Selective Gas-Phase Mass Tagging via Ion/Molecule Reactions Combined with Single Analyzer Neutral Loss Scans to Probe Pharmaceutical Mixtures

Abstract

We have demonstrated the use of a simple single ion trap mass spectrometer to identify classes of compounds as well as individual components in complex mixtures. First, a neutral reagent was used to mass tag oxygen-containing analytes using a gas-phase ion/molecule reaction. Then, a neutral loss scan was used to indicate the carboxylic acids. The lack of unit mass selectivity in the neutral loss scan required subsequent product ion scans to confirm the presence and identity of the individual carboxylic acids. The neutral loss scan technique reduced the number of data-dependent MS/MS scans required to confirm identification of signals as protonated carboxylic acids. The method was demonstrated on neat mixtures of standard carboxylic acids as well as on solutions of relevant pharmaceutical tablets and may be generalizable to other ion/molecule reactions.

Introduction

Ion/molecule reactions see wide use in mass spectrometry. Ambient reactions of protonated water clusters (which are themselves produced by ion/molecule reactions) are used for ionization of nebulized neutrals in atmospheric pressure chemical ionization (APCI) [1, 2]; plasma-based ionization sources can be used to probe surfaces for analytes of interest using ion/molecule reactions [3,4,5,6,7,8]. Within the vacuum system, ion/molecule reactions can be used to ionize neutrals via chemical ionization [9].

Another set of ion/molecule reactions includes selective reaction of neutral reagents with analyte ions [10,11,12]. Often these reactions are performed in a quadrupole ion trap or in a Fourier transform ion cyclotron resonance cell. Among the many capabilities of ion trap mass spectrometers in conjunction with ion/molecule reactions are differentiation of sugar-phosphate isomers [13], “counting” of functional groups in oxygen-containing molecules [14,15,16,17,18], differentiation of isomeric glucuronides [19], determination of enantiomeric excess of pharmaceutical compounds [20], deconvolution of mass spectra of polymers [21], discrimination of stereoisomers [22], ion activation (e.g., ozone-induced dissociation) [23], protein conformation determination (H/D exchange) [24], and identification of various molecular functionalities including primary N-oxides [25], carboxylic acids [16], sulfones [26], phosphates and phenoxides [27], phenols [28], and sulfoxides [29]. In this work, we focus on the last capability: determining the molecular functionality of an ion through chemoselective ion/molecule reactions. The usual procedure is that the reaction is followed by product ion scan tandem mass spectrometry (MS/MS) using collision-induced dissociation (CID) to characterize those ions possessing the functional group of interest.

In this paper, we demonstrate an augmented workflow in which data-independent neutral loss scans are utilized after reaction between mixtures and a selected neutral reagent in order to “sort” through the complexity of the resulting mass spectrum after reaction, thereby simplifying the subsequent analysis by data-dependent MS/MS. That is, data-dependent MS/MS is conducted only on the ions detected in the neutral loss scan. The reagent—trimethylborate (TMB)—is known to react selectively with oxygen-containing compounds [15, 17]. It is used here to give [carboxylic acid + TMB−MeOH + H]+ adducts which all give a fixed neutral loss (90 or 118 Da) upon CID; other functionalities (e.g., aldehydes, alcohols, etc.) do not, which is why TMB was chosen for this study. After the ion/molecule reaction, a neutral loss scan in the ion trap then detects only those ions which dissociate into an ion and a neutral with the specified mass, removing much of the chemical noise from the mass spectra and simplifying the subsequent analysis by data-dependent MS/MS. This procedure may be particularly useful for small and resource-constrained mass spectrometers (e.g., portable quadrupole ion traps [30,31,32]) which often have limited sample quantity or limited instrument mass and power and therefore benefit from more efficient workflows.

