Adduct-ion formation in trapped ion mobility spectrometry as a potential tool for studying molecular structures and conformations
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Recent developments in the field of ion mobility spectrometry provide new possibilities to explore and understand gas-phase ion chemistry. In this study, hyphenated trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) was applied to investigate analyte ion mobility as function of adduct ion formation for twelve pharmaceutically relevant molecules, and for tetrahydrocannabinol (THC) and its isomer cannabidiol (CBD). Samples were introduced by direct infusion and ions were generated with positive electrospray ionization (ESI+) observing protonated and sodiated ions. Measurements were performed with and without addition of cesium-, lithium-, silver- and sodium ions to the samples. For the tested compounds, metal adduct ions with the same m/z but with different mobility and collision cross section (CCSs) were observed, indicating different molecular conformations. Formation of analyte dimers was also observed, which could be associated with molecular geometry of the compounds. By optimizing the range and speed of the electric field gradient and ramp, respectively, the separation of THC and CBD was achieved by employing the adduct formation. This study demonstrates that the favorable resolution of TIMS combined with the ability to detect weakly bound counter ions is a valuable means for rapid detection, separation and structural assignment of molecular isomers and analyte conformations.
KeywordsTrapped ion mobility spectrometry Dimers Adduct ions Protomers Molecular conformations
Over recent times, ion-mobility spectrometry (IMS) has brought new insights into the behavior of gas-phase ions and complexes of small molecules, biomolecules and polymers [1, 2, 3]. The IMS separation mechanism, which is based on ion charge, size and shape, may facilitate the separation of isomers and recognition of different ion conformations. Additional structural identification often is achieved by hyphenating IMS to (high-resolution) mass spectrometric (MS) detection. The past twenty years has seen rapid development of new IMS devices, in which a considerable improvement, especially in resolving power, was achieved [4, 5, 6, 7]. Still, separation of structural isomers with IMS can be quite challenging.
A common approach to separate isomers, which exhibit similar collision cross section (CCS (Å2)), is the use of mono- and divalent cations, which form distinguishable adduct complexes with the target analytes, and has led to successful separations of isomeric carbohydrates, lipids, and peptides [8, 9, 10, 11, 12, 13]. The improved resolution was due to mobility shifts in one of the isomeric forms as a result of conformational changes induced by cation adduct formation. Other common approaches to shift the mobility of ions involve the use of mixed carrier gases, shift reagents (SR) and/or changes in temperature of the buffer gas [14, 15, 16, 17].
Huang & Dodds  and Clowers & Hill  reported unpredictable changes in conformations of analytes upon use of different adduct ions, which can depend on the structure of the analytes and the ionic radius of the cations. These molecular species could then result in improved mobility separation. Clowers & Hill  studied the separation of flavonoids diglycosides isomers using dual gate-ion mobility quadrupole ion trap mass spectrometry. They showed multiple gas phase conformations for narirutin and hesperidin upon sodium, potassium and/or silver adduct formation. Similar observations were made for carbohydrates, where a trisaccharide maltotriose with cesium adduct showed more than one mobility peak . In the latter study, a Waters Synapt G2 HDMS quadrupole time-of-flight hybrid mass spectrometer equipped with travelling-wave ion mobility (TWIM) cell was used. In both cases, the appearance of the new gas phase conformations of the molecules was explained as a result of different binding sites of the cation and the target compound.
Recently, Morrison, Bendiak, & Clowers  investigated the influence of cation adduct formation with tetrasaccharides using atmospheric pressure dual-gate drift ion mobility coupled to a Thermo linear ion trap mass spectrometer. They noticed that for some glycans the coordination of a metal ion lead to formation of dimers, which was observed as a second mobility peak appearing at the same nominal mass as the monomeric form of the analyte. The dimers were formed at a very low cation concentration, and adducts of bivalent metals were more prone to form dimers than adducts of monovalent cations.
