Alen,

You have to use a lower flow rate for the anthocyanidins, otherwise you will have bad peakshapes, we use 0.1ml/min on a column that is usually used at 0.3-0.4ml/min for other applications. We use a Waters BEH UPLC column, if you don't have a UPLC you can try the Phenomenex Gemini or Waters Xbridge or any other column with good pH stability. We clean these columns sometimes with 50% formic acid, they don't die that easily!

What is the flow rate you are using?

It's probably going to affect your ultimate vacuum, so you'll lose a bit in sensitivity. Of course, with the anthocyanins that shouldn't be an issue. I haven't had any issues running my LC-MS with up to 1% formic (Agilent 1100MSD), but have never tried 5%. If it was a salt I'd shy away, but I think you'll be able to get away with it in the acid form.

It would help if you specified your ionization mode (pos or neg). Expect to see ionization suppression (lower response) in negative ion ESI when using HCOOH as compared to using no acid. There is always a trade off between better chromatography separation and ESI effeciency when using pH adjustments. pH to give increased chromatographic separation normally is the inverse of pH of best ionization effeciency. Running 1% HCOOH in positive ion mode should pose no suppression issues. Running at 1% HCOOH will not have any negative consequenses on your equipment. 5% HCOOH seems excessive for ESI.

It might have ion suppression issues if the formic concentration is that high. However, a split of HPLC flow and post-column compensation before the API, might get around this issue.

Hi Alen.

Should not use a silica-based LC column. Get a RP-18 type polymer based one. Should also be carefull with the potential you use in the ion source. In order to separate anthocyanins use a grandient program starting with high percentage of water.

There will not be any problem to the LC-MS system, this is a weak acid of low Ka. Only a slight decrease in the useful life of the column, however, you can use the gradient to their analysis.

From my experience, you get a strong suppression using concentration higher than 0.5% of FA in ESI neg mode. Usually a 0.1% concentration is high enough to provide a good peak shape. But if you get a lower quality of separation and need MS detection, think to change solvent or column.

A high acid concentration in the mobile phase flow can lead to very high ionic strength which can lead to a short circuit which disables the high voltage.However if it is possible to reduce the flow of mobile phase that reaches the electrospray source via a postcolumn split of an 20% of the total flow, I think could work smoothly with good stability of the ion current. Good luck

I would not increase the concentration of FA more than 1%. If the chromatographic separation is not succesful enough under these conditions, try to use HILIC column which is great for the separation of polar compounds. In this case you have to change eluents, ....one comment to above mentioned suggetions, I would never use TFA as a mobile phase modifier for LC-MS where many other analyses are used to run. It remains in the system for forever because it is very hard to flush it away.

I completely agree with Alexandra, never use TFA on a LC system that may be coupled with ESI-MS, because you'll never get a clean and sensitive system again.

Hi Alen, please note that if you don't add FA to your organic modifier, the pH will change during the gradient run as less and less of the FA in the water component is mixed in at higher organic modifier levels.

I agree Kerry Peru's points

The use of 1% to 5% formic acid is to secure ion suppression however try to replace formic acid by trifluoroacetic acid but with a much lower concentration than 1% since it is a very strong acid. I suggest to use 0.01% as a trial and see the results.

Aloso use grandient elution as suggested by some collegeaues.

Yes i agree with the above comments on high(%) use of formic acid in MS.General recomendations of HCOOH is less than 1% as the system sensitivity decreases in long runs.Better to use TFA(0.1%) in positive ionisation.

We use up to 7.5% formic acid for methods to analyse anthocyanidins in eSI positive, TFA does not give better results. The big draw back of high amounts of formic acid is that the impurities in formic acid is much more prominent and you should be aware of them. ESI negative will give you a much weaker signal and you will most probably not see formate adducts and a much weaker M-H.

Thank you all for your expertise. I forgot to mention that I am working with positive ESI at 0.6 mL/min. I have also never used FA at a concentration higher than 1% that is why I was somewhat surprised when very high concentrations (10% @ 0.4 mL/min) of this acid are readily used in LC-MS for the separation of anthocyanins (e.g. K. Nagy et al., Anal. Chem., 2009, 81, 6347-6356). I know that ordinary silica C18 column would not give a very long column life, that is why I was thinking about using the Hypercarb column from Thermo, which has a working pH range 1-14, but 5% or 10% of FA, respectively, and MS just don't feel very compatible from my point of view. I dislike using TFA in combination with MS because, as some of you suggested, it can be a pain to remove it after the analysis.

