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Controlling Na and K Adducts in LC-MS

Adduct ions are prevalent in LC-MS analyses and can come from any number of sources.  An adduct ion is any ion formed by adduction of an ionic species to a molecule, and can be present under all modes of ionization (e.g. ESI, APCI etc.) and under different ionization polarities (positive or negative ionization).

The commonly observed protonated molecule, [M+H]+ is technically an adduct ion.  However, adduct ions which originate from alkali metals, solvents, or other metal species can cause problems when identifying the molecular ion and interpreting mass spectra (Figure 1, Tables 1 and 2).

Adduct ions in APCI must be volatile which means ammonium, chloride, and water adducts can occur but metal adduct ions cannot.

Figure 1: Negative ESI spectrum of α-naphthoic acid.
 
Observed Explanation Mass
[M-H]- Deprotonation M-1
[M-H-nH2O]- Deprotonation and loss of H2O M-1-(nx18)
[M+Cl]- Ion attachment M+35 (37)
[M-2H+Na]- M + Na adduct M+21
[M-H-CO2]- Carbon dioxide loss M+45

Table 1: Typical adduct ions encountered in ESI negative ion mode.

 
Observed Explanation Mass
[M+H]+ Protonation M+1
[M+NH4]+ Mainly when using CH3NH4 M+18
[M+H+nH2O]+ Water cluster M+1+(nx18)
[M+H+H2O]+ M + H2O adduct M+19
[M+Na]+ M + sodium adduct M+23
[M+K]+ M + potassium adduct M+39
[2M+H]+ Analyte dimerization (2xM)+1
[M+H+CH3CN]+ In presence of CH3CN M+42
[M+H+CH3CN+nH2O]+ Water-acetonitrile cluster M+42+(nx18)

Table 2: Typical adduct ions encountered in ESI positive ion mode.

The sources of adduct ions are prolific, but an understanding of these sources can help to eliminate or reduce the occurrence of adduct ions.  Sodium and potassium adducts are some of the most common LC-MS adducts produced.  Sodium adducts will appear 22 m/z units above the protonated molecule and potassium adducts will be 38 m/z units above (Figure 2). 

 

Figure 2: Mass spectrum illustrating sodium and potassium adduct ions to an analyte with a molecular mass of 376.

 

The most common source of these species is from glass due to the salts used in the manufacturing process.  If excess adduct formation is seen due to the use of laboratory glassware consider switching to MS certified glass or high quality plasticware.  However, the use of plastic comes with its own problems due to plasticizer contamination (e.g. phthalates). 

The use of high quality phthalate free plastic can reduce this type of contamination, and as the m/z values will be at fixed values they may be easier to discount during spectral interpretation - unlike adduct ions which will have varying m/z values based on the analyte’s mass.  Biological samples can exhibit pronounced levels of alkali metal adduction as there is a high endogenous concentration of various salts, with others being added during sample preparation. 

Sample clean up processes, such as solid phase extraction, are effective in remove matrix compounds from biological samples, which will have the added benefit of reducing possible ion suppression.  Mobile phase design is important for all LC-MS applications, in particular the use of volatile components to avoid contamination of the ion source.  Avoiding sodium- and potassium-based pH and ion-pair reagents will help to reduce the presence of alkali metal adducts. Handling samples and labware without wearing gloves can also transfer enough sodium to produce significant adduct ions.        

In some cases, the protonated molecule may be completely absent with only metal adduct species present.  There are two strategies which can be used to deal with alkali metal adducts.

  1. Lower the pH - this is the preferred method for dealing with unwanted alkali metal adducts.  An organic acid, such as formic acid, is added to provide an excess of protons relative to metal ions which drives all or a major portion of the ion formation to the protonated molecule [M+H]+.  Additionally, lowering the mobile phase pH should improve ionization efficiency providing better limits of detection for the protonated molecule.

  2. Add potassium or sodium acetate to the mobile phase - this approach can be used if lowering the pH does not eliminate the majority of the metal adduct species.   A large excess of metal ion produces nearly exclusive formation of the metal adduct ion species.  The reagents employed must be volatile so as to avoid precipitation of involatile salts in the interface which can cause blockages and contamination.

For more information on dealing with metal adducts and contamination in LC-MS applications watch the webcast Troubleshooting LC-MS »

 

Useful link Mass Spectrometry Adduct Calculator »

 

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Dr. Dawn Watson
 

This article was written by Dr. Dawn Watson.

Dawn received her PhD in synthetic inorganic chemistry from the University of Strathclyde, Glasgow. The focus of her PhD thesis was the synthesis and application of soft scorpionate ligands. As well as synthetic skills, this work relied on the use of a wide variety of analytical techniques, such as, NMR, mass spectrometry (MS), Raman spectroscopy, infrared spectroscopy (IR), UV-visible spectroscopy, electrochemistry, and thermogravimetric analysis.

Following her PhD she spent two years as a postdoctoral research fellow at Princeton University studying the reaction kinetics of small molecule oxidation by catalysts based on Cytochrome P450. In order to monitor these reactions stopped-flow kinetics, NMR, HPLC, GC-MS, and LC-MS techniques were utilized.

Prior to joining the Crawford Scientific and CHROMacademy technical team she worked for Gilson providing sales and support for the entire product range including, HPLC (both analytical and preparative), solid phase extraction, automated liquid handling, mass spec, pipettes, and laboratory consumables.

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