2.1. Effect of Physical State on Thiamine Degradation
The physical states of all solid dispersions, before and after storage treatments, were documented using powder x-ray diffraction (PXRD). Consistent with results from a previous study [
15], and as shown in
Figure 1 and
Figure 2, it was possible to stabilize TClHCl in the amorphous form in the presence of both poly(vinylpyrrolidone) (PVP) and pectin (PEC) polymers. A minimum of 60%
w/
w PVP was needed to amorphize TClHCl (
Figure 1), with less PVP resulting in TClHCl crystallization during or immediately after lyophilization (
Figure 1A). While different ratios of PEC dispersions were not prepared in this study, a minimum of only 40%
w/
w PEC was needed to amorphize TClHCl [
15]. No evidence of thiamine crystallization was found in any of the amorphous vitamin:polymer dispersions, including both PVP and PEC, that were stored at 11% relative humidity (RH) and temperatures ranging from 30–60 °C for 8 weeks (the duration of the chemical stability study) (
Figure 2). Therefore, these environmental conditions were chosen to explore differences in the chemical stability of amorphous versus crystalline thiamine.
As shown in
Figure 3A, significant differences (
p < 0.05) in thiamine degradation were found between TClHCl:PVP solid dispersions containing decreasing vitamin:polymer ratios. As the amount of PVP in the solid dispersion increased, thiamine stability decreased. This trend was most evident in dispersions containing 10% or less of thiamine, wherein the percentage of thiamine remaining differed by over 15% between the dispersions with 10% and 1% thiamine after 56 days (82% and 65% thiamine remaining, respectively). Dispersions in which the TClHCl had crystallized (containing ≤ 70% PVP) were the most stable, supporting the hypothesis that crystalline thiamine is more stable than amorphous thiamine. The TClHCl that had been lyophilized in the absence of a polymer was the most stable and was found to have crystallized during or immediately following lyophilization.
In general, thiamine degraded more when amorphous than when crystalline, with degradation rate depending on vitamin proportion in the dispersion (
Figure 3). Although completely crystalline thiamine was not expected to degrade over the duration of the study, some degradation was still found in samples in which amorphous thiamine had crystallized. These samples, however, had stability trends in which increasing TClHCl content resulted in less thiamine degradation. Although the percentage of thiamine crystallinity was not quantified in this study, differences in PXRD peak intensities between crystalline samples (
Figure 1A) indicated that it was possible that partial crystallization may have been the cause for this observation, with greater amounts of thiamine (and therefore less PVP) resulting in a greater percentage of the less chemically labile crystalline thiamine. That thiamine was most labile and chemically unstable in dispersions containing the lowest vitamin proportions is concerning for foods and supplements, wherein vitamin contents tend to be low in proportion to polymers and other ingredients.
Thiamine degradation has been reported to follow first-order reaction kinetics in foods and solutions [
12,
17,
18], which is consistent with what was found for thiamine degradation in amorphous TClHCl:PVP dispersions in this study (
Figure 3B). The observed reaction rate constants (k
obs) calculated from linear regressions (R
2 = 0.92−0.96) are reported in
Table 1. The k
obs values were influenced by the proportion of PVP in the solid dispersion, with k
obs increasing as the proportions of PVP increased. The k
obs values in this study varied from those in previous studies of reaction kinetics of amorphous thiamine degradation, likely due to different temperature and humidity storage conditions as well as different excipients in the amorphous matrix [
17,
19,
20,
21]. The t
90 values, or time when 90% of thiamine remained (10% thiamine had degraded), ranged from 17 to 70 days, increasing as TClHCl concentration increased from 1 to 20% in the dispersions (
Table 1).
