Acid-catalyzed inversion of sucrose in the amorphous state at very low levels of residual water

Article abstract of DOI:10.1023/A:1007517526245

Purpose. Factors affecting the solid-state acid-catalyzed inversion of amorphous sucrose to glucose and fructose in the presence of colyophilized citric acid, with less than 0.1% w/w residual water, have been studied. Methods. Samples of citric acid and sucrose were lyophilized at a weight ratio of 1:10 citric acid:sucrose from solutions with initial pH values of 1.87, 2.03, and 2.43, as well as at a weight ratio of 1:5, at an initial pH of 1.87. Glass transition temperatures, Tg, were measured by DSC and the presence of any possible residual water was monitored by Karl Fischer Titrimetry. The inversion of sucrose was measured by polarimetric analysis after reconstitution of solid samples stored at 50°C under P₂O₅. Results. Samples of 1:10 citric acid:sucrose at an initial pH of 1.87, 2.03, and 2.43 exhibited the same Tg. The initial rate of reactivity was affected at a 1:10 ratio by the solution pH before lyophilization in the order: 1.87 > 2.03 > 2.43 and by citric acid concentration at pH 1.87 in the order 1:5 > 1:10. Conclusions. Sucrose, colyophilized with an acid such as citric acid, undergoes significant acid-catalyzed inversion at 50°C despite the very low levels of residual water, i.e., <0.1% w/w. At the same ratio of citric acid to sucrose (1:10), and hence the same Tg, the rate of reaction correlates with the initial solution pH indicating that the degree of ionization of citric acid in solution is most likely retained in the solid state. That protonation of sucrose by citric acid is important is shown by the direct relationship between maximum extent of reaction and citric acid composition. It is concluded that colyophilization of acidic substances with sucrose, even in the absence of residual water, can produce reducing sugars capable of further reaction with other formulation ingredients susceptible to reaction with reducing sugars.

