All reagents were used as supplied unless otherwise noted. Water was purified using a Hydroservices system (Levittown, PA) and then polished using a Millipore Simplicity UV purification unit (Billerica, MA); HPLC grade acetonitrile (ACN) was obtained from Burdick and Jackson (Morris Plains, NJ).

VTS-270 (Vtesse, Inc., Gaithersburg, MD) was supplied in 14 separate synthetic batches for comparison. Two separate synthetic batches of Trappsol Cyclo (CTD Holdings, Inc., Alachua, FL) were used for comparison. HPβCD solutions were prepared as a 1 mg/mL stock in water and infused at either a 200-fold dilution in 1:1 water:ACN or a 400-fold dilution in water:80% (v/v) ACN. Based on the chemical structure and stock solution, a 200-fold dilution represents a concentration of approximately 5 μM.

An Agilent Model 6560 Ion Mobility Quadrupole Time-of-Flight Mass Spectrometer (Agilent Technologies, Santa Clara, CA) was used for all measurements. The instrument has been described previously [18]. In brief, electrospray-generated ions were formed by direct infusion of HPβCD solutions using a nano-electrospray ionization (ESI) source with a flow rate set at 600 nL/min, V_cap potential of 1500 V, and sheath gas flow at 5.0 L/min at 150^°C. With this instrument, ions are sampled into the drift tube through a series of ion funnels, the last of which serves as a trapping ion funnel to deliver ions at 80-msec intervals to the ion mobility drift tube. RF amplitudes in the trapping ion funnel were adjusted to optimize ion intensities. The drift tube was operated with a N₂ bath gas pressure at 3.94 Torr and 27^°C as well as 1450 V potential across the tube. Upon exiting the drift tube, ions passed through another series of optics and were mass analyzed by a high-resolution Q-TOF mass spectrometer. Ion signals were collected for 3-minute intervals prior to analysis and measurements were made in duplicate each day for a total of 3 days over 3 weeks (ie, 12 replicate measurements). All spectra collected were analyzed using Agilent Mass Hunter software (version 7.01) for visualization and manually interpreted and annotated. Mass assignments were based upon accurate mass measurement. Ion collision cross sections were determined using software supplied in this same package.

Under conditions of low drift fields in an appropriate pressure regimen, as defined by Mason and McDaniel [19] and approximating both an ion and the buffer gas as hard spheres, the collision cross section, Ω, can be calculated in units of Ų from the expression: Ω=(18π)1/216ze(kBT)1/2[1mi+1mB]1/21K01N* where k_B is the Boltzmann constant, z and e are the charge state and electronic charge, respectively, N^* is the gas number density in the drift tube, m_i is the ion mass, and m_B is the mass of the buffer gas. Widespread usage has allowed the spherical assumptions to be extended to what are clearly non-spherical ions and to non-spherical, polarizable drift gases. K₀ is determined from measurements of apparent ion drift times that are corrected for passage time in the instrument outside of the drift tube region and transformed to STP conditions.

Initial experiments compared the ion mobility profile of the two HPβCD products. Fig 2 shows “heat map” false color representations of the instrument response in ion mobility-space of VTS-270 (upper panel) and Trappsol Cyclo (lower panel). Ions that have larger three-dimensional structures have longer drift times than ions with smaller structures [20]. Ions with the same m/z values but different charge states have different drift times, with higher charge state ions having shorter drift times [20]. These components of the ion mobility response give rise to the several “families” of ion responses corresponding to ions with related structures and different charge states seen in the two panels of Fig 2. Considering that the two materials are infused under the same preparation conditions and at the same concentration, it is apparent that there is a much greater level of non-specific chemical “noise” associated with Trappsol Cyclo compared with VTS-270, particularly in the lower m/z regions over drift times up to approximately 45 msec. In addition, the Trappsol Cyclo heat map shows the presence of ions at m/z >2000 and drift times between 30 and 40 msec, which are not observed in VTS-270. These species correspond to aggregates of multiple HPβCDs at high charge states.