2.2. Relationship of T−Tg with Thiamine Stability
Thiamine was found to be more stable in crystalline TClHCl physical blends with PVP or PEC than in amorphous solid dispersions at the same vitamin:polymer ratio (
Figure 4A), which is further evidence that amorphous thiamine is more labile and therefore less stable than crystalline thiamine; of note, differences in thiamine stability were also found in different amorphous dispersions (
Figure 3). Differences in chemical stability in different amorphous matrices have been attributed to differences in T
g, wherein a lower T
g is generally associated with decreased stability due to increased molecular mobility, especially when the storage temperature is above the T
g [
14,
22]. This has been shown to affect thiamine stability due to the effect of the excipient on resulting solid dispersion T
g [
19]. Additionally, the magnitude of difference between storage temperature and T
g (T−T
g) also affects molecular mobility: as T−T
g increases, molecular mobility increases, which usually correlates to decreased stability in terms of both physical change and chemical reactivity [
22,
23].
Immediately following lyophilization, the T
g values of samples containing ≤80% PVP were below 60 °C, further decreasing as the amount of PVP decreased because the T
g of the polymer was greater than that of amorphous thiamine (
Table 2). The presence of the PVP polymer inhibited thiamine crystallization (
Figure 2A), even above the T
g for some of the vitamin:polymer ratios studied [
15]. Even when the samples were exposed to 11% RH, which increased the moisture content of the samples and thereby decreased the T
g, PVP still inhibited thiamine crystallization. The scope of this study therefore included formulations and storage conditions that enabled comparisons of thiamine degradation in the amorphous state in both glassy and supercooled liquid matrices. For example, the initial T
g of the 5TClHCl:95PVP solid dispersion was above 60 °C, the highest temperature used in this study, while the T
g following equilibration at 11% RH and 60 °C was lowered to 47 °C, indicating the change of the solid dispersion to the supercooled liquid state (
Table 3). This same dispersion type equilibrated at 11% RH and 30 °C had a T
g of 54 °C and therefore remained in the glassy state (
Table 3). Increasing storage temperature increased thiamine degradation in these dispersions: those stored at 60 °C had the most thiamine degradation after 56 days (76% thiamine remaining), followed by those stored at 50 °C (88% thiamine remaining) (
Figure 4C). Less thiamine degraded in the samples stored at temperatures below the T
g, with 94% thiamine remaining in the samples stored at 30 °C at the end of the study. These results are consistent with the general theory that the glassy state is more stable than the supercooled liquid state of a matrix and the increase of T−T
g further decreases stability [
19,
22].
However, despite having the lowest T
g, thiamine was the most chemically stable in the amorphous 40TClHCl:60PVP dispersions when comparing the samples containing ≤30% TClHCl, and thiamine stability further decreased as the vitamin:polymer ration decreased (
Figure 3). In fact, the dispersion with the highest T
g (1TClHCl:99PVP) (
Table 2) had the highest rate of thiamine degradation (
Table 1). This indicates that factors other than T
g are presumably influencing chemical stability behaviors of amorphous thiamine. Generally, the correlation between sample T
g and chemical stability can only be analyzed in identical samples. If the samples are not identical to each other (e.g., samples prepared with different vitamin:polymer ratios or using a different polymer), other factors, including intermolecular interactions and contact surface area between compounds, could be the major determinant for chemical stability, rather than T
g. Numerous publications have shown that a higher T
g does not always result in increased chemical stability [
24,
25,
26,
27]. For example, Bell and Hageman [
24] investigated aspartame stability in two different PVP polymers possessing different T
g values in systems with similar moisture contents and water activities (
aw). Although the PVP T
g values were different, no difference was found in degradation rate constants for aspartame [
24]. Moreover, a recent study has shown that ascorbic acid stability was actually higher in the samples with lower T
g values than those with higher T
g values, wherein chemical degradation occurred in the glassy state [
27]. Therefore, the T
g values of the solid dispersions in this study were not considered to be the driving factor for differences in the chemical stability of thiamine across formulations, but T−T
g did correlate with the effect of storage temperature on thiamine stability in the same matrix.
2.3. Effect of Polymer Type on Thiamine Degradation in Amorphous Solid Dispersions
In addition to differences in thiamine chemical stability between amorphous and crystalline matrices, as well as differences in stability due to storage conditions, the type of polymer present in the amorphous matrix was also found to have a significant effect on thiamine stability in the amorphous state (
Figure 4). Significantly more (
p < 0.05) thiamine degraded in the presence of PVP than when formulated with PEC in the solid dispersions. A series of studies was undertaken to better understand why thiamine was more stable in dispersions with PEC than in dispersions with PVP, encompassing pH, moisture content, T
g, and intermolecular interactions.
