Unexpected Peaks in Tandem Mass Spectra Due to Reaction of Product Ions with Residual Water in Mass Spectrometer Collision Cells

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. for MS/MS measurement as above. Spectra were acquired in Tandem mass spectra show that the [M + H]+ ions of these ’ ’ centroid mode, whereby signals within each individual time compounds undergo loss of the glutamic acid residue interval in a given spectrum were centered and integrated (147 Da) to form the product ion [M + H–147]+ (or [M + H– + C5H9NO4] ). Representative spectra obtained with folic and tetrahydrofolic acids are shown in Figs. 1 and 2. These aCertain commercial equipment, instruments, or materials are identiﬁed in this document. Such identiﬁcation does not spectra, as well as the spectra of other compounds listed in – + imply recommendation or endorsement by the National Table 1, show that, in addition to the [M + H 147] product – + Institute of Standards and Technology, nor does it imply that ion, a peak corresponding to [M + H 129] is observed. The

2646 the products identiﬁed are necessarily the best available for abundance of this ion varies with collision energy and is the purpose. dependent on instrument conditions. It is unclear how this

Table 1. Folic acid and related compounds, their precursor ions (p = [M + H]+), and main MS2 peaksa

m/z m/z m/z + – + – + Compound Structure [M + H] [M + H Glu] [M + H Glu + H2O]

1 Folic acid 442 295 313

2 Dihydrofolic 444 297 315 acid

3 Tetrahydrofolic 446 299 317 acid

4 Methopterin 456 309 327

5 Methotrexate 455 308 326

6 Raltitrexed 459 312 330

7 N-(4-Aminobenzoyl)- 267 120 138 L-glutamic acid 2647 (Continues)

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Table 1. (Continued)

m/z m/z m/z + – + – + Compound Structure [M + H] [M + H Glu] [M + H Glu + H2O]

8 Rimonabant 463 363 (M + H-C5H12N2) 381

aThe spectra were recorded with the QqQ, IT, and Orbitrap mass spectrometers. The latter instrument provides high- resolution spectra, which help conﬁrm peak annotation. The m/z values in the table are the nominal values, observed in all the instruments. Column 4 lists the m/z values of the precursor ion [M + H]+, column 5 lists the m/z values of the product ion formed by loss of the glutamate residue (or the C5H10NNH2 residue for entry #8), and the last column lists the m/z values of the product of reaction with water. Peaks observed at higher collision energies are not listed.

Figure 1. Electrospray ionization MS/MS spectra obtained Figure 2. Electrospray ionization MS/MS spectra obtained with protonated folic acid: (a) high-resolution HCD spectrum, with protonated tetrahydrofolic acid: (a) high-resolution collision voltage 8 V; (b) low-resolution IT spectrum, HCD spectrum, collision voltage 13 V; (b) low-resolution IT normalized collision energy 35%. The precursor [M + H]+ ion spectrum, normalized collision energy 35%. The precursor at m/z 442 undergoes complete fragmentation in the ion [M + H]+ ion at m/z 446 undergoes complete fragmentation trap and is not visible in spectrum (b); it is seen in the in the ion trap and is not visible in spectrum (b); it is seen in HCD spectrum (a) and its intensity varies with collision the HCD spectrum (a) and its intensity varies with collision voltage. Loss of a glutamic acid residue from the precursor voltage. Loss of glutamic acid residue from the precursor ion yields the m/z 295 ion, which reacts with H2O to yield ion yields the m/z 299 ion, which reacts with H2O to yield the m/z 313 ion. the m/z 317 ion.

product ion can be formed directly from the precursor ion. ion. The simpler analogous compound 7, N-(4-aminobenzoyl)- Exact mass measurements show that the m/z difference L-glutamic acid, exhibits a similar behavior. These ﬁndings between the two product ions corresponds to the mass suggest that the acylium ion formed by loss of the glutamic

