Improved Mass Determination of Poly(ethylene glycols) by Electrospray Ion-Mobility Time-of-Flight Mass Spectrometry Coupled with Ion–Molecule Reactions - Pharmaceutical Technology

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Improved Mass Determination of Poly(ethylene glycols) by Electrospray Ion-Mobility Time-of-Flight Mass Spectrometry Coupled with Ion–Molecule Reactions
The authors developed a method to accurately measure the average molecular weight of large poly(ethylene glycols) (PEGs) using ion-mobility time-of-flight mass spectrometry coupled with gas-phase ion–molecule reactions.

Pharmaceutical Technology

Results and discussion

Figure 2a & 2b
Analysis of PEG 4450 before charge reduction. An ESI-TOF analysis of PEG 4450 before the reaction with superbase DBU results in a complex ESI spectrum (see Figure 2a). As shown in the inset of Figure 2a, the instrument resolving power at the collected m/z window is sufficient to provide isotopic resolution for peaks in the spectrum. The isotopic resolution permits the determination of the number of charges that each oligomer holds as well as the charge states distribution in the spectrum. The data show that several charge states, from 2+ to 4+, are observed in Figure 2a, suggesting the attachment of multiple cations to each PEG oligomer. On the basis of these calculations, it is concluded that charge carriers (cation adduction) are not completely contributed by one type of metal ions (e.g., Na+). Some oligomeric ions are possibly generated by the attachment of protons (and also possibly K+), yielding many different ion series in the spectrum. As a result, the average molecular weight cannot be readily determined.

Figure 2b shows the drift time and m/z distributions for the PEG 4450 sample analyzed in the TOF-mobility mode (IMS–MS). In these experiments, the time required for IMS separations is less than 20 ms. As shown in Figure 2b, ions with different charge states, or different conformers of the same m/z value, were resolved by IMS. In comparison with the ESI-TOF spectrum of this sample (see Figure 2a), at least four resolvable charge envelopes (from 1+ to 4+) are observed in the IMS analysis. Individual oligomers can be clearly resolved not only in the m/z dimension but also by their mobility drift time. The separation greatly simplifies the complexity of the spectrum such that some of the minor components in the samples that can not be observed otherwise can be easily identified from the analyses. For example, without the mobility separation, the 1+ envelope from PEGs with a shorter chain length is not observable in the mass spectrum (see Figure 2a), demonstrating an increase in dynamic range when ion mobility is coupled with MS. Several higher charge-state envelopes are also present in the spectrum, but they are not well resolved from each other, thereby making complete characterization difficult.

Analysis of PEG 4450 with charge reduction. To further simplify the mass spectrum, gaseous DBU was introduced into the trap cell. Upon the reaction, some of the protons are stripped from the multiply charged PEG ions, and the charge-state distribution is reduced to primarily a single charge state (2+) as illustrated in Figure 3. In this example, the multiply charged PEG ions were reacted with the DBU in the trap cell, and a symmetric distribution of doubly charged ions at a mass-to-charge interval of 22 m/z was generated. Coexisting with the 2+ charge state ion species are ion series with the charge state of 1+. The MWs of these PEGs are estimated to be much lower than the one from the charge envelope of 2+, indicating that these peaks are possibly generated by the impurities in the sample.

Figure 3
When the data from the series of doubly charged ions from the ion–molecule reaction between PEG 4450 with DBU (see Figure 2b) are fit to a Gaussian function, the mean (x o) and peak variance (w) of the best fit Gaussian function are 2240.2 and 240, respectively. The dots in the inset of Figure 3 show the data points (peak maxima) that are used for the fitting process. As can be seen from the inset, the data fit the distribution well. The quality of fit is also measured by the correlation coefficient, which has a value of 0.9977. Because this is a doubly charged envelope, these values correspond to an average MW and MWD of 4480.4 and 720 Da, respectively, for the PEG 4450. In comparison, the average MW provided by the supplier, as was determined by GPC, is 4450. The value obtained by the current work is in good agreement with the value measured with an orthogonal method.

Analysis of PEG-aldehyde 20K with and without charge reduction. The ESI-TOF spectrum of PEG-aldehyde 20K (see Figure 4a) is more complex compared with that of PEG 4450. Individual oligomers are not discernible in the spectrum, and the spectrum comprises relatively broad "noisy" bands, which are attributed to the overlapping of the charge states, the polydispersity of the oligomers and the compression of the m/z range by the high-charge states. As noted previously, a PEG sample consists of a mixture of oligomers, each of which can have various charge states. The number of possible combinations of oligomer size and charge state increases rapidly with increasing nominal molecular weight. At a MW of 4450, there are as many as four or more discernible ions within an interval of one unit of m/z at the scale nearby m/z 1000. It is believed that as many as 28 charges can be placed onto an oligomer chain with a mass of 20 kDa (6). Under the resolving power of a TOF analyzer, such complexity results in spectral congestion (see Figure 4a). Consequently, it is not possible to extract molecular weight information from the data.