In IMS devices which use high electric fields for the separation of ions, such as TWIMS, heating of ions may result in analyte fragmentation, as observed for e.g. p-methoxybenzylpyridinium (m/z 200)  and a leucine enkephalin dimer (m/z 1112) . Ion heating may also cause disintegration of ion adducts and dimers in the mobility cell. Trapped ion mobility spectrometry (TIMS), a recent IMS development commercialized by Bruker Daltonics (Bremen, Germany), operates at low electric field, preventing ion heating, thus offering optimal conditions for studying non-covalent complexes and relatively loosely bound adducts. Additionally, TIMS offers mobility separations with up to a resolving power (R) of 400 , which was successfully applied in fast separations of isobars and isomers [4, 21, 22].
During exploring TIMS experiments in our laboratory in which the ortho, meta and para isomers of dimethylphtalate were separated, we observed various sodium adduct ions (data not shown). The main objective of the present study was to gain a more fundamental understanding of adduct-ion formation in TIMS and to further evaluate this phenomenon to distinguish isomeric analytes which could not be separated as protonated molecule. Fourteen pharmaceutically relevant compounds with a molecular weight ranging from 270 to 645 Da were used as test compounds, systematically studying several cationic counter ions. The effect of adduct-ion formation with the target analytes on the appearance of new conformations and dimers, was investigated by TIMS, and correlations with the analyte molecular structures were made. Additionally, gas phase separation of isobaric tetrahydrocannabinol (THC) and cannabidiol (CBD) by TIMS was studied employing cation adduct formation.
Materials and methods
Amoxicillin (AMX), carvedilol (CVD), alpha-naphthoflavone (ANF), lincomycin hydrochloride (LM), amiodarone hydrochloride (AMD), fluvoxamine (FVX), rutaecarpine (RUT), loperamide hydrochloride (LOP), ketoconazole (KET), tolbutamide (TBM), sodium chloride (NaCl) and formic acid (FA) were obtained from Sigma-Aldrich (Zwijndrecht, The Netherlands). Cetirizine (CTZ) was kindly provided by DSM Materials Science Center. Ellipticine was a kind gift from Dr. Jan Commandeur from the Division of Molecular and Computational Toxicology (VU Amsterdam, The Netherlands). Tetrahydrocannabinol (THC) and cannabidiol (CBD) were obtained from Echo Pharmaceuticals (Weesp, The Netherlands). Lithium chloride (LiCl), silver nitrate (AgNO3) and acetonitrile (ACN) of LC-MS grade, were from Merck (Darmstadt, Germany), and cesium chloride (CsCl) was from Riedel de Haen (Zwijndrecht, The Netherlands). Acetic acid was from J.T.Baker (Deventer, The Netherlands). High purity water of MS grade was used for samples and solvents preparation.
TIMS operational mode
Where v g , is the gas velocity, E is the electric field at which an ion elutes, A is the calibration constant that can be obtained from measuring calibration solution with known CCS. The difference between the V elution (elution voltage) and V out (voltage applied to the last electrode in the analyser section in TIMS) defines the mobility K of an ion.
All analyte solutions were analyzed by direct infusion at a flow rate of 180 μL/h using a KDS100 syringe pump (KD Scientific Inc., MA, USA). TIMS settings depended on the analytical question. For most pharmaceuticals, the EFG was from −20 to −120 V using 1000 TOF pulses per accumulation and 500 accumulations, whereas for ELL and AMD it was −5 to −160 V and −20 to −150 V, respectively with 1000 TOF pulses per accumulation and 500 accumulations. For the analysis of the two isobaric compounds THC and CBD, initially, the EFG was from 0 to −165 V, enabling analysis of ions within a large mobility range. For isomer separation studies, a narrower EFG was used (from −30 to −80 V) to improve the resolving power of TIMS. The resolving power in TIMS can be improved by reducing the EFG range and to a lesser extent by increasing the number of TOF pulses, which determines the V ramp and t ramp, respectively. In both of the analyses (with a narrow and wide EFG range) 600 TOF pulses per IMS scan with 400 accumulations and 20 repetitions were used. Nitrogen was used as buffer gas at temperature of 300 K, and a default pressure of 1.7 mbar. The mobility resolving power was calculated following equation: R = CCS/ΔCCS.
Experimental CCS and K0 calculations
Where ze is the charge of the ion, k B the Boltzman’s constant, N is the number density of the buffer gas, T is the gas temperature, and m i and m b are the molecular mass of the ions and buffer gas, respectively.