2648 of a water molecule. A mechanism that can account for the acid residue may react with residual water in the collision cell – + – + [M + H 129] ion is the addition of H2O to the [M + H 147] of the mass spectrometer, as observed for the acylium ions

wileyonlinelibrary.com/journal/rcm 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. Reactions of product ions with water produced from protonated 3-formylchromone.[15] We tested (m/z 120) from 2-aminobenzamide (Table 2). In all these cases, this hypothesis by studying simpler acylium ions produced the acylium ions do not react with water but undergo loss of by losses of small molecules such as NH3 or amines. CO and the resulting phenyl cations do react with water. Protonated N-methylphthalimide (15)(m/z 162) undergoes loss of CH2 = NH to form the m/z 133 ion, which is probably Aroyl cations + a benzoyl cation with an ortho-formyl group (OCHC6H4CO ). The MS/MS spectra (Fig. 3) recorded with protonated This product undergoes two successive losses of CO groups benzamide (m/z 122) show loss of NH3 to produce the to form the phenyl cation which then reacts with H2O, a benzoyl cation (m/z 105), but no reaction of this cation with reaction that is detected only in MS3 experiments. H2O was detected. Instead, this ion undergoes loss of CO to Replacing benzene with pyridine introduces an additional produce a phenyl cation (m/z 77), which then reacts with factor in the fragmentation route of [M + H]+, i.e. whether the water (to give the m/z 95 ion). Similar behavior was observed proton remains on the pyridine ring or is removed within the with the benzoyl cation produced from methyl hippurate (10) lost neutral molecule. Experiments with nicotinuric acid (16), and mebendazole (11), the 4-hydroxybenzoyl cation (m/z 121) nifenazone (17), and N,N-diethylisonicotinamide (18) show from nifuroxazide (12), the 4-chlorobenzoyl cation (m/z 139) that the pyridyl ion remaining after several neutral losses can + from moclobemide (13), and the 2-aminobenzoyl cation be observed in three forms: protonated pyridine (C5H5NH , + + m/z 80), protonated pyridyl (C5H4NH or C5H5N , m/z 79), + and the pyridyl cation (C5H4N , m/z 78). Only the latter reacts with water, but it is the least abundant of the three ions. Therefore, the reaction with water has negligible signiﬁcance with pyridine derivatives compared with benzene derivatives.

Comparison of simple aroyl cations with those from folic acid and derivatives If the simple benzoyl and related cations in Table 2 do not react with water, it is puzzling that the MS/MS spectra of the compounds in Table 1 show product ions corresponding to the benzoyl-type cations produced by loss of a glutamic acid residue as well as the products of addition of water to these ions. It may be speculated that the latter product ions are produced not by intermolecular reactions of the benzoyl cations with residual water in the collision cell, but rather by an intramolecular reaction, whereby a carboxyl group of glutamic acid concomitantly inserts its OH group at the site of the amide bond during its dissociation. This pathway is supported by the following ﬁndings. The MS2 spectrum of protonated folic acid (m/z 442) recorded in the IT mass spectrometer shows formation of the [M + H–147]+ (m/z 295) as well as the [M + H–129]+ ion (m/z 313), the latter with abundances of ~8%. The same precursor ion also undergoes loss of water to produce the m/z 424 ion (with low abundance). When the MS3 spectrum of the m/z 424 product ions is recorded with the IT instrument, the major product is the m/z 295 ion, while the m/z 313 ion was barely observable. Moreover, the MS3 spectrum of the m/z 295 ion recorded in the IT instrument shows no observable product ion with m/z 313. Parallel results were obtained with dihydrofolic acid. These ﬁndings suggest that the glutamic acid residue may be responsible for the production of the [M + H–129]+ ion by an intramolecular mechanism, and, when the glutamyl residue Figure 3. Electrospray ionization MS/MS spectra obtained undergoes loss of water, it can no longer participate in such with protonated benzamide: (a) high-resolution HCD a process. This mechanism of formation of the [M + H–129]+ spectrum, collision voltage 21 V; (b) HCD spectrum of the ion via intramolecular reaction with the glutamyl residue m/z 105 ion, collision voltage 15 V; and (c) low-resolution IT + resembles the mechanism of formation of [bn-1 +H2O] ions spectrum, normalized collision energy 35%. The precursor in the fragmentation of protonated peptides.[17,18] On [M + H]+ ion has m/z 122. Ion trap CID (c) shows only loss the other hand, spectra recorded with the QqQ mass of NH3 (m/z 105) and loss of CONH (m/z 79). Higher energy C-trap dissociation (a) shows also the secondary loss of CO spectrometer show somewhat different results. When the from the m/z 105 product ion to form the m/z 77 peak, m/z 295 product ion from folic acid is produced in the ion ’ ’ corresponding to the phenyl cation, and addition of H2Oto source (by increasing cone voltage) and then selected into 3 this product gives the m/z 95 ion. The product ions of m/z 77 the collision cell to obtain a pseudo-MS spectrum, an 2649 and m/z 95 are more intense in the HCD MS3 spectrum (b). abundant product ion at m/z 313 is observed at low collision