Experimental

Chemicals

All chemical standards as well as the neutral reagent trimethylborate were purchased from Sigma-Aldrich (St. Louis, MO). Oxygen-containing standards were diluted to a concentration of 500 μM in 50:50 methanol/water with 0.1% formic acid added to improved ionization efficiency. HPLC-grade methanol was purchased from Fisher Scientific (Hampton, NH). Purified water was obtained from a Direct-Q 3 UV water purification system from EMD Millipore (Billerica, MA). Meijer brand naproxen sodium tablets and ibuprofen tablets were purchased from Meijer in West Lafayette, IN.

Ionization

Analytes were ionized (protonated) in the positive ion mode using a commercial Ion Max atmospheric pressure chemical ionization source from Thermo Fisher (Waltham, MA). Solutions were flowed into the source at a rate of 10 μL/min. The source parameters were set as follows: vaporizer temperature, 300 °C; sheath gas flow rate, 25 (arbitrary units); auxiliary and sweep gas flow rates, 5 (arbitrary units); discharge current, 5 μA; capillary temperature, 300 °C; capillary voltage, 10 V; tube lens voltage, 60 V.

Instrumentation

Ion/molecule reactions and mass spectrometry experiments were conducted on a Thermo LTQ linear ion trap mass spectrometer (San Jose, CA, USA) equipped with an external reagent mixing manifold heated to 100 °C. The reagent was introduced into the manifold by using a syringe pump at a flow rate of 5 μL/h and diluted with helium at a flow rate of 500 mL/min. The mixture was directed into a variable leak valve that allowed part of the mixture into the ion trap at a flow rate of 2 mL/min, and the excess was directed to an exhaust line.

The LTQ’s rf frequency was tuned to 1.166 MHz. After ion injection and cooling, the ions were allowed to react with the neutral reagent TMB for ~ 100 ms. Subsequently, the rf voltage was raised to a desired level and fixed during the neutral loss scan. The LTQ has been modified to perform orthogonal double-resonance excitation [33] (i.e., dipolar ac signals can be coupled onto both the x and y rods simultaneously for ion excitation), allowing the ion trap to conduct neutral loss scans as described previously [34]. Neutral loss scans were performed at constant rf voltage by applying three inverse Mathieu q scans (nonlinear ac frequency sweeps with a linear relationship between m/z and time) simultaneously to the x and y rods of the ion trap. The frequency sweeps, which were calculated in Matlab as described in detail previously [35], were triggered by the LTQ’s built-in trigger functionality (in the ‘Diagnostics’ menu) and were generated by two synced Keysight 33612A arbitrary waveform generators (Chicago, IL, USA) with 64 megasample memory upgrades. As described in ref. [34], a first frequency sweep was applied to the y rods to mass-selectively fragment the precursor ions in the trap while a second frequency sweep—with a particular trigger delay with respect to the first sweep—applied in the x dimension (where there are two detectors) ejected product ions whose m/z values satisfied a selected neutral loss relationship with the precursor ions. Because m/z is proportional to time, if an inverse Mathieu q scan is used (see ref. [35]), the trigger delay between the excitation sweep and the ejection sweep is directly proportional to the neutral loss mass. A third “artifact rejection” frequency sweep was also applied to the y rods and had a trigger delay approximately half of the trigger delay between the two previously described waveforms. This waveform is used to neutralize on the y rod precursor ions that survive activation. Without this waveform, surviving precursor ions would also be detected as undesired artifacts by the product ion ejection frequency sweep. Excitation frequency sweeps had an amplitude of 150 mVpp while product ion ejection and artifact rejection frequency sweeps had an amplitude of 500 mVpp. The frequency sweeps started at Mathieu q = 0.908 and ended at q = 0.15 600 ms later. Because unconventional frequency sweeping methodology was utilized in this study, mass spectra were calibrated externally via a linear fit of m/z vs. time. For an example, calibration for Fig. 2c, see Fig. S1 in the supplemental information.

One important note is that ion intensities can only be compared across particular spectra. Because the LTQ reports different ion counts with automatic gain control (AGC) “on” vs. “off,” they cannot be compared. However, AGC “on” spectra can be compared and AGC “off” spectra can be compared. AGC was on for Fig. 2a, b as well as Fig. 3a but off otherwise.

