My mobile phase is acetonitrile and water (0,1% formic acid) and two compounds (Methotrexate and Ciprofloxacin - cytostatic drugs) show double peaks but these peaks are far from each other. eg: Methotrexate have peaks in tr=5.27 min and 1.25 and both are proportional and Ciprofloxacin have peaks in tr=5.46 and 1.28). The method uses MRM mode. These double peaks result in poor limits of detection.
Hi! have you performed a google search with "HPLC", "troubleshoot", "double peaks"? it seems to be associated to a large void volume (column collapse). Inspect your LC system carefully (especially at the column level. If not, I would suggest do a longer conditioning and a longer elution step and see how that affect your chromatography. Try other compounds as well to see if it is unique to this particular compound.
Good Luck
T
Hello Thierry, thanks for the suggestion. I will try to change the column. The column used was a C18 (5um x 2.1 x 100mm). Other pharmaceutical compounds don't have double peaks and this problem is associated with split peaks usually in the HPLC troubleshoot. Interestingly, These compounds with double peaks have good peak shape.
Best regards,
Are you sure that the peaks come from the same respective structure? Or is it possible that they are isomers eluting at different times (I don't know by heart if they are chiral)?
Anyway, I've read multiple times that reversing the column can help if all other things fail, but I have no personal experience with this (read: http://www.chromatographyonline.com/peak-shape-problems-0?id=&pageID=1&sk=&date=)
best of luck!
Hi Rodrigo,
ok if other compounds don't generate any doublepeaks then the LC should be fine, then the other component is your molecule. I quite like Hannes suggestion.
T
This could be as previously suggested isomers. Or it might be impurities with the same molecular weight and fragmentation patterns. Have you run known pure standards and optimized the transitions using those standards? I would also decrease the pH as this will minimize secondary interactions with the column. Ther are many options for this however, I think the first step is to determine if that truly is your compound. Also what is your solvent that your compound is dissolved in. If this is a lot stronger organic than you intial gradient you could see this. The samples should be in similar matrix to your intial gradient or a small injection that won't affect your gradient.
what's the volume ratio of the acetonitrile and water? did you try different ratio of the mobile phase or the different pH value? the peak maybe come from the impurity or the ionized sample during the separation.
If changing the column doesn't solve your problem, try one run using the same conditions but without MRM and see how they look. You may have an adduct and the MRM may be picking some ions produced in the fragmentor corresponding to the "original" molecules.
hello,
your problem can have many causes and solutions.
I think that this molecules are partially ionized, some molecules are in the neutral form and the others in the ionized form. I think interesting to increase the concentration of formic acid on the mobile phase, and increase formic acid on the injection solvent.
good luck with your analysis!!! tell me if you have a good results with my opinion.
best regards
Janclei
As per above, what's the composition of your sample buffer? Are your compounds dissolved in polar, organic solvents? Are they dissolved in water? Are they dissolved in a mixture of both? Based on your column dimensions, I sense that you are losing some of your analytes in the void volume. Long story short, change the composition of your sample buffer to contain less organic solvent (if its in organic solvent). If its not in org. solvent, check the pKa of your analytes and try to load at a pH that favours a neutral compound. I took a quick look at your compounds, acidic side chains, some alcohols... Try water. or something like 80:20 water:methanol. Good luck.
I am 100% agree with scott but before this kindly share are you getting the same peaks in LC also or you are not getting the two peaks in HPLC.
The separation in the peaks is not related to mass it ispurely depends on the HPLC column and your mobile phase.
What HPLC system are you using? Could be you are getting carry over from injection to injection. Try running a blank right after running your high standard. Are you seeing theses peaks? If so you might want to incorporate a strong wash into your LC program. Also look at your sample loop, it may be to large, or small, for your injection amount. Try to match it as closely as possible. Also try to match your standards to initial gradient conditions. Are you injecting 100% organic? If you start LC at 20% ACN, make sure your standards are prepared at 20%ACN. You will see immediate improvement if you follow this simple rule.
Hi,
Thanks for all the suggestion and Scott Farrow was right, I changed the solvent of the sample for 80/20 (water/methanol) and the additional peaks disappeared (consequently increased L.D). My initial solvent was 100 methanol and i tested different proportions of water/methanol and 80/20 was great.
Peter Philbrook, I used methanol because it's the elution solvent.
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