Chromatography Forum

Please help had following gradient and column for ATP detection.

Colum: waterxterra,C8,2.1x50mm.
5micron

Mobile Phase A:10mM DMHA( made by mixing 1.73 ml of DMHA(mw:129) in 998ml water) Adusted the Ph to 7.0 by dil HCl.
Mobile phase B: Methanol

ATP was made in water 50uM

I see ion at 130(ESI+VE) and 295(ESI+VE) all over the run more than 10 million count. nothing else. UV-VIS shows ATP comes out at 1.5 min. but dont see any in ion at 508 or neg 506 in -ve mode.

What happed to mass spec ? is 130 is due to DMHA..i dont understand what is 295 ?

Please help..How can I attache the file to the post? I wanted to post chromatograma...any idea

I really appreciate your help

DMHA? dimethylhippuric acid? That's kind of an odd ball buffer. Why not try simple ammonium acetate instead?? (unless you need exactly pH 7.00)

Did you try opening your m/z window on acquisition? Acquire 500-550 m/z to see if you can observe any adducts?

I found this paper:
Title: AN LC-MS Method for a Hexokinase Inhibitor Study Based on Adenosine 5'-Triphosphate
Determination and Application to the Anticancer Mechanism of Momordica cochinchinensis
Author(s): Pan, L; Yu, YQ; Sun, LW
Source: CHROMATOGRAPHIA Volume: 72 Issue: 5-6 Pages: 425-430 Published: 2010


You could try to replicate their mobile phase and see what happens..

Dimethyhexylamine not dimethylhippuric acid. I read a paper they used the same mobile phase..i dont know whats wrong with my lcms. I wanted to use volatile buffer so thats why i chose amine buffer

Are you operating in positive ion mode? If so, amine containing buffers such as DMHA or ammonium acetate are often a source of ion suppression. Available protons (and there aren't that many at pH 7) largely go toward formation of the much more abundant ammonium ion rather than to your analyte. We see much better results, and more consistent results, with simple organic acids, such as formic or acetic, to adjust to lower pH for positive ion mode. It's best to avoid all amines in the mobile phase for positive ion.

If, in fact, you are operating in negative mode, I would go to higher pH to ensure you are abstracting all available acidic protons.

Bottom line-- I think you need to experiment with adjustment of the pH one way or the other.

Fred Klink
San Ramon, California
www.mass-spec-training.com

Ok, sorry for the misnomer on DMHA (just not familiar with it). Whoops I made a fool of myself

Secondary and tertiary amines are proton-abstracting modifiers and, like fredklink explained, they will cause signal suppression in positive mode. You might have better luck using plain old 0.1% ammonium hydroxide to help your signal instead of DMHA... but a buffer is best as you have complete control of the pH.

I copied the LC/MS conditions of the paper I mentioned previously and pasted them below. These conditions (or modification thereof) are definitely worth a shot.

LC–MS Instrumentation and Conditions
An Agilent (Palo Alto, USA) 1100 MSD single quadrupole mass spectrometer equipped with an ESI source operating in the positive ion mode was used as a detector...[clip] The selected ion monitoring (SIM) mode was used for quantification with m/z of 508 for ATP and 428 for ADP. The ESI–MS operating variables used in this study were as follows: capillary voltage, 3.0 kV; source temperature, 300 °C; drying gas flow, 10 L/min; fragment voltage, 100 V; and nebulizer pressure, 30 psi.
The LC separation was performed using an Agilent Extend C-18 column (3.1 x 100 mm, 3 um). The mobile phase consisted of 5 mM ammonium acetate (pH 7.5) and methanol (60:40, v/v), and it was delivered at a flow rate of 0.3 mL/min; the column temperature was set at 20 °C.


One question, do you make your ATP solution fresh?

130 in positive mode is almost certainly DMHA. It's also probably got some tendency to behave like an ion pair reagent, having hydrophobicity and the right positive charge to stick to your negative analyte. It will therefore (I think, probably) increase the retention time of ATP on your column relative to a non-ion-pairing buffer of the same pH.

Does the 295 peak in positive mode also have some isotope peak at 297? I'm not experienced with DMHA, but wonder about cluster ions such as [2DMHA + HCl + H](+). But who knows, someone else might have a better idea.

You may find it hard to get rid of 130 from your system.

In negative mode, do you see any ions associated with the ATP peak? Remember, ATP can easily have more than one charge, so you may have to look for a doubly charged ion. You may also find that the phosphates have exchanged hydrogens for sodium.

I dont see any peak in negative ion mode either..only when i switched to formic acid then i saw peak not good but then i can not retain ATP on my C8 column. I am going to try ammonium acetate to see if i can get better results.

I would also advise doing some injections of reasonably large amounts of ATP with no column at all, so you can check it ionises in your conditions. You may need to reduce source temperatures as it is quite labile.

I though I could use uv for high conc samples cause i do have nice uv peak 4.0 with DMHA and then spend time on mass spec to fix the issue , but when i injected ATP diluted in 100mM ammonium bicarbonate the peaks comes really early now 0.9 min instead of 4.0 min..what is this buffer doing now?..i tried it with ammonim acetate still the same..ATP diluted in water comes at 4.0 but ATP diluted in buffer elutes at 4.0min

The phosphates really get in the way with ATP retaining on a column....... you need an ion-pairing agent to help sometimes.

There are so many possibilities out there... here's another one: 0.1% formic acid and 0.01% trifluoroacetic acid in the mobile phase. The trifluoroacetic acid will act as an ion-paring agent to aid in retention on the column, but will cause ion suppression (+ve mode) on the mass spec. When you add the formic acid, it can sometimes help to counter-act the suppression.

Also, what about adding 0.1% formic acid as a post-column infusion with your current mobile phase? Don't know if it would work for DMHA-induced suppression....... Just shooting out ideas now.

MK12, I just came across your post. I hope my response is not too late...

The peak at 295 is most likely [2DMHA+Cl+H]+, i.e. an adduct of protonated DMHA and DMHA hydrochloride salt. You can easily confirm that by looking at the M+2 isotope satellite of this peak. It should have quite intensive M+2 satellite (ca. 25%) due to the 37Cl contribution. DMHA is used here as an ion-pairing additive in order to increase ATP retention on the column. The use of chloride as counter ion is not appropriate for ms detection, though. Because the HCl salt of DMPA is not very volatile, it give rise to intensive array of clusters with masses [(n DMPA.HCl)+DMPA+H]+, where n = 0, 1, 2...n. You would get similar array in the negative mode [(n DMPA.HCl) + Cl]-. Those intensive signals would kill your sensitivity completely. For this reason people usually use volatile organic salts, such as acetate (most ofter) or formate or trifluoroacetate, i.e. DMHA and acetic acid in your case. Also, if you adjust your pH bellow 7 (i.e. ca. 5) you will increase the ion-pair defectiveness of DMPA, because of better protonation, without significant decrease of the sensitivity in the negative mode. Another disadvantage of the non-volatile buffers, like chlorides in lcms is that they quickly contaminate the instrument's entrance and ion optics.