LC-MS of THC and CBD
In order to better understand the dimerization of THC and CBD observed, and to compare the dimer formation of both compounds (as discussed in the results section), solutions of THC and CBD (5 μL injected) were analyzed by LC-MS using an Agilent Technologies 1200 series LC system (Waldbronn, Germany) equipped with an Acquity UPLC BEH C18 (2.1 × 50 mm; particle size, 1.7 μm) analytical column (Waters, Milford, MA). Seven different concentrations between 0 and 20 μM were prepared from THC and CBD stock solutions (3.2 mM in methanol), by serial dilution in 80% ACN. Mobile phases A and B were pure water and ACN, respectively. The gradient used started with 20% B for 1 min, rose to 95% B in 8 min, was isocratic at 95% B for 4 min, and in 0.1 min decreased to 20% B. Column equilibration was set for the next 7.9 min at 20% B. The total run time was 18 min at a flow rate of 0.3 ml/min and the column oven was set to temperature of 40 °C. To form silver adducts, post-column addition of a silver nitrate in water (250 μM) was performed in a flow ratio of 1:4 leading to a final concentration of silver ions of 50 μM. MS detection was performed with a Micro-TOF II instrument (Bruker Daltonics, Bremen, Germany) employing ESI+ with a needle voltage of 4.5 kV and a drying gas temperature and flow rate of 240 °C and 8 L/min, respectively.
Results and discussion
Separation and structural identification of isobaric and isomeric molecules and their conformations can be a challenging task. Adduct-ion formation has shown to specifically influence molecular conformation, potentially allowing separation of isomeric compounds by TIMS. In the present work, the cation-adduct formation of twelve pharmaceutically relevant compounds and its effect on molecular conformation and dimer formation was studied by measuring their mobilities using TIMS-MS. Additionally, TIMS separation of the two isomers THC and CBD was investigated using their cation adducts.
Conformational changes in small molecular ions upon cation adduct formation
In addition to protonated molecules, the usually observed sodium adducts were encountered in the acquired electrospray mass spectra. Interestingly, also in the mobilograms of several sodiated analytes multiple mobility peaks were observed. Cation adducts indeed are known to be able to alter structural conformation of gas phase ions as shown for example, for carbohydrates [9, 26]. Therefore, we studied the effect of several cations on the IM behavior of the test analytes by mixing the compounds with salts of alkali (LiCl and CsCl) and transition metals (AgNO3). The metal ions tend to form non-covalent adducts with target analytes via electrostatic interactions, such as ion-dipole interactions. Silver ions also have affinity towards double bonds, as has been employed, for example, in the LC separation and MS detection of fatty acids (FA) and triacylglycerols (TGA) [27, 28].
For six of the pharmaceuticals, more than one conformation upon cation adduct formation was observed. These were AMX (+Na+, +Ag+, +Li+), RUT (+Ag+), AMD (+Ag+), TBM (+Na+, +Ag+, +Li+), LOP (+Ag+) and ANF (+Na+). The mobilograms for the ions for which multiple conformations were observed are shown in Fig. 2. For most of these compounds (AMX, RUT, AMD, LOP and TBM) an additional conformation was observed upon addition of silver ions, which could be due to the earlier mentioned affinity of silver to double bonds (Fig 2e–i). When the compound possesses multiple double bonds and has high density of π electrons, binding of a silver ion can occur at more than one site in a molecule, and formation of multiple conformations can result . Indeed, AMX, RUT, AMD, TBM and LOP, comprise at least three double bonds. It should be noted that KET and FVX, which have double bonds, do not show detectable multiple conformations in IM, indicating that silver affinity is compound specific. In addition, other ionic interactions obviously can play a role in the induction of multiple conformations. Other cations that also lead to the appearance of multiple mobility peaks were sodium (in the case of TBM, ANF and AMX, Fig. 2J–L, respectively) and lithium (TBM and AMX, Fig. 2l, m, respectively).