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Table 2. Compounds forming aroyl cations, their precursor ions (p = [M + H]+), and their MS2 peaks involving addition of watera

m/z m/z m/z m/z + + + + + Compound Structure [M + H] [ArCO] [Ar] [Ar] [Ar + H2O]

+ 9 Benzamide 122 105 C6H5 77 95

+ 10 Methyl hippurate 194 105 C6H5 77 95

+ 11 Mebendazole 296 105 C6H5 77 95

+ 12 Nifuroxazide 276 121 HOC6H4 93 111

+ 13 Moclobemide 269 139 ClC6H4 111 129

+ 14 2-Aminobenzamide 137 120 H2NC6H4 92 110

> + 15 N-Methyl phthalimide 162 133 - 105 C6H5 77 95

+ 106 C5H4N 78 96 + 16 Nicotinuric acid 181 107 C5H5N 79 - + 108 C5H5NH 80 -

+ 17 Nifenazone 309 106 C5H4N 78 96 + 107 C5H5N 79 - 2650 (Continues)

Table 2. (Continued)

m/z m/z m/z m/z + + + + + Compound Structure [M + H] [ArCO] [Ar] [Ar] [Ar + H2O]

+ 106 C5H4N 78 96 + 18 N,N-Diethyl isonicotinamide 179 107 C5H5N 79 - + 108 C5H5NH 80 -

aSee footnote in Table 1. Column 4 lists the m/z values of the precursor ion [M + H]+, column 5 lists the m/z values of the aroyl cations produced by loss of various residues, column 6 gives the structures of the product ions formed after loss of CO from the aroyl cations, column 7 lists their m/z values, and the last column lists the m/z values of the product of reaction with water. Other peaks observed at higher collision energies are not listed.

energies, which is clearly due to reaction of the aroyl cation [M + H–147]+ with water in the collision cell. The same results were obtained with dihydrofolic acid. These findings indicate that the intramolecular mechanism is not the only pathway for producing the [M + H–129]+ ions from these compounds and that different mechanisms may be operative under different conditions. In the IT instrument the precursor or product ions are collected in the ion trap and then allowed to undergo CID under 0.35 mTorr of He. In the QqQ instrument the ions are produced in the ion source and immediately selected into the collision cell under 1.6 mTorr of Ar. Thus, in the latter case the ions have more immediate access (microseconds vs milliseconds) to higher concentrations of water (due to higher pressure in the cell) and can attach a water molecule quickly. In the IT, however, the ions may rearrange to a non-reactive form before they encounter water molecules. Another factor affecting the probability of reaction of the acylium ions with water is the stability of this ion towards rearrangement or fragmentation. While the simple benzoyl cation (Table 2) undergoes loss of CO in preference to attaching a water molecule, the more complex acylium ions from tetrahydrofolic acid (Fig. 2) or rimonabant (Fig. 4) (Table 1) are more stable and can attach a water molecule with high fi Figure 4. Electrospray ionization MS/MS spectra obtained ef ciency. Thus, the reaction of product ions with water takes with protonated rimonabant: (a) high-resolution HCD place in competition with other reactions of these ions and, spectrum, collision voltage 32 V and (b) low-resolution IT 2 therefore, observation of the water addition products in MS spectrum, normalized collision energy 35%. The precursor spectra depends on the balance among all the reactions [M + H]+ ion at m/z 463 undergoes complete fragmentation involved. In MS3 spectra, addition of water may be observed in the ion trap and is not visible in spectrum (b); it is seen in at low collision energies whereas at higher energies the ions the HCD spectrum (a) and its intensity varies with collision undergo fragmentation. The IT instrument (LTQ) allows us to voltage. Loss of C5H12N2 from the precursor ion yields the trap product ions at zero collision energy and at extended m/z 363 ion, which reacts with H2O to give the m/z 381 ion. activation times so that we can monitor their reaction with water in the absence of CID. The relative rates for the water significant abundance in the MS3 spectra. To extend these addition reactions will be discussed in section 2 of the results. studies on aryl ions we examined other compounds whose [M + H]+ ions may fragment by various pathways to form aryl cations (not via decarbonylation of aroyl cations). Aryl cations Protonated dapsone (19), sulfabenzamide (20), and The results in Table 2 suggest that simple benzoyl-type ions sulfamethoxazole (21) (Table 3) produce the 4-aminophenyl do not react with water to any appreciable extent that can cation (m/z 92) which reacts with water to yield the m/z 110 be observed in the tandem mass spectra but rather undergo ion. Similarly, protonated p-toluenesulfonic acid (22) loses

