Acetic acid additive best for negative ESI

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  • Published: Dec 13, 2017
  • Author: Ryan De Vooght-Johnson
  • Channels: HPLC
thumbnail image: Acetic acid additive best for negative ESI

Questions remain on additives for negative ion ESI

LC-MS using negative ion mode ESI is the best detection/identification method for many types of compound. Additives such as ammonium salts, pyridinium salts or organic acids are required in order to give better peak shape in the HPLC and adequate response from the mass spectrometer; however, there is no consensus as to the best ones to use. Traditionally, ammonium acetate or ammonium hydroxide have been used in many laboratories, but other authors have recommended using organic acids, usually acetic acid.

The Concordia University researchers determined the best additive for a range of human lipids, looking at acetic acid, ammonium hydroxide and ammonium acetate. Among the classes of lipid examined were: phosphatidylglycerol (PG), phosphatidylcholine (PC), lysophosphatidylcholine (LPC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (PA), sphingomyelin (SM), lysosphingomyelin (LSM) and ceramide (Cer). Mixtures of lipid standards were examined, along with lipid extracts from human plasma, isolated after protein precipitation.

Acetic acid investigated for negative ion ESI of lipids

Different additives were investigated with a mixture of lipid standards using an Agilent 1200 LC system fitted with a Waters XSelect CSH C18 column. Mass spectrometry was carried out with a Thermo Fisher LTQ Orbitrap Velos instrument, fitted with a heated ESI source (HESI). The mobile phases consisted of 60:40 water:methanol (A) and 90:10 isopropanol:methanol (B). The proportion of B was raised from 20 to 95% in a series of gradients. Three additive systems were examined: 0.02% acetic acid, 10 mM ammonium acetate and a mixture of the two. For six out of nine classes of lipid, PI, LPC, PG, PS, PE and SM, acetic acid gave a much stronger response than ammonium acetate or the mixture of the two additives. For one class of lipids (PA), ammonium acetate was somewhat better than the others, while for another cla

ss (Cer) the mixture of the two additives proved superior. For the final class, LSM, no signals were seen with acetic acid, while the effect of ammonium acetate and the additive mixture was similar. Overall, acetic acid was shown to be superior to ammonium acetate or a mixture of the two for the standards mixture; this trend was also seen when examining lipids from human plasma.

Acetic acid was compared to ammonium hydroxide using an Agilent 1290 Infinity II UHPLC instrument fitted with a Phenomenex Kinetex EVO C18 column. Mass spectrometry employed an Agilent 6550 iFunnel Q-TOF instrument with a Dual Agilent Jet Stream (AJS) ESI source. The mobile phases were as before, but the amount of B was taken from 40 to 95% during the run. The following additive systems were examined: 0.02% acetic acid; 0.014%, 0.02% and 0.05% ammonium hydroxide. Again, acetic acid proved the most effective additive, giving stronger peaks for a standards mixture. Ammonium hydroxide tended to act as an inhibitor, often giving lower intensity peaks the higher its concentration. With the mixture of plasma lipids, more signals were seen with acetic acid compared to ammonium hydroxide.

Further Q-TOF experiments were carried out in which no additives were present in the mobile phase, but they were instead injected directly into the ESI source. In most cases, ammonium hydroxide gave clear inhibition of the MS signals, while acetic acid gave strong signals. Hydroxide has a greater proton affinity than the acetate anion, but these results show this factor is not crucial.

Acetic acid superior to NH4OH and NH4OAc for lipid LC-MS

These results clearly show that acetic acid is a superior additive for negative ion ESI for many lipids. The work could usefully be extended by examining a wider range of additives under the same conditions. In some laboratories, ammonium hydroxide is always used for negative ion ESI because ‘You need a base to remove a proton’, but it is certainly worthwhile to see whether acetic acid gives better performance.

Related Links

Rapid Communications in Mass Spectrometry, 2017, Early View Paper. Monnin et al. Comparison of the influence of AA and AmOH: Improving negative ESI-LC-MS lipidomic analysis of human plasma using acetic acid as a mobile phase additive.

Analytical Chemistry, 2004, 76, 839-847. Wu et al. Favorable effects of weak acids on negative-ion electrospray ionization mass spectrometry.

Wikipedia, Electrospray Ionization

Article by Ryan De Vooght-Johnson

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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