For Research Use Only. Not for use in diagnostic procedures.
Using Differential Mobility Spectrometry for the Detection of Intramolecular Hydrogen Bonding
J. Larry Campbell*1; Chang Liu1; J.C. Yves Le Blanc1; Gilles H. Goetz2; Jefry Shields2; John Janiszewski2; Ahdia Anwar3; W. Scott Hopkins3
1SCIEX, Concord, ON, Canada; 2Pfizer, Inc., Groton, CT, USA; 3Department of Chemistry, University of Waterloo, Waterloo, ON, Canada
MATERIALS AND METHODS
ABSTRACT
By using a differential mobility spectrometry (DMS)-based method, we could determine the presence of
intramolecular hydrogen bonds (IMHBs) among isomeric drug molecules. IMHBs are valuable features for many
drug molecules, as their presence may assist in passive cellular membrane transit.
Figure 3. Structures for the compounds examined in this
DMS study. They are separated into five sets that set apart
the isomeric forms within each group of compounds. These
compounds had been examined previously a part of the
EPSA study that correlated the presence of IMHBs with
shorter SFC retention times.[Goetz et al., 2014] It is
important to note here that, while neutral compounds and
non-ionic IMHBs were features of the EPSA/SFC
study,[Goetz et al., 2014] this DMS-based study employed
the protonated forms for each of these species. For each
isomer pairing, the site of protonation was the same for
each molecule. For example, in the set of IMHB-7, IMHB-8,
and IMHB-9, the calculated lowest-energy structures placed
the site of protonation at the pyridine ring N, even though its
relative position with respect to the rest of the molecule
changed between the isomeric species of this set.
These IMHB-derived differences are determined based upon relative shifts in the compensation voltage (CV) shifts
in the presence of gas-phase water or methanol. This provides IMHB information in a fraction of the time of other
analytical methods and traditional cell permeability assays.
INTRODUCTION
A drug molecule’s overall utility depends upon a number of factors, one of which is its cell permeability. Can the drug
molecule successfully enter the cells targeted for the specific therapeutic agent? Recent studies [Rafi et al., 2012;
Shalaeva et al., 2013] have demonstrated that drug molecules that contain IMHB(s) are generally more cell
permeable than isomeric forms that lack an IMHB; essentially, the polar regions of IMHB-containing molecules are
purported to be less exposed to the solvent environment, promoting desolvation and lipophlicity.
However, verifying the presence of IMHBs in drug molecules can be a time- and labor-intensive process, especially
when cell permeability assays, such as the RRCK assay, [Di et al., 2011] are required. Alternative analytical
techniques, such as supercritical fluid chromatography,[Goetz et al., 2014] have shown success in highlighting the
presence of IMHBs in ~20 min experiments (Figure 1).
RESULTS & DISCUSSION
RESULTS & DISCUSSION
Figure 5. Dispersion plots for the five sets of isomeric drug molecules; each isomer in a set differed only by subtle
structural changes that promoted or hindered formation of IMHB(s). Each species is labeled with its IMHB number,
the measured EPSA value [Goetz et al., 2014], and its calculated lowest energy structure (HF/3-21g). Yellow circles
mark the presence of IMHBs, with a red X (for IMHB-2) denoting the absence of an IMHB for that site. For each set
of isomers, the ions with the lower EPSA values exhibit more positive CV shifts (blue traces). Both of these findings
(EPS and CV shifts) suggest these species exhibit lower degrees of solvation (lower binding energies with methanol
molecules), and the correlation between EPSA and DMS behavior is quite promising.
Figure 6. The presence of IMHBs in analyte ions has been observed previously.[Lintonen et al., 2014]
(a) Schematic diagram of the ESI(-) of a mixture of glycerophospholipids (GPLs), where DMS has been used to
differentiate lipids based upon their respective head groups’ ability to form IMHBs. The greater the propensity to
form IMHBs, the less intermolecular clustering occurs between the GPL anions and the chemical modifiers in the
DMS cell, the more positive the CV shift. (b) Energy-minimized structures [B3LYP/6-311++G(d,p)] [Becke, 1993;
Lee et al., 1988] for six model GPLs, reveling the different extents of intramolecular hydrogen bonding between the
GPL head group and the phosphate anion. As the IMHBs become more prominent, the binding between the analyte
ion and the chemical modifier in the DMS cell is reduced, resulting in more positive CV shifting.
(a)
(b)
Here, we demonstrate another analytical method, based upon DMS (Figure 2) to highlight the presence of IMHBs in
isomeric drug molecules. These findings support our recent lipidomics work that demonstrated the potential
importance of IMHBs in the DMS-based separation of glycerophospholipid classes.[Lintonen et al, 2014]
MATERIALS AND METHODS
Sample Preparation:
The various groups of isomeric molecules (Figure 3) were prepared in-house (Pfizer) .
DMS-MS/MS Conditions:
A DMS system (Figure 2) [Schneider et al., 2010] was mounted in the atmospheric region between the mass
spectrometer’s sampling orifice and an ESI source (+5500 V). In each experiment, an individual IMHB solution (100
ng/mL each in 50/50 H2O/ACN + 0.1% formic acid) was infused into the ESI source (7 μL/min). The DMS
temperature was maintained at 150 °C, and the nitrogen curtain gas was operated at 30 psi. When desired, volatile
solvents (chemical modifiers) [Schneider et al., 2010; Campbell et al., 2012] were added to the curtain gas at 1.5%
(v/v). As the DMS’ Separation Voltage (SV) was stepped from 0 to 4000 V (in 500-V increments), the Compensation
Voltage (CV) was scanned from -30 V to +15 V in 0.15-V increments. Dispersion plots (CV versus SV, Figures 4, 5)
[Levin et al., 2014] reveal the extent of the protonated drug molecules’ solvation in the DMS.[Campbell et al., 2014]
RESULTS & DISCUSSION
Figure 4. Dispersion plots obtained for IMHB-1 and IMHB-2 under three different DMS cell conditions: No modifier
(i.e., N2 only), 1.5% water, and 1.5% methanol (MeOH). While no differences in DMS behavior were observed
using only N2, the presence of water and methanol in the DMS cell enable the moderate and complete separation of
these isomers, respectively. Note that the presence of the amidine functionality essentially guarantees that this will
be the site of protonation for these specific molecules. However, the other 9 compounds required a survey of many
possible sites of protonation to ensure that the lowest-energy protonated structures were considered.
CONCLUSIONS
Within each isomeric pair of drug molecules, the presence of IMHBs were highlighted based upon the observed DMS
behaviors (that correlated well with the respective EPSA values) determined for each species. Essentially, the DMS
behavior for the protonated molecules postulated to exhibit IMHBs displayed lower degrees of ion/molecule clustering
in the DMS cell, as exhibited by more negative shifts in CV compared to the non-IMHB containing isomers.
Expansion of these experiments could lead to a greater understanding of the solvation of drug molecules in bulk
solution and the possible benefits that such a measurement could bring (i.e., physicochemical property prediction).
Figure 1. Example of the SFC-based separation of IMHB-1
(red) and IMHB-2 (blue) developed by Goetz and coworkers
(2014). Note that the isomer containing an IMHB is less
retained on the SFC column due to that structural feature.
Figure 2. Key components of the differential mobility
spectrometry-mass spectrometer (DMS-MS)
employed in this study.
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