rapid loss of CO to form the corresponding aryl ions. These H2O and SO2 to form the 4-methylphenyl cation (m/z 91) 2651 latter ions do react with water to form product ions with which then yields the m/z 109 ion by reaction with water.

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Table 3. Compounds forming aryl cations, their precursor ions (p = [M + H]+), and their MS2 peaks involving addition of watera

m/z m/z m/z + + + Compound Structure [M + H] Product ion [Ar] [Ar + H2O]

19 Dapsone 249 p-H2NC6H4SO2H92110

20 Sulfabenzamide 277 p-C6H5CONHSO2H92110

21 Sulfamethoxazole 254 p-C4H6N2O3S92110

22 p-Toluenesulfonic acid 173 p-H2SO3 91 109

- p-NH3 105 23 2,6-Xylidine 122 p-NH3-C2H4 77 95

24 2-Aminopyridine 95 p-NH3 78 96

25 2-Aminopyrimidine 96 p-NH3 79 97

26 2-Chloroquinoline 164 p-HCl 128 146

27 Crimidine 172 p-HCl 136 154

28 6-Mercaptopurine 153 p-H2S 119 137

29 Trigonelline 138 p-HCO2H92110 2652 (Continues)

Table 3. (Continued)

m/z m/z m/z + + + Compound Structure [M + H] Product ion [Ar] [Ar + H2O]

30 Flumequine 262 p-H2O-C3H6 202 220,238

31 Meclizine 391 p-C12H18N2-HCl 165 183

32 Azamethiphos 325 p-C4H8NO5PS 112 130

aSee footnote in Table 1. Column 4 lists the m/z values of the precursor ion (p = [M + H]+), column 5 shows the overall neutral loss to produce the aryl (or heteroaryl) cation from p, column 6 lists the m/z values of the aryl cations, and the last column lists the m/z values of the product of reaction with water. Other peaks observed at various collision energies are not listed.