Results and Discussion

Reaction Between Carboxylic Acids and Trimethylborate

Gas-phase reactions of trimethylborate (TMB) are well known [15, 17]. In some cases, TMB has been used as a chemical ionization reagent [36, 37], and in others it has been used as the neutral reagent in ion/molecule reactions of phosphorylated sugars [13, 38,39,40], polyols [14, 15], phospho- and sulfocarbohydrates [41], and other oxygen-containing ions [15,16,17,18]. Kenttämaa’s group has shown that TMB can be used to differentiate oxygen-containing ions such as carboxylic acids, ketones, alcohols, and amides through ion/molecule reactions of protonated analytes with TMB in both an FT-ICR [15] and a linear quadrupole ion trap [16, 17]. Almost all of the protonated oxygen-containing analytes investigated reacted with TMB to produce a diagnostic reaction product 72 Th higher in m/z than the protonated precursor ion. Collision-induced dissociation was then used to differentiate the individual oxygen-containing molecule classes through distinct neutral losses or product ions.

An example reaction between protonated hexanoic acid and TMB is shown in Scheme 1, top. From the observation of a reaction product 72 Th higher in mass-to-charge (middle), it can only be deduced that the precursor ion contains a protonated oxygen atom; using CID, the exact functional group can be determined. For example, upon CID TMB-derivatized carboxylic acids tend to lose HO-B(OCH3)2, a neutral loss of 90 Da (Scheme 1, middle, for the hexanoic acid adduct), and alpha-benzylic carboxylic acids such as ibuprofen tend to lose 118 Da (an additional CO compared to the neutral loss of 90 Da, Scheme 1, bottom). By contrast, aromatic alcohols tend to form O=BOCH3 (58 Da) neutral fragments and aldehydes tend to lose either O=BOCH3 (58 Da) or O=BOCH3 and CH2O (88 Da total), thus allowing differentiation based on product ion m/z (and hence the neutral loss mass) [17]. Two examples of product ion spectra of reaction products of aromatic and non-aromatic carboxylic acids with TMB are shown in Fig. 1. To obtain Fig. 1a, a hexanoic acid standard was ionized by APCI and subjected to reaction with TMB, after which the TMB-acid adduct was isolated and fragmented by CID. As predicted, the hexanoic acid adduct [M + TMB−MeOH + H]+ shows a primary neutral loss of 90 Da (approximately 13% of the total MS/MS intensity). In contrast, in a similar experiment, ibuprofen was used (Fig. 1b) and was found to have a neutral loss of 118 Da because it can form a highly stable benzylic cation (this was the case for the ions that had α-benzylic carbons) which makes up nearly 100% of the total MS/MS intensity.

Scheme 1
scheme1

(Top) Gas-phase ion/molecule reaction between protonated hexanoic acid and trimethylborate (TMB) forms a (middle, left) protonated acid-TMB adduct minus methanol with mass-to-charge 72 Th higher than the original ion’s m/z value. (Middle, right) CID of the adduct generates a product ion via neutral loss of 90 Da or (bottom) 118 Da in the case of alpha-benzylic acid adducts like that formed from reaction of ibuprofen with TMB. NL = neutral loss

Figure 1
figure1

Representative product ion MS/MS scans of ion/molecule reaction products ([M + H + TMB−MeOH]+) of (a) hexanoic acid and (b) ibuprofen with trimethylborate. Note the prominent neutral losses of 90 Da and 118 Da

Ion/molecule reactions usually generate dozens of reaction products in the gas phase, which makes sorting through a mixture tedious when relying solely on data-dependent MS/MS (i.e., successively isolating and fragmenting each ion of interest). In this work, we implemented neutral loss scanning prior to data-dependent MS/MS in order to reduce the number of MS/MS scans performed on non-diagnostic product ions. First, we use trimethylborate as a gas-phase “mass tag” to tag oxygen-containing precursor ions with a 72 Da label (TMB−MeOH) and subsequently, we use a neutral loss scan to identify all of the diagnostic oxygen-containing reaction products of interest, e.g., those satisfying neutral loss of 90 or 118 Da for carboxylic acids. Because the ion trap neutral loss scan does not have unit selectivity on the product ions [34, 42], we finally implement unit-resolution data-dependent product ion MS/MS to confirm the presence of particular carboxylic acids. Because only precursor ions that were identified based on the 90 or 118 Da mass transition in the ion trap neutral loss scan are interrogated via conventional product ion MS/MS, the analysis is simplified and other non-diagnostic product ions (as well as most unreactive ions) are ignored entirely.

