binary hplc started working. I managed this problem with the hplc specialist.
Bye
Hi there,
Do you load exactly the same volume for the three samples? Are they exactly identical in composition except for lactate and pyruvate (ie. what is the counterion if any)?
About TBA, you should check as it is about ion pairing: in the paper it is monobasic TBAP which means you have 1:1 stoechiometry between TBA and phosphate; for your TBA sulfate you should precise if it is hydrogenosulfate (in this case the 1:1 stoechiometry is OK) or just sulfate (in that case you would get 2 TBA per sulfate and therefore 6mM TBAsulfate would actually be 12mM in TBA)...
Matrix is not an issue because they are the same. I would repeat the exact condition as said in the paper to see if you can repeat before modify it.
Compare the black line shift of the UV-based chromatogram and the other two. It looks like a solvent gradient starts to increase earlier in the mixture run compared to the single compound runs (however you run it isocratical, don't you). This would also describe that the differences of standard retention times remain the same in the single compound experiments and the mixture.
Did you use different prepared solutions for that runs?
Did you do a repeat injection of the same std several time (single and mixed) and was it precised?
how many times did you inject the same vial. May be the column was not equilibrated yet.
I would start with Narongs suggestion. The chromatography of the lactate by itself is good. The chromatography of the pyruvate not so much. The chromatography of the combine standards roughly the same as the pyruvate but shifted. Could be the column is not equilibrated, could be the Channel A is acting up.
In my experience ion paring can be very touchy to the sample preparation. What typically reduces the issue is to put a slightly higher concentration of the ion pairing compound into the actual samples and standards. This will be critical for samples that may contain a high amount of matrix and/or salts that could interfere with the ion pairing.
I have also had many issues with the use of ACN that could be solved by substituting methanol as the non-polar solvent. Methanol tends to solvates ions better than ACN which leads to better peak shapes, faster column re-equilibration between runs (and this can also be a factor when coming back to water from ACN with ion pairing), and often better separation.
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