fl Protonated 2,6-xylidine (23)(m/z 122) loses NH3 and the Protonated umequine (30) undergoes loss of H2O and product ion (m/z 105) barely reacts with water. However, then loss of C3H6 to produce the m/z 202 ion, which reacts this product ion loses C2H4 to form a phenyl cation (m/z 77), with two water molecules in succession to form the m/z 220 which reacts with water. Although most phenyl cations ion and then the m/z 238 ion. Protonated meclizine (31) bearing various substituents react with water, there is a undergoes loss of C12H18N2 (the 3-methylbenzylpyrazine significant effect of the substituent on the rate and extent of moiety) and then loss of HCl to form the m/z 165 product reaction, which will be discussed in section 2. ion. This ion can react with water to produce the m/z 183 Protonated 2-aminopyridine and 2-aminopyrimidine (Table 3) ion. The reaction occurs with high abundance in MS4 2 undergo loss of NH3 and the product ions react with water. experiments, although MS spectra show only low Protonated 2-chloroquinoline and crimidine (27)undergoloss abundance of the m/z 183 ion. Protonated azamethiphos (32) of HCl and the product ions react with water. Protonated undergoes loss of C4H8NO5PS to form a chloropyridyl cation, 6-mercaptopurine undergoes loss of H2Stoformthem/z 119 m/z 112, which reacts with water to form the m/z 130 ion. ion, which reacts with H2Otoproducethem/z 137 ion. A parallel reaction is the loss of HCN from the ring, but this is Phosphorus-centered ions not followed by reaction with water. These heterocyclic product ions, which react with water, are similar to the phenyl cation in In Table 4 we summarize results for other types of product that one of the ring carbons is trivalent (after losing its bonding ions that are found to react with water in the mass to the N, Cl, or S atom), and this is the driving force for their spectrometer. The first group involves product ions with a reaction with water. positively charged phosphorus center. For example, Protonated trigonelline (29) undergoes loss of CO2 to protonated methylphosphonic acid (m/z 97) undergoes loss form the m/z 94 ion and parallel loss of HCO2Htoproduce of water to produce the m/z 79 ion with a positively charged the m/z 92 ion; only the latter ion reacts with water to form phosphorus center. This product ion regains a water molecule the m/z 110 ion rapidly and completely. The m/z 94 ion and reverts to the precursor ion, a reaction that is not 2 3 produced by loss of CO2 is essentially the N-methylpyridinium apparent in MS spectra but is demonstrated in MS ion, which is not expected to attach a water molecule. To form experiments. Protonated diethyl methylphosphonate (34) the m/z 92 ion, two hydrogens have to be removed along with (m/z 153) produces the same m/z 79 ion by loss of C2H5OH (or in addition to) the CO2. Although protonated aliphatic and C2H4. The reaction of the m/z 79 ion with water to carboxylic acids are known to undergo loss of HCO2H, it is produce the m/z 97 ion is clearly observable in this case, but not clear how the m/z 92 ion is formed from protonated the results also show that some of the m/z 97 ions are trigonelline. Nevertheless, its reaction with water is efficient produced directly from the m/z 153 precursor by loss of two

and the m/z 110 ion produced by this reaction is highly C2H4 molecules. Protonated hexamethylphosphoramide (35) 2653 2 abundant in the MS spectrum. (m/z 180) undergoes loss of (CH3)2NH to form the m/z 135

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Table 4. Various compounds, their precursor ions (p), and their MS2 peaks involving addition of watera

Product Precursor Product ion, ion + H2O, Compound Structure ion, m/z Product ion m/z m/z

A P-centered p = [M + H]+

33 Methylphosphonic acid 97 p-H2O7997

34 Diethyl methylphosphonate 153 p-C4H10O7997

p-C2H7N 135 153 35 Hexamethyl-phosphoramide 180 p-C4H12N2 92 110

p-C3H8S 247 265 36 Sulprophos 323 p-C5H12S 219 237,255

37 Fenamifos sulfoxide 320 p-C5H13N 233 251

B Si-centered p = [M + H]+

p-CH4 281 299 38 Octamethyl-cyclotetrasiloxane 297 p-2CH4 265 283 p-3CH4 249 267

39 Decamethyl-cyclopentasiloxane 371 p-CH4 355 373 p-C5H16Si 267 285

À + C Sn-centered p = [M + H H2O]

40 Cyhexatin 369 p-C6H10 287 305 p-C12H20 205 223

2654 ((Continues)Continues)

Table 4. (Continued)

Product Precursor Product ion, ion + H2O, Compound Structure ion, m/z Product ion m/z m/z À D Negative ions p = [MÀH]

41 Rhein 283 p-CO2 239 257

ﬂ 42 8-Carboxy-3-methyl avone 279 p-CO2 235 253

aSee footnote in Table 1. Column 4 lists the m/z values of the precursor ion, which is deﬁned in the subheadings in rows A, B, C and D, column 5 shows the overall neutral losses to form the product ions from p, column 6 lists the m/z values of those product ions, and the last column lists the m/z values of the product of reaction with water. Other peaks observed at various collision energies are not listed.