Ion Trap Neutral Loss Scans

Figure 2a shows a full scan of a mixture—before reaction with TMB—of eight singly protonated carboxylic acids, namely hexanoic acid (m/z 117), benzoic acid (m/z 123), m-toluic acid (m/z 137), m-methoxybenzoic acid (m/z 153), 4-phenylbutyric acid (m/z 165), 5-methoxysalicylic acid (m/z 169), caffeic acid (m/z 181), and 2-chlorophenoxyacetic acid (m/z 187), using APCI on an LTQ linear ion trap mass spectrometer. After a 100-ms reaction with TMB, the spectrum in Fig. 2b was obtained. Note the many diagnostic reaction products with m/z values 72 Th higher than the unreacted species. However, a significant amount of chemical noise is also present, some of which can be attributed to side products which are not necessarily diagnostic of the acid functionality. One way to simplify the analysis is to identify those reaction products which undergo a specified mass transition indicative of carboxylic acids. For carboxylic acid adducts, the most common transition is 90 Da (HO-B(OCH3)2), as discussed previously. A neutral loss scan of 90 Da—performed in the ion trap using recently developed methodology [34, 42, 43]—reveals seven of the carboxylic acid adducts added to the original mixture as prominent peaks in the mass spectrum with a significant reduction in noise. In the case of the 2-chlorophenoxyacetic acid adduct, the product ion at m/z 169 has very low intensity even in the product ion MS/MS spectrum (not shown) and so is not detected by the neutral loss scan. The identification of seven of the eight carboxylic acids using a single mass scan and reduction in noise demonstrates the advantages of this approach. Product ion MS/MS scans confirmed that each of the carboxylic acids detected lost 90 Da upon CID in the ion trap. This approach reduced the total instrument runtime by eliminating analysis on the many peaks in Fig. 2b that did not meet the CID criterion.

Figure 2
figure2

(a) Full-scan APCI mass spectra of eight protonated carboxylic acids, hexanoic acid (m/z 117), benzoic acid (m/z 123), m-toluic acid (m/z 137), m-methoxybenzoic acid (m/z 153), 4-phenylbutyric acid (m/z 165), 5-methoxysalicylic acid (m/z 169), caffeic acid (m/z 181), and 2-chlorophenoxyacetic acid (m/z 187). (b) Mass spectrum after ion/molecule reaction with trimethylborate in the ion trap. (c) Neutral loss scan of 90 Da. All labeled ions are protonated. The intensity of (a) should not be compared to panels (b) and (c), but (b) and (c) can be compared

Carboxylic Acid Selectivity

The selectivity of the method for carboxylic acids was tested using a mixture of various oxygen-containing groups: an aldehyde, a ketone, an amide, a carboxylic acid, and an alcohol. The full scan before and after reaction of protonated benzaldehyde, 2-decanone, p-toluamide, 4-phenylbutyric acid, and 1-octanol with TMB is shown in Fig. 3a, b. Some of the precursor ions are more prominent than others due to differences in ionization efficiency.

Figure 3
figure3

(a) Full scan of five-component mixture of oxygen-containing compounds (benzaldehyde, 1-octanol, p-toluamide, 2-decanone, and 4-phenylbutyric acid) before reaction with TMB. (b) Full scan after reaction with TMB. (c) Neutral loss scan of 90 Da. Intensities in (a) and (b) can be compared but cannot be compared to (c)

Scheme 2 shows the expected diagnostic reaction products and also product ions observed upon CID of each TMB adduct (the spectra of which are shown in Fig. 4). Since the ion trap neutral loss scan does not have unit selectivity (because the product ions are generated and ejected at varying Mathieu q values), the neutral loss scan in Fig. 3c detects not only the carboxylic acid adduct (4-phenylbutyric acid) but also the benzaldehyde adduct (neutral loss of 88 Da), p-toluamide adduct (neutral loss of 89 Da), and the 2-decanone adduct (neutral loss of 90 Da). Note that the amide was buried in the noise in the full scan of the reaction mixture but is recovered in the neutral loss scan because of the generally observed increase in signal-to-noise when using MS/MS.