Sample pH has been reported to be an important factor affecting thiamine stability in solutions [
12,
18]. As shown in
Table 4, the pH levels of pre-lyophilized solutions of 5TClHCl:95PVP and 5TClHCl:95PEC were 3.8 and 3.5, respectively, which are both below the food-relevant pK
a of thiamine (4.8). A speciation plot of thiamine illustrates that the more stable protonated (pyrimidine N1) species was dominant at both of these pH values [
12]. Although the fraction of protonated species was slightly higher in 5TClHCl:95PEC solutions (~95%) than in 5TClHCl:95PVP solutions (~91%), this slight difference in fraction of protonated species was not likely to have caused the substantial difference in chemical stability between PEC- and PVP-based dispersions, with the dispersions containing 96% and 76% thiamine, respectively, following 56 days of storage at 11% RH and 60 °C (
Figure 4). Additionally, it has been reported that pre-lyophilized solution pH does not always correlate to pH of the amorphous solid dispersion [
28]; thus, pH was not considered the major factor influencing the differences in thiamine stability between PEC- and PVP-based dispersions.
Moisture sorption isotherms of individual ingredients, TClHCl:polymer solid dispersions, and physical mixtures of TClHCl with polymers generated at 25 °C are compared in
Figure 5A–C. When considering the individual ingredients, significantly more moisture was absorbed by PVP when compared to PEC, especially above 30% RH, and the difference in sorbed moisture increased as RH increased (
Figure 5A). Solid vitamin:polymer dispersions followed the same general trend as individual polymers for moisture sorption, wherein PVP dispersions sorbed significantly more moisture than PEC dispersions, regardless of the polymer amount used (
Figure 5B). This can be attributed to the more hygroscopic nature of PVP compared to PEC. At low-RH conditions such as those used in this study (11% RH), however, no significant differences in moisture sorption between the solid dispersions were found. For example, significantly more thiamine (20% more) degraded in 5TClHCl:95PVP dispersions than 5TClHCl:95PEC dispersions at 11% RH and 60 °C after 56 days of storage, but no significant difference in the amount of moisture gained by the dispersions of 5TClHCl:95PVP and 5TClHCl:95PEC at 11% RH was found. However, moisture sorption profiles were conducted at 25 °C, so the increased temperature should also be considered. Based on the moisture sorption findings in this study, no direct relation was found between the chemical stability of thiamine and hygroscopicity of polymers/dispersions at the low-RH condition studied.
Different polymers have different T
g values, with PVP having a higher T
g (134 °C) than PEC (90 °C) [
15]. Based on these values, the T
g values of PVP dispersions were expected to be higher than T
g values of PEC dispersions of the same vitamin:polymer ratios prior to storage. However, TClHCl degraded more in PVP dispersions than in PEC dispersions (
Figure 4A). Additionally, more thiamine degraded in PVP dispersions with the highest T
g values (and highest proportions of polymer) (
Table 1 and
Table 2;
Figure 3A). Therefore, differences in T
g were not considered to be the reason for the differences in the chemical stability of TClHCl in these solid dispersions.
Chemical structures and properties of polymers and TClHCl were also considered for their relations to chemical stability. Structures of thiamine and the polymers (
Figure 6) were extensively reviewed by Arioglu-Tuncil, Bhardwaj, Taylor, and Mauer [
15] in terms of functional groups (and hydrogen bond acceptor group strengths based on the p
KBHX scale), which may be involved in hydrogen bonding. Thiamine is comprised of a thiazole and a pyrimidine ring, which are connected to each other via a methylene bridge. Free hydroxyl and NH
2 groups of TClHCl can act as both hydrogen bond donors (HBDs) and acceptors (HBAs). In addition, the nitrogen atoms on the pyrimidine ring can interact via hydrogen bonding as weak HBAs [
15]. PEC possesses a variety of functional groups, including hydroxyl, carboxylic acid, ether, and ester groups, that can all participate in hydrogen bonding. On the other hand, the only functional group on PVP able to hydrogen bond is the amide carbonyl group, which acts as a strong HBA [
15].