ion and then loss of CH3N=CH2 to form the m/z 92 ion. These are required to understand which structures of negative ions product ions both react with water to produce the m/z 153 formed by loss of CO2 from the carboxylate ion are capable and m/z 110 ions, respectively, but the latter appears with a of reacting with H2O. It appears that delocalization of the higher abundance. Protonated sulprophos (36)(m/z 323) negative charge to other sites within the molecule is necessary undergoes loss of C3H7SH to form the m/z 247 ion and then to promote the reaction with water. In the case of positively loss of C2H4 to form the m/z 219 ion, both of which react with charged product ions, however, it appears that localization water. Protonated fenamifos sulfoxide (37) undergoes parallel of the charge on C, P, Si, or Sn is necessary to drive the losses of C2H4 and C3H7NH2 to produce the m/z 233 ion with reaction with water, but if the charge is transferred to a positively charged phosphorus, which then reacts with water. different site (along with transfer of protons) the reaction with These are examples of compounds whose protonated ions water may not take place. undergo losses of various molecules to yield product ions with a positively charged P center, which then react with water. Rate of reaction with water The LTQ ion-trap mass spectrometer permits us to obtain Si- and Sn-centered ions multistage MS3 and MS4 spectra, which confirm observations The second group in Table 4 includes protonated cyclic with the QqQ instrument, where the reaction with water was 3 siloxanes (38, 39). These ions lose a CH4 molecule and then observed in pseudo-MS spectra of fragment ions produced additional CH4 or larger fragments. The product ions of these in the ion source. The LTQ also permits us to follow the fragmentations have a positively charged Si which reacts reaction with water by isolating the fragment ion in the with water. The next example in Table 4 is cyhexatin (40). absence of collision energy and varying the activation time The [M + H]+ precursor ion was not observed in this case between 0.1 ms and 10 s. Such measurements show, for + because it underwent rapid loss of H2O to form (C6H11)3Sn example, the gradual and complete conversion of the phenyl (m/z 369). This ion loses two cyclohexene molecules in cation (m/z 77) into protonated phenol (m/z 95) in a time- + fi fi succession to form (C6H11)2SnH (m/z 287) and then dependent manner. The data points in Fig. 5(a) t rst-order + À1 C6H11SnH2 (m/z 205). These product ions both react with kinetics and give a rate constant of 1.2 s for the reaction + → + water to form the m/z 305 and m/z 223 ions, respectively. with H2O. Although the reaction C6H5 +H2O C6H5OH2 is second order, the kinetic plot shows first-order behavior because the concentration of water is in large excess over Negative ions the concentration of the ions, and, when the concentration While all the ions discussed above are positively charged, the of water is increased, the observed rate constant increases last two entries in Table 4 are the negative ions of rhein (41) accordingly. fl and 8-carboxy-3-methyl avone (42), which lose CO2 and then While the phenyl cation is converted completely into attach a water molecule. This type of reaction, however, is not protonated phenol, the reaction of other ions with water

observed with simple aromatic carboxylic acids, nor with may not proceed to completion. For example, the 2655 chromone- and coumarin-carboxylic acids. Further studies 4-aminophenyl cation (m/z 92), produced from 19, 20, and