Additional differences between VTS-270 and Trappsol Cyclo can be seen in Fig 3, which presents the mass spectra extracted from Region 1 denoted on Fig 2. These mass spectra exhibit resolutions of 20,000 or greater and peak assignments are consistent with 10 ppm or better mass accuracy when compared with expected values. This region of the heat maps covers the mass range 1200 to 1700 Da and drift times between 25 and 50 msec. Fig 3 represents a “compression” of all the ion intensities in this drift region and therefore combines the intensities of multiply charged ions of the same m/z, namely, monomeric singly charged and doubly charged ions formed from two HPβCD molecules as either two molecules of a single DS (homodimers) or two different degrees of substitution (heterodimers). The most intense ions in the spectra are those formed by NH₄⁺ adduction to neutral HPβCD species with smaller contributions from protonated adducts. The spectra show a distribution of ion intensities corresponding to the degrees of substitution of the two materials studied, and demonstrate clear differences between them. The numbers above the most intense ions of each spectrum show the DS of the ammonium adduct for that species (eg, nominal mass 1384 corresponds to the ammonium adduct of a DS = 4). Close examination of the isotope clusters for each of the major ions shows both doubly charged homodimers of ammonium adducts (ie, [DS_n + NH₄]₂⁺² of the ammonium adducts). In the case of Trappsol Cyclo, doubly charged dimers of the protonated ions are observed. Both materials show doubly charged homodimers from adduction of both a proton and ammonium; one of these is noted in the upper panel of Fig 3. Doubly charged heterodimers ([DS_n + DS_n-1 + 2NH₄]⁺² are noted for the case of degrees of substitution of 6 to 7 at nominal mass 1530) are formed in both materials but are far more intense in Trappsol Cyclo than in VTS-270. Finally, we determined that all of these differences were maintained at ~2.5 μM concentrations (400-fold dilution) in 80% ACN, indicating that these dimers have strong intermolecular associations and that this does not suggest a concentration dependence in ion formation at the working concentrations studied.

Figs 2 and 3 represent a portion of the characteristic “fingerprint” of these molecular mixtures. Additional differences in the fingerprints are shown for VTS-270 and Trappsol Cyclo in Fig 4. This figure shows mass spectra from the region of Fig 2 over a mass range from ~990 to 1100 Da and drift times of ~25 to 27 msec. Here, compositional differences between the two materials are also readily apparent. Ions of the form of triply charged dimers of both homo and hetero origin (ie, [DS_n +2NH₄ +H]⁺³ and [DS_n+DS_n-1+2NH₄+H]⁺³; labeled A and B, respectively, in Fig 4) are visible in both panels but less intense in the VTS-270 sample than in the Trappsol Cyclo sample.

Variations in the relative intensities of dimeric ions are presented in Fig 5. The fraction of total ions represented by homodimers observed as a function of DS for both VTS-270 and Trappsol Cyclo are shown in Fig 5A. Similar fractional intensity changes as a function of DS are seen for both materials, but the relative fractional intensities of these dimers appear to be greater for Trappsol Cyclo than for VTS-270. Fig 5B shows the fraction of total ions represented by heterodimers as a function of DS for both materials. The difference between the two is more apparent in this case as the degree of heterodimer formation is seen to be much greater for Trappsol Cyclo than for VTS-270, particularly at higher DS. While these data were collected at 5 μM solution conditions, it is important to note that these observable differences were preserved under conditions of a 400-fold dilution in 80% ACN.

Collision cross section (Ω) areas of the major ions of HPβCD were calculated using the VTS-270 and Trappsol Cyclo data as described and presented in Tables 1–6. These novel data were generated and included to add to the growing publicly available characterization of the ion collision cross sections measurements of various molecular species. While there are differences between VTS-270 and Trappsol Cyclo in the various ions present as described above, the ion collision cross sections for each of the ions present in both materials were similar, as expected.

Tables 1–6 show values of Ω for a series of different ions with varying DS observed in the m/z region between 1200 and 1620 Da. As seen in the mass spectra in Figs 3 and 4, these ions consist of a mixture of adducts of protons as singly and doubly charged species. As also seen in the spectra, not all ions of each adduct type are present in both VTS-270 and Trappsol Cyclo; however, when both species are present, the values calculated for their collision cross sections are in close agreement as noted in Tables 1–6.