This difference in hydrogen bonding potential between PEC and PVP with TClHCl has been shown previously using Fourier transform infrared spectroscopy (FTIR), as related to amorphization capabilities of the polymers [
15]. Briefly, peak shifts to lower wavenumbers in FTIR spectra of vitamin:polymer dispersions compared to polymers alone indicate stronger/more extensive intermolecular interactions. In the mentioned study, TClHCl:PEC dispersions were found to have peak shifts up to 148 cm
−1 in the hydroxyl region, indicating very strong hydrogen bonding interactions between PEC and TClHCl [
15]. Moreover, TClHCl:PEC dispersions were also found to have peak shifts up to 18 cm
−1 in the carbonyl region compared to only 1 cm
−1 peak shifts in the TClHCl:PVP dispersions, indicating comparatively stronger intermolecular interactions between TClHCl and PEC than between TClHCl and PVP [
15]. TClHCl also has the potential for ionic interaction with the carboxylic group in PEC. The stronger hydrogen bonding and/or ionic interactions in TClHCl:PEC dispersions compared to PVP led to greater physical stability of amorphous thiamine shown in that study [
15]. The intermolecular interactions between thiamine and polymers in the solid dispersions were therefore also proposed as the explanation for the greater chemical stability of TClHCl:PEC amorphous solid dispersions compared to TClHCl:PVP dispersions, wherein PEC protected thiamine against chemical degradation by restricting the molecular mobility of TClHCl (higher molecular mobility resulted in an increased degradation rate). A similar observation was also reported by Ismail and Mauer [
29], who found that stronger intermolecular interactions between PVP and ascorbic acid than between PVP and sodium ascorbate were likely a contributing factor for the higher stability of ascorbic acid in these PVP-based amorphous solid dispersions. Due to lack of interactions between TClHCl and PVP in the amorphous solid dispersions in this study, thiamine was significantly less stable in PVP dispersions than in PEC dispersions that had more vitamin–polymer intermolecular interactions.
2.4. Chemical Stability of Thiamine Related to Polymer Proportion in a Solid Dispersion
Thiamine had decreased stability in solid dispersions containing higher proportions of PVP compared to those with less polymer. For example, solid dispersions containing 20TClHCl:80PVP, with a t
90 of 70 days, were more stable than those composed of 1TClHCl:99PVP, which had a t
90 of 17 days (
Table 1). This finding is consistent with results from a study of ascorbic acid in which the chemical stability of amorphous ascorbic acid in PVP dispersions was found to decrease during storage at 11% RH and 60 °C as the relative proportion of PVP in the dispersions increased [
27]. This observation was attributed to a kinetic model developed by Waterman et al. [
30] in which solid state degradation is directly related to the relationship between the drug and the excipient. This kinetic model proposes that if the numbers of drug and excipient particles are comparable to each other, drug concentration does not play a role in its degradation rate. In contrast, if the excipient particles are found in excess in the system, the drug-to-excipient ratio has an effect on the drug degradation rate due to greater surface area of contact between the drug and the excipient (degradation rate increases with increasing contact surface area) [
30]. The increased surface area, in turn, enables a greater extent of drug–excipient interactions, which are detrimental to chemical stability. This mechanism can be applied to the PVP-based solid dispersion chemical stability data, in which thiamine degradation increased in TClHCl:PVP dispersions as the number of PVP molecules increased relative to that of TClHCl. The increase in PVP content occurs concurrently with more thiamine–polymer interactions, at the expense of thiamine–thiamine interactions, where such interactions are clearly detrimental to thiamine chemical stability. Thus, the chemical stability of thiamine in systems containing different proportions of thiamine to polymer was presumably largely dictated by the extent of molecular interactions between thiamine and polymer.