Scheme 2
scheme2

Characteristic reaction products of various oxygen-containing compounds with trimethylborate in the gas phase as well as prominent product ions from CID of the analyte-TMB adduct: (a) benzaldehyde, (b) 1-octanol, (c) p-toluamide, (d) 2-decanone, and (e) 4-phenylbutyric acid adducts. Product ion structures are tentative

Figure 4
figure4

Product ion MS/MS spectra of various oxygen-containing analytes with TMB and notable neutral losses: (a) benzaldehyde, (b) 1-octanol, (c) p-toluamide, (d) 2-decanone, and (e) 4-phenylbutyric acid

Table 1 lists the reaction products observed after isolation and reaction of each protonated precursor ion in the mixture with TMB. In all cases, multiple reaction products are observed, increasing the complexity of the mass spectrum (especially in cases when the precursor ion is not isolated) despite also producing a diagnostic TMB adduct. Data-dependent MS/MS applied to this mixture would therefore be inefficient (especially on a portable mass spectrometer with ion/molecule reaction capabilities [44, 45]); many scans would be wasted on the non-diagnostic reaction products. The neutral loss scan therefore serves as an initial survey scan to limit the number of possible precursor ions for product ion MS/MS since only the ions which satisfy the specified MS/MS transition (90 Da neutral loss in this case) are further investigated.

Table 1 Reaction Products for Various Oxygen-Containing Analytes with TMB

Figure 4 shows the product ion MS/MS spectra that were acquired showing the different neutral losses that distinguish the ketone, aldehyde, and amide from the carboxylic acid. In the case of the ketone, a small neutral loss of 90 Da was observed. A previous report documented that upon CID ketone-TMB adducts lose the larger side chain, which was also observed here (neutral loss of 112 Da for 2-decanone). In contrast, it is clear that p-toluamide loses 89 Da upon CID and benzaldehyde loses 88 Da upon activation, thus differentiating them from the carboxylic acid which has a 90 Da mass transition.

Analysis of Pharmaceutical Tablets Using Gas-Phase Mass Tags

We applied the neutral loss scanning methodology to two pharmaceutical mixtures. Naproxen sodium and ibuprofen tablets were purchased from a local supermarket, and small amounts were dissolved in 50:50 methanol/water with 0.1% formic acid. The full APCI mass spectra after reaction with TMB are shown in Fig. 5a, c. The naproxen- and ibuprofen-TMB adducts occur 72 Da higher in mass than their protonated precursors. Because both naproxen and ibuprofen are α-benzylic carboxylic acids, their TMB adducts form stable benzylic cations upon CID. A neutral loss scan of 118 Da (90 Da + mass of CO) for each mixture (Fig. 5b, d) then recovers the adducts of the active ingredients at m/z 303 and m/z 279. In the case of the naproxen tablet, a significant reduction in noise is observed, increasing S/N and reducing the number of peaks one would need to interrogate via product ion MS/MS to confirm the carboxylic acid functionality. MS/MS of the naproxen adduct is shown in Fig. S2, with m/z 185 (neutral loss of 118 Da) as the most prominent fragment ion, as expected. The MS/MS spectrum of the ibuprofen adduct was identical to Fig. 1b.

Figure 5
figure5

(a) Reaction of analytes from a naproxen tablet ionized by APCI with TMB and (b) neutral loss scan of 118 Da. (c) Reaction of analytes from an ibuprofen tablet ionized by APCI with TMB and (d) neutral loss scan of 118 Da

Limitations

The limitations of the current method include the fact that single analyzer ion traps do not have unit mass selectivity in the neutral loss scan mode. Not only are carboxylic acids detected, but so are other reaction products which have neutral losses of similar mass (within a few m/z [42]). Similarly, unreactive ions with neutral losses close to 90 Da will also be detected. This problem was avoided here by using the two-step neutral loss/product ion scan procedure.