@ Stander, do you have any problems with the stability of the spray current at this high FA concentration, which Jose Rios also implied? what flow rate do you use?

Alen,

You have to use a lower flow rate for the anthocyanidins, otherwise you will have bad peakshapes, we use 0.1ml/min on a column that is usually used at 0.3-0.4ml/min for other applications. We use a Waters BEH UPLC column, if you don't have a UPLC you can try the Phenomenex Gemini or Waters Xbridge or any other column with good pH stability. We clean these columns sometimes with 50% formic acid, they don't die that easily!

I suggest you this paper of Giordano L et Al: Development and validation of an LC-MS/MS

analysis for simultaneous determination of

delphinidin-3-glucoside, cyanidin-3-glucoside and

cyanidin-3-(6-malonylglucoside) in human plasma

and urine after blood orange juice administration

I agree with those who express concerns about signal suppression and increased impurities. We have found that the formic acid in sealed ampules is actually very impure. If you're using SRM, though, that might not be much of an issue. I also agree with the person that suggested 0.05% TFA + 0.1% formic acid. I've had good luck with this combination.

@ Anna, thank you for your reference. I read the article but the validated method is unfortunately developed for only four compounds whereas in our case we have as much as 20 different anthocyanins in our sample (natural sample). But I see that in this case 5% HCOOH was also used for the LC-MS/MS analysis.

anthocyanin, U need ion-paring agent to improve the resolution. please try TFA at low concentration

I find your question ver interesting as you have answered it yourself in the question.

My colleague used 0.2% TFA and 1% formic acid. You can try the same, perhaps first lower the amount of formic acid slightly.

TFA and formic acid were used simultaneously

I know TFA is a good ion-pairing reagent, but at the same time it also causes severe ion suppresion and most people agree it should be avoided in MS. What are your thoughts on that?

Yes, the conc. of TFA should be kept as low as possible

because you have to use very acidic condition of keep the peak shape. You can switch to tfa if you want, but probably won't help much on MS

TFA (0.1%) suppressed our mass spec (HR-QTOF) sensitivity. Although many researchers are using, as per my experience, TFA is not a good mobile phase. Double check with your mass spec  company.

Formic acid at 1% is a fairly normal use, 5% is excessive.  TFA is ion suppressive and is rarely, if ever used with small molecules. 

Hi Bela,

I know 5% is excessive (and not normally used), but why? I dont get much ion suppression compared to 1%, so why not use it?

If in your work you are not getting much ion suppression then use it.....

Two main reasons why high concentration of acid in ESI positive mode causes the ion suppression is the possibility for neutral ion pairs and the competition for available droplet surface. The location near the droplet surface is important for an effective ejection of analyte from the droplet. Our studies have not observed strong TFA suppression effects (Liigand et al J. Am. soc. Mass Spectrom 28(3) 461-469; Ojakivi et al ChemistrySelect 3(1) 12394-12397 ) Kostiainen and Kauppila review 2009 suggested that ESI point of view the additive should not exceed 10mM.

Hi Alen, I have the same question. Could you please tell me how did you solve this problem? Thanks! Alen Albreht

Hi Long, I just went with 5% formic acid, no problems with the MS so far. Just make sure that your capillary voltage is low enough (assuming you are using ESI) to avoid high ESI current (and arcing), which causes an unstable ESI signal (and could damage the MS in the long run).

Hope this helps...

Agree, we are also using 5% formic acid with our anthocyanin methods

I forgot to mention that it does not work well in negative ESI due to the obvious reasons...

Thank you very much Alen. Much appreciated! :) Alen Albreht

Perhaps of some interest to those interested at this RG thread. ― About calculating the pH of either formic acid aq. sol. and of formic acid / sodium formate buffer aq. sol. at this forum: https://www.researchgate.net/post/pH_calculation_of_a_mixture_of_formic_acid_NaOH_and_water