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a small extent of reaction of a product ion with water but may not be able to determine whether the rate of reaction is slow or the reaction occurs only to a small extent (speciﬁc examples will be discussed). The phenyl cation (m/z 77) reacts with water rapidly and is converted completely into protonated phenol (m/z 95) (Fig. 5(a)), but other fragment ions undergo only partial reaction with water (Figs. 5(b) and 5(c)). Partial reaction with water may be due to an equilibrium state involving the reverse reaction in which the water adduct loses that water molecule. Such an equilibrium is not likely in the present LTQ experiments since the water adducts are found to be stable in the ion trap and will lose a water molecule only when the collision energy is raised. It is likely that partial reaction indicates that the ion exists in two forms, distinguished by shifts of protons, and only one of these forms reacts with water. For example, the 4-aminophenyl + cation (m/z 92) may exist 25% in this form (H2NC6H4) which reacts rapidly with water, while 75% are converted into a form that does not react with water, such as phenylaminium + ( HNC6H5). The rearrangement may occur rapidly to create an equilibrium mixture of products or it may occur during, and in competition with, the reaction with water. In certain cases, the MS2 spectra obtained in collision cell instruments exhibit substantial reaction of a product ion with water but, when this product ion is collected in the ion trap, in an attempt to follow its reaction with water as a function of activation time, very little reaction is observed. The apparent discrepancy between such results may be due to the different concentration of water in the different instruments and to the timing of the observations. In the collision cell, the product ions are formed and can directly react with the residual water in the cell (within microseconds). In the ion trap, however, those same product ions are ﬁrst trapped (for milliseconds) and then are examined for their reaction with water; as a result, the ions may isomerize into a more stable unreactive form. This situation is pronounced with some folic acid derivatives (Table 1). The product ion formed from tetrahydrofolic acid (3), by loss of the glutamate residue, reacts with water almost completely, whereas the product ions formed by similar fragmentations from folic acid (1) and some other compounds Figure 5. Reaction of product ions (solid circles) with water (2, 4, 5, 6) in Table 1 react with water only to a very small to form the adduct ions (open circles) in the ion trap of the extent within the observable time range. This raises the LTQ mass spectrometer as a function of activation time with question whether the reaction with water in the latter cases relative collision energy set at zero: (a) phenyl cation from is very slow or the extent of reaction is low because of benzamide (Tables 2 and 5); (b) 4-aminophenyl cation from isomerization of the ion (by proton transfer). When we dapsone (Tables 3 and 5), partial reaction because some of the ions are changed into an unreactive phenylaminium; examine the results for folic acid (1) and methopterin (4)in and (c) the product ion from sulprophos (Tables 4 and 5) more detail, we realize that the reaction may be fast but only where the structure with a positive charge on P reacts with a small fraction of the product ions undergo this reaction. water but another structure, probably with the charge on S, The two types of behavior of the compounds in Table 1 are does not react. probably related to the extent of conjugation between the pteridine heterocycle and the p-aminobenzoyl group. When the pteridine is fully aromatic, the positive charge initially 21 (Table 3), reacts with water with the same rate constant, located on the carbonyl group of the p-aminobenzoyl moiety À k =1.2 s 1, but the extent of reaction is only 25% (Fig. 5(b)). can transfer to the pteridine so that only a small fraction Another example (Fig. 5(c)) is the reaction with water of the of the ions retain the charge on the carbonyl and react m/z 247 fragment ion produced from protonated sulprophos with water. On the other hand, when the conjugation (36) (Table 4), which proceeds to 30% conversion with a rate between the pteridine and p-aminobenzoyl is disrupted by À constant k = 2.6 s 1. Since the LTQ activation time is limited saturation of the double bonds (as in tetrahydrofolic acid)

2656 to 10 s, one cannot effectively follow reactions with rate most of the positive charge remains near the carbonyl group À constants <0.1 s 1. Therefore, in some cases we may observe where the reaction with water can take place. In the case of

Table 5. Rate and extent of reaction of product ions with water

À1 Compound Cation m/z k(+H2O), s % conversion

9 Benzamide 10 Methyl hippurate 77 1.2 >90

12 Nifuroxazide 93 2.9 >90

19 Dapsone 20 Sulfabenzamide 92 1.2 25 21 Sulfamethoxazole

26 2-Chloroquinoline 128 2.5 >90

27 Crimidine 136 2 80

28 6-Mercaptopurine 119 0.9 30

29 Trigonelline 92 0.7 >90

30 Flumequine 202 0.7 >90

32 Azamethiphos 112 2.7 >90

((Continues)Continues) 2657

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Table 5. (Continued)

À1 Compound Cation m/z k(+H2O), s % conversion

33 Methylphosphonic acid 79 1.9 >90 34 Diethyl methylphosphonate

36 Sulprophos 247 2.6 30

37 Fenamifos sulfoxide 233 1.6 >90

38 Octamethylcyclotetrasiloxane 281 2.7 >90

1 Folic acid 295 ~0.6 ~3

4 Methopterin 309 ~0.6 ~3

3 Tetrahydrofolic acid 299 0.17 >90

8 Rimonabant 363 1.9 >90

rimonabant (8), the aroyl cation reacts with water rapidly The reaction of product ions with water depends on the and completely, indicating that the charge remains mainly concentration of water in the mass spectrometer. We have

2658 at the carbonyl group. The rate constants are summarized shown previously that the abundance of the water addition in Table 5. products is dependent on the pressure of the collision gas

