Research Article Received: 22 August 2014 Revised: 12 September 2014 Accepted: 15 September 2014 Published online in Wiley Online Library Rapid Commun. Mass Spectrom. 2014, 28, 2645–2660 (wileyonlinelibrary.com) DOI: 10.1002/rcm.7055 Unexpected peaks in tandem mass spectra due to reaction of product ions with residual water in mass spectrometer collision cells Pedatsur Neta*, Mahnaz Farahani†, Yamil Simón-Manso, Yuxue Liang, Xiaoyu Yang and Stephen E. Stein Biomolecular Measurement Division, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA RATIONALE: Certain product ions in electrospray ionization tandem mass spectrometry are found to react with residual water in the collision cell. This reaction often leads to the formation of ions that cannot be formed directly from the precursor ions, and this complicates the mass spectra and may distort MRM (multiple reaction monitoring) results. METHODS: Various drugs, pesticides, metabolites, and other compounds were dissolved in acetonitrile/water/formic acid and studied by electrospray ionization mass spectrometry to record their MS2 and MSn spectra in several mass spectrometers (QqQ, QTOF, IT, and Orbitrap HCD). Certain product ions were found to react with residual water in collision cells. The reaction was confirmed by MSn studies and the rate of reaction was determined in the IT instrument using zero collision energy and variable activation times. RESULTS: Examples of product ions reacting with water include phenyl and certain substituted phenyl cations, benzoyl-type cations formed from protonated folic acid and similar compounds by loss of the glutamate moiety, product ions formed from protonated cyclic siloxanes by loss of methane, product ions formed from organic phosphates, and certain negative ions. The reactions of product ions with residual water varied greatly in their rate constant and in the extent of reaction (due to isomerization). CONCLUSIONS: Various types of product ions react with residual water in mass spectrometer collision cells. As a result, tandem mass spectra may contain unexplained peaks and MRM results may be distorted by the occurrence of such reactions. These often unavoidable reactions must be taken into account when annotating peaks in tandem mass spectra and when interpreting MRM results. Published in 2014. This article is a U.S. Government work and is in the public domain in the USA. In the course of expanding the NIST library of MS/MS compounds, some of which are drugs, pesticides, or spectra[1] for use in metabolomics, we recorded the spectra metabolites, and some are simple compounds studied to of many precursor ions using electrospray ionization in gain mechanistic insights. conjunction with several types of mass spectrometers. As for Earlier mass spectrometric studies of protonated guanosine the NIST/EPA/NIH EI Library, collected spectra are showed unexpected peaks in the tandem mass spectra and manually evaluated[2] before they are added to the library. revealed that certain product ions react with water or This quality control process often involves comparison of methanol in the collision cell.[3,4] Subsequent studies with spectra from different mass spectrometers and assignment several other protonated molecules also detected addition of – of each peak in the spectrum to a reasonable product ion. water, alcohol, or acetonitrile to certain product ions.[5 8] Peak assignment is further enhanced by the use of Similar water addition reactions were also reported for instruments with ppm-level mass accuracy. In recent product ions from sodiated benzene dicarboxylate salts[9] measurements we noted the presence of unexpected and some sodiated chalcones.[10] Some negatively charged product ions arising from water adduction and undertook product ions were also found to attach a water molecule. a more detailed study of this fragmentation process. For example, collision-induced dissociation of deprotonated Herein we report our findings on several groups of guanine led to formation of certain product ions which then reacted with water[11] and the iodide ion formed from iodobenzoate anion was detected as the water adduct.[12] These findings provide possible explanations for * Correspondence to: P. Neta, Biomolecular Measurement unexpected product ions in tandem mass spectra and suggest Division, National Institute of Standards and Technology, careful considerations in interpretation of multiple reaction Gaithersburg, MA 20899, USA. monitoring (MRM) results. For example, studies of E-mail: [email protected] protonated quinolone drugs raised questions about the † [13] Present address: Office of Generic Drugs, Food and Drugs validity of MRM results which we then explained by 2645 Administration, Silver Spring, MD 20993, USA. finding a reversible water loss and water addition reaction Rapid Commun. Mass Spectrom. 