Figure 4
Figure 4b shows the mass spectrum resulting from reacting PEG 20 k with DBU in the trap cell. Two dominate charge state envelopes, 4+ and 5+, are observed. Detailed examinations of the spectrum also indicate that peaks corresponding to the individual oligomers of the polymer are baseline resolved. However, unlike the data for PEG 4450, isotopic resolutions for these PEG 20K ions are not observed.

The appearance of the discernable PEG oligomers makes it feasible to determine the average MW of the PEG. To determine an average MW of the polymer by the curve-fitting method, an overall MWD represented by the total ion abundance of each oligomer is needed. Because 4+ and 5+ charge states dominate the charge-stripping spectrum, the summation of ion abundance from both charge states for each oligomer should naturally yield a close reflection of the original MWD of the polymer. For this purpose, the m/z value at each peak under the charge envelops of both 4+ and 5+ charge states was first converted to "zero-charge" distributions by multiplying with its respective charge states and subtracting the mass of sodium multiplied by the charge. This mathematical calculation is based on the assumption that the observed charge envelopes resulted from the sodium ion attachment to the PEG oligomers, as has been reported in the literature (15). The final assembly of the ion abundance was achieved by summing the abundances of the corresponding peaks of the same oligomer from these "zero-charge" distributions for both the 4+ and 5+ envelopes. The zero-charge distribution obtained by combining the distributions from the 4+ and 5+ charge envelopes was fitted to a Gaussian (dashed line, see Figure 4c), which provides an average MW of 21,552 Da and a MWD of 3228 Da. This MW value is in close agreement with the literature value measured by similar MS approach as well as an NMR method (21,920 Da) (15).

It is noteworthy that MWs of polymers measured by mass spectrometric methods can potentially deviate from values obtained by other methods. Mass bias, ionization suppression, and instrument tuning conditions were cited as possible sources of errors in the mass measurement (6, 15). In addition, inhomogeneous ion–molecule reaction across many different PEG oligomer ions could bring in an additional source of deviation for the current approach. However, the good agreement between the measured values in current work and GPC values suggest that the potential impact of possible variables was minimal in our experimental setup, and accurate MW values for high-mass PEGs can be obtained.

Several observations can be made from the ion–molecule reactions. First, the final charge states as well as the charge-state distribution depends on the partial pressure of DBU vapor in the cell. Because the transit time for PEG ions passing through the trap cell is limited by their collisions with the gas molecules, higher gas pressures imply more collisions taking place in the trap cell before ions exit out. Higher gas pressures obviously favor longer reaction time, and thus lower charge states could be obtained. Second, stronger bases are required to reduce the charge to the same terminal charge state for longer polymer chains. This observation is likely caused by a combination of two factors: First, the longer polymer chains have a higher initial charge state, requiring more efficient charge stripping reaction to reduce to the same charge state. In addition, longer polymer chains can retain an additional charge more effectively, which would make an additional charge more difficult to remove.


This work provides a method for improving mass determination of high molecular weight poly(ethylene glycol) (PEG) by means of an ion–molecule reaction inside a Synapt high definition mass spectrometer. The improved performance results from reductions in the spectral complexity of polydisperse PEG materials. The reduction in spectral complexity yielded resolved oligomers and allowed the average molecular mass and polydispersities to be determined for the polymers. In addition, the combination of this ion–molecule reaction methodology with ion mobility separations and TOF mass spectrometry has provided enhanced sensitivity and specificity for characterizing PEG on the basis of chain lengths and for differentiating the low molecular weight impurities in the PEG materials. All the data demonstrate this method has great potential to rapidly characterize PEG materials to the extent unachievable by other methods.

The work also introduces a simple and flexible method for modifying a commercial MS instrument with off-the-shelf components. The modifications have effectively coupled the high-performance tandem mass spectrometer with gas-phase ion–molecule reactions together without sacrificing any high performance attributes of the original instrumentation. With many unique capabilities of the instrument, such as ion–mobility separation, the ability to perform ion–molecule reaction inside Synapt has truly expanded the applicability of the instrument. It is conceivable that this configuration can be readily applied to many challenging analytical tasks in pharmaceutical industry.


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