(since higher pressure permits higher water content).[15] water depends on their detailed structure (compare Tables 1 However, the concentration of water may vary over time and 2). The simple benzoyl cation undergoes loss of CO and may change when the mass spectrometer undergoes rather than reacts with water; only more stabilized aroyl preventive maintenance service. Therefore, the rates of cations, which do not undergo decarbonylation, react with reaction with water may vary and the values given in Table 5 water. However, these complex aroyl cations may rearrange may not be reproducible in different instruments or at different into unreactive forms, and it is difficult to predict which times. For example, the reaction of the phenyl cation (m/z 77) structures will exhibit reaction with water. In the case of produced from benzamide to give the m/z 95 ion was found to product ions with a positive charge on P, Si, or Sn, it appears proceed to near completion but the rate constant was that if the charge is localized on these atoms the reaction with À À k =1.2s 1 in one measurement and k =1.5s 1 when measured water takes place. On the other hand, if a positive charge is several weeks later. In another example, the p-aminophenyl localized on N or S atoms the ions do not react with water. À cation (m/z 92) produced from dapsone (19)showedk =1.1s 1 Therefore, it is advisable that any peak in the MS/MS À and several weeks later k =1.8s 1; the extent of reaction was spectrum, which is not assignable to a simple fragmentation 25% in the first case and 29% in the later measurement. product, be considered for the possibility of being a product Because of these variations, the rate constants summarized of reaction of another ion with water. Mass accuracy in the in Table 5 should be assumed to have a wide margin of ppm range is critical for confirming such an assignment. uncertainty, probably around 50% when compared in the The abundance of the ions resulting from reaction with same mass spectrometer under similar conditions, but higher water in the MS/MS spectra depends on the experimental than that when compared in different instruments. Therefore, conditions, mostly as these conditions relate to the Table 5 is given only as a general guideline and it presents one concentration of water in the mass spectrometer, and also set of results which should be internally consistent to permit vary with collision energy. The abundance may be as low as us to compare the behavior of different ions in their reaction several percent of the base peak (as in Fig. 1(a)), sometimes with water. It is clear from Table 5 that the phenyl cation barely observable, or as high as becoming the most intense reacts with water rapidly and completely, presumably peak in the MS2 spectrum (as in Fig. 4(a)). Small peaks in forming protonated phenol. Other cations with similar the MS2 spectra, which are due to reaction with water, may behavior are those in which an aromatic or heterocyclic ring become more abundant in MS3 spectra and may help confirm is missing one of the ring hydrogens and include the occurrence of the reaction with water. Formation of the 4-hydroxyphenyl, 2-quinolinyl, and chloropyridyl. However, water addition product ions, and their subsequent when the phenyl cation bears an amino group the rate of dissociation in the collision cell, may lead to observation of reaction is similar but the extent of reaction is diminished MS/MS spectra with unexpected product ion peaks and by a factor of four, probably due to partial rearrangement of may distort MRM results. We have outlined several types of the ion into an unreactive form, as discussed above. Product product ions that can react with water and we expect that ions with the positive charge on P or Si atoms also can react additional types will be detected. Therefore, the possibility with water rapidly and completely. Comparison of the results of reaction of product ions with water should be taken into obtained with sulprophos (36) and femamifos sulfoxide (37) account when interpreting tandem mass spectra and should suggests that the ion from 37 has a positive charge localized be carefully considered when using MRM measurements. on P (and undergoes complete reaction with water) but the The reaction with water can change the relative abundances positive charge in the product ion derived from 36 is only of product ions and can lead to formation of product ions partly localized on P and mostly localized on S, thus which cannot be formed directly from the original undergoing only partial reaction with water. And, finally, compound. MRM measurements are more reliable if all the we note the difference between, on one hand, folic acid and peaks in the MS/MS spectrum of the precursor ion can be methopterin, where the pteridine ring is fully aromatic and ascribed to CID products or if an isotopically labeled internal the reaction of the [M + H–Glu]+ ions occurs to a very small standard is used. extent, and, on the other hand, tetrahydrofolic acid, where partial saturation of that ring leads to complete reaction of – + the [M + H Glu] ions with water. REFERENCES

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