2014, 28, 2645–2660 Published in 2014. This article is a U.S. Government work and is in the public domain in the USA. P. Neta et al. [14] while the loss of CO2 was irreversible. More recently, we by the instrument data system. Typically, m/z values were found that certain protonated aldehydes undergo loss of H2 within 0.2 m/z units of the theoretical m/z values throughout to form ketene cations and that these cations can react with the m/z range of interest. water to produce the protonated carboxylic acids.[15] To examine the influence of type of collisional excitation, Therefore, the MS2 spectrum of the protonated aldehyde MS/MS spectra of the ions were also measured by ion-trap may contain the peak of the corresponding protonated (IT) fragmentation (LTQ, Thermo Fisher Scientific, Waltham, carboxylic acid and its fragmentation products, again raising MA, USA) with 0.35 mTorr helium as collision gas, using concern in interpretation of MRM results. another beam-type collision cell (a quadrupole time-of-flight Water appears to be present in varying concentrations in instrument (model 6530; Agilent Technologies, Santa Clara, the collision cells of most electrospray ionization mass CA, USA) with 0.02 mTorr N2, and using HCD (higher- spectrometers. Its reaction with product ions appeared more energy C-trap dissociation) in an orbital ion trap (OIT) pronounced with increasing collision gas pressure.[15] It is instrument (Orbitrap Elite, Thermo Fisher Scientific, also possible that water is adsorbed and desorbed from metal Waltham, MA, USA) with 0.5 Pa (3.75 mTorr) N2. All gases surfaces in the mass spectrometer. Experiments using D2O used in the mass spectrometer collision cells were of ultrahigh 2 3 4 instead of H2O in the solvent mixture showed that the water purity grade (99.999%). Both MS and authentic MS and MS reacting with product ions does not originate in the solvent spectra were obtained with the LTQ, and this instrument was used in the electrospray.[15] Since water is a common liquid also used to determine the rate and extent of reaction of the chromatography (LC) fluid, its precise origin is not various product ions with water as outlined in the second necessarily clear. In any case, since attachment of water was section of the results. While the QqQ and IT instruments detected in product ions of various compounds, we provide low-resolution spectra, the OIT instrument provides embarked on a search for guidelines to anticipate the HCD spectra with high accuracy m/z values, which help in occurrence of such reactions. We studied folic acid and similar confirming peak assignments. The accuracy of the m/z values compounds, which form aroyl cations and their water is reflected in the annotation of the peaks from the various adducts, other compounds forming aroyl cations, compounds instruments; only the HCD spectra are annotated with four forming aryl cations, compounds forming cations with significant figures. The spectra presented in the figures are positive charge on P, Si, or Sn, and some compounds forming the average of 20 to 100 individual spectra. Noise peaks are negative ions. The rate and extent of reaction of product ions removed if they appear in <20% of the individual spectra. with water are also presented. Further details of quality control procedures are summarized in a recent publication.[16] EXPERIMENTALa RESULTS AND DISCUSSION The compounds were obtained from various sources for inclusion in the NIST MS/MS library. The latest release of this During measurement of MS/MS spectra of many compounds library (2014) includes many of the MS2 and MSn spectra for inclusion in the NIST tandem mass spectral library, and discussed in this paper. The compounds were dissolved in while attempting to annotate all the product ion peaks acetonitrile/water/formic acid (50:50:0.1), and in some cases obtained with high mass accuracy instruments, we came the acetonitrile was replaced with methanol or propanol. across a number of peaks that could be clearly ascribed to For initial studies, electrospray ionization mass spectrometry products of reaction with water. Representative compounds was carried out with a Micromass (Waters Corp., Milford, exhibiting this behavior were studied in more detail for MA, USA) Quattro Micro triple quadrupole instrument inclusion in this paper and spectra of other compounds are (QqQ). First the mass spectra were recorded at different cone included in the MS/MS library. The spectral observations voltages to optimize the abundances of the precursor ions. are discussed below and the rates of reaction with water will Then, the precursor ion at the optimal cone voltage was be discussed in the subsequent section. selected for fragmentation in the collision cell, with 0.21 Pa (1.6 mTorr) argon as collision gas, and the MS/MS spectrum Spectral observations was recorded at 20 different collision voltages. The range of collision voltage spanned from near 0 V up to a value where Folic acid and related compounds 3 no precursor ion remained. Pseudo-MS spectra were Folic acid (1), its dihydro (2) and tetrahydro (3) derivatives, measured by using a high cone voltage to produce the and the drugs methotrexate (5) and raltitrexed (6) (Table 1) fragment ion in the cone region and this ion was then selected have a glutamic acid residue attached to an aroyl group.