A second limitation is that molecules with multiple functional groups will be protonated at the most basic site, usually nitrogen. In this case, reaction with TMB will not proceed and oxygen functionalities will not be identified [17]. This is a limitation of the reaction, not the neutral loss methodology. Multifunctional compounds with more complex chemistry and fragmentation patterns could also pose an interference risk. Ethers react with TMB to form adducts minus methanol, which through CID give a neutral loss of 90 Da, as documented previously [17]. However, in our experiments, both a linear ether (allyl propyl ether) and a cyclic ether (tetrahydrofuran)-TMB adduct gave very low intensity of product ions corresponding to neutral loss of 90 Da (Fig. 6). In contrast, most of the carboxylic acids tested gave a highly abundant neutral loss of 90 Da (hexanoic, benzoic, caffeic, palmitic, stearic, and 4-phenylbutyric acids as well as ibuprofen gave this or a 118 Da neutral loss with 100% relative abundance). Hence, ethers may not be significant interferences in this method.

Figure 6
figure6

MS/MS spectra of [M + TMB-MeOH + H]+ for (a) tetrahydrofuran and (b) allyl propyl ether. The peaks correlating to neutral loss of 90 Da are 0.000003 and 3% relative abundance

Lastly, the neutral loss scanning methodology is only useful for reactions which generate adduct ions that fragment similarly within a molecular class but fragment differently between molecular classes. That is, trimethylborate was chosen in this study because (1) it reacts nearly exclusively with oxygen-containing ions in the gas phase; (2) carboxylic acid-TMB adducts consistently have neutral losses of 90 or 118 Da through CID, which can therefore be interrogated efficiently and selectively using a neutral loss scan; and (3) adducts of TMB with other molecular classes largely do not have neutral losses of 90 or 118 Da.

Conclusion

We have demonstrated a gas-phase mass tagging method wherein oxygen-containing analytes from mixtures were selectively tagged with a neutral reagent. A neutral loss scan was then used to indicate the ions in the mixture most likely to be carboxylic acids. The lack of unit selectivity in the neutral loss scan required subsequent product ion scans to confirm the presence of carboxylic acids. The neutral loss scan technique reduces the number of MS/MS scans required to confirm which peaks in the spectrum are from protonated carboxylic acids. The method was demonstrated on neat mixtures of standard carboxylic acids as well as on solutions of relevant pharmaceutical tablets but may be generalizable to other ion/molecule reactions. Although this work was conducted using a benchtop linear ion trap mass spectrometer, it is most applicable where instrument power or sample volume is limited, in which case workflows must be rapid, efficient, and effective.

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Acknowledgements

The authors acknowledge funding from Merck & Co., Inc. and from NASA Planetary Sciences Division, Science Mission Directorate (NNX16AJ25G). This work was also funded by a NASA Space Technology Research Fellowship (DTS). Ryan Hilger and Mark Carlsen (Jonathan Amy Facility for Chemical Instrumentation) are thanked for modifications to the LTQ instrument for orthogonal double resonance capabilities. The authors finally thank Dr. Joann Max (Eli Lilly) for help with ion/molecule reactions and Rob Schrader for the table of contents graphic.

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Correspondence to R. Graham Cooks.

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Snyder, D.T., Szalwinski, L.J., Pilo, A.L. et al. Selective Gas-Phase Mass Tagging via Ion/Molecule Reactions Combined with Single Analyzer Neutral Loss Scans to Probe Pharmaceutical Mixtures. J. Am. Soc. Mass Spectrom. 30, 1092–1101 (2019). https://doi.org/10.1007/s13361-019-02149-y

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Keywords

  • Ion/molecule reaction
  • Linear ion trap
  • Neutral loss scan
  • Carboxylic acid