As mentioned earlier, we expected rotational bonds to facilitate the formation of other conformations upon cation binding. Interestingly, multiple conformations were also observed in planar compounds, i.e. RUT and ANF (Fig. 2f, k, respectively). However, it is not clear what causes the change in the shape of the molecule in the gas phase, since in the case of RUT the additional conformation was observed only upon cation adduct formation with silver. This could suggest that the size of the silver ion, which has an ionic radius of 1.15 Å , has a considerable impact on the overall shape of the complex, causing the mobility shift even in absence of rotating bonds. The behavior could also be explained by silver residing on different positions. Depending on the position, silver ion could cause changes in the shape of the molecule and consequently changing its CCS.
In most cases of multiple conformations only one additional peak was observed, which had a lower mobility and thus a higher CCS than the primary conformation of the molecule. Here we define the primary conformation as the one which has a mobility closest to the protonated form. This means that the second conformation has a more open or elongated structure. The two planar molecules RUT and ANF (Fig. 2f, k, respectively) appear to follow the common trend, in which the second conformation of the molecule is more open than the primary one. Interestingly, in the protonated species the abundance of the peak with the higher mobility (smaller CCS) tends to be lower in intensity than the peak with the lower mobility. A different behavior was observed in the analytes upon cation adduction, where the first peak (higher K0 and smaller CCS) was observed to be more abundant. This means that the conformation of the ion complex which was observed to have a higher K and lower CCS was present in the gas phase at a higher concentration than the one with a lower K and higher CCS.
Changes in the mobility of molecular ions after formation of cation adduct complexes
Surprisingly, a substantial shift in the mobility between some of the ion adducts was observed for ELL, FVX and RUT. In order to assess the mobility shift, the mobility difference between the mobility value of the protonated species and the adduct of interested was calculated. The protonated species was taken as a reference. Mobility values of all complexes are reported in Table S2. For ELL, the difference in CCS for the silver-ion adduct [M + Ag]+ was 23.5 Å2 (Fig. 3). For RUT, the difference for the [M + Ag]+, [M + Cs]+ and [M + Li]+ CCS was 17.9 Å2, 72.8 and 24.2 Å2, respectively (Fig. 3). In FVX, a clear mobility difference of 6.9 Å2 was observed in the [M + Ag]+ and in LOP a difference of 11 Å2 was observed between the [M + Cs]+ (Fig. 3). The largest difference in the CCS induced by cationization of the twelve small molecular ions was observed in RUT and ELL, which are planar molecules with no freely rotating bonds. This could indicate that in the case of flexible molecules a cation adduct is capable of affecting the structure of the molecule in a way that the CCS does not significantly differ from the CCS of the protonated form. For the more rigid structures, the adducted cation increases the CCS of the analyte molecule.
Dimer formation in molecular ions with limited number of rotational bonds
Dimers in the case of isomers: THC and CBD
Separation of isomers employing cation adduct formation
In this study, we used TIMS to investigate the behavior of twelve pharmaceutically relevant compounds upon cation adduct formation. The selected compounds varied in regard to their molecular shape and flexibility, which resulted in differences in their behavior in the gas phase upon cation adduct formation. In this study four interesting observations of gas phase ion behavior were made: 1) cation adduct can generate different conformation of molecules; 2) for most compounds the correlation between the cross section and the radius of the cation was observed; 3) more planar and rigid molecules tend to form dimers more readily than molecules exhibiting greater flexibility due to the presence of rotating bonds; 4) the separation of structural isomers, THC and CBD, was facilitated by the formation of lithium, cesium and silver adducts. The study presented here confirms previous findings on the cations adduct formation with target analytes and enhances our understanding of the behavior of ions in the gas phase, which is related to the molecular structure of the analytes. The knowledge gained from our observations should prove to be particularly valuable in characterization of unknown molecular structures and in analyzing complex mixtures in which the additional selectivity provided by TIMS is required. Considering the impact that the cations have on the small molecular ions, this study provides a base for future studies on structural isomers, for example, drug metabolites present in complex mixtures.
The authors acknowledge ARIADME, a European FP7 ITN Community’s Seventh Framework Programme under Grant Agreement No. 607517. We are grateful names, (Bruker) for providing the opportunity to work with a prototype TIMS instrument, and their useful support. Thanks for Prof. Michel Nielen from the Wageningen University for providing us with THC and CBD.
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