Full text of DOI:10.1023/A:1007517526245

Pharmaceutical Research, Vol. 17, No. 3, 2000
Research Paper
appeared to occur; however, with the sucrose system, the origi-
nal white cake became progressively colored and eventually
collapsed to a very small volume. Based on previously reported
studies with acidic solutions of sucrose (1), and particularly
with freeze-dried sucrose at acidic pH conditions and in the
presence of residual water (2–4), it was concluded that sucrose
most likely had undergone acid-catalyzed inversion to glucose
and fructose, both reducing sugars. The coloration during this
process was believed to be the result of additional reactivity
between the drug and reducing sugars that produced non-enzy-
matic browning (5,6). The acidity needed to catalyze this reac-
tion most likely came from the acidic nature of the drug.
That such a reaction can occur in the presence of acidic
species for amorphous sucrose at low levels of water, as most
likely observed above, was shown by Karel & Labuza with
lyophilized formulations of sucrose, citric acid, and microcrys-
talline cellulose stored at 55ЊC with a water content as low
as 0.22% w/w, in the weight ratio of sucrose:microcrystalline
Acid-Catalyzed Inversion of Sucrose
in the Amorphous State at Very Low
Levels of Residual Water
Evgenyi Y. Shalaev,1 Qun Lu, Maria Shalaeva,
,2
3
1,2
and George Zografi1
,4
Received July 28, 1999; accepted December 7, 1999
Purpose. Factors affecting the solid-state acid-catalyzed inversion of
amorphous sucrose to glucose and fructose in the presence of colyophi-
lized citric acid, with less than 0.1% w/w residual water, have been
studied.
Methods. Samples of citric acid and sucrose were lyophilized at a
weight ratio of 1:10 citric acid:sucrose from solutions with initial pH
values of 1.87, 2.03, and 2.43, as well as at a weight ratio of 1:5, at
an initial pH of 1.87. Glass transition temperatures, Tg, were measured cellulose:citric acid of 30:2:1 (6). In the absence of citric acid
by DSC and the presence of any possible residual water was monitored negligible reaction took place at 55ЊC over the same time period.
by Karl Fischer Titrimetry. The inversion of sucrose was measured by It was concluded that at amounts of residual water well below
polarimetric analysis after reconstitution of solid samples stored at
the so-called “BET-monolayer” level, as estimated from water
5
2 5
0ЊC under P O .
vapor sorption isotherms (7), water still played an important
role in the reaction mechanism. How this might occur at such
low levels, and how citric acid might be mechanistically
involved was not considered further.
Results. Samples of 1:10 citric acid:sucrose at an initial pH of 1.87,
.03, and 2.43 exhibited the same Tg. The initial rate of reactivity was
2
affected at a 1:10 ratio by the solution pH before lyophilization in the
order: 1.87 Ͼ 2.03 Ͼ 2.43 and by citric acid concentration at pH 1.87
in the order 1:5 Ͼ 1:10.
Water in such an amorphous system can be expected to
Conclusions. Sucrose, colyophilized with an acid such as citric acid, have at least 3 important functions in this reaction. It can act
undergoes significant acid-catalyzed inversion at 50ЊC despite the very as a reactant, as a plasticizer to increase molecular mobility
low levels of residual water, i.e., Ͻ0.1% w/w. At the same ratio of and as a medium supporting acid-base reactions involved in
citric acid to sucrose (1:10), and hence the same Tg, the rate of reaction
correlates with the initial solution pH indicating that the degree of
ionization of citric acid in solution is most likely retained in the solid
not have expected that such an extensive chemical change could
state. That protonation of sucrose by citric acid is important is shown
by the direct relationship between maximum extent of reaction and
citric acid composition. It is concluded that colyophilization of acidic
substances with sucrose, even in the absence of residual water, can
produce reducing sugars capable of further reaction with other formula- even in the presence of 0.1–0.2% water, storage at 50ЊC would
the overall chemical mechanism (8). Given the very low levels
of water in our system, Ͻ0.1%, relative to sucrose, one might
occur due to reaction with water over a relatively short time
period, i.e., 2–4 weeks at around 50ЊC. Given that amorphous
sucrose has a glass transition temperature of about 74ЊC (9),
tion ingredients susceptible to reaction with reducing sugars.
be at a temperature well below the glass transition temperature,
Tg, (spirapril HCl has a Tg of about 85ЊC and would be expected
to slightly raise the Tg in a mixture with sucrose (10)). Thus,
we would not expect a significant role of water at such low
levels as a plasticizer. This suggests that “pH” effects at such
low levels of water in these systems are most likely the most
important factors. This suggestion is supported by the work of
Richards who reported studies on the thermal degradation of
sucrose in the melt over the temperature range of 120ЊC to
KEY WORDS: amorphous; sucrose; acid-catalyzed; residual water;
solid-state degradation.
INTRODUCTION
During the course of carrying out experiments with rela-
tively dry (Ͻ0.1% w/w water) freeze-dried amorphous mixtures
of an organic drug molecule, spirapril HCl, (1,4diatha-7-
azaspirol[4,4]nonane-8-carboxylic acid, 7-[2[[1-ethoxycarbo-
nyl)-3-phenylpropylamino]-1-oxopropyl]-monohydrochloride)
and either sucrose or trehalose in a 1:10 weight ratio of drug
to sugar, certain interesting observations were made after storing
these samples at 50ЊC for up to about one month. In the case of
the trehalose system, no apparent physical or chemical changes
180ЊC for a period of about 400 minutes in the complete absence
of water (11). It was concluded that traces of initial degradation
reactions produced acidic substances, such as acetic, formic, and
levilinic acids, which in turn protonated sucrose and facilitated
degradation. Thus, it seems reasonable that in the present case
the role of any acid present might be enough to explain the
results of studies carried out in the range of 50Њ–55ЊC in the
absence of any significant amounts of water, if there was suffi-
cient molecular mobility of sucrose and acid at the temperature
of the reaction.
1
School of Pharmacy, University of Wisconsin-Madison, 425 N. Char-
ter St., Madison, Wisconsin 53706.
Present address: Pfizer, Inc., Groton, Connecticut.
Present address: Pharmacia & Upjohn, Co., Kalamazoo, Michigan.
To whom correspondence should be addressed. (e-mail: catalyzed inversion at 50ЊC under the conditions of very low
2
3
To examine the tendencies of sucrose to undergo acid-
4
gzografi@facstaff.wisc.edu)
water content and to examine the role of any acid present,
0
724-8741/00/0300-0366$18.00/0 ᭧ 2000 Plenum Publishing Corporation
366

Acid-Catalyzed Inversion of Sucrose in the Amorphous State
367
we have replaced spirapril with citric acid. We have prepared (Seiko Instruments, Horsham, PA). The instrument was cali-
samples at a citric acid:sucrose weight ratio of 1:10 by lyophili- brated for temperature using the melting point of high purity
zation with different solution concentrations of citric acid and indium, tin, and gallium. Samples of 3–13 mg were placed
sucrose so as to bring the initial pH to values of 1.87, 2.05, into hermetically sealed pans under dry-box conditions of low
and 2.43 while keeping the composition of citric acid and relative humidity and a nitrogen atmosphere. Typically, samples
sucrose at the 1:10 level. This was of interest since it had were cooled to Ϫ20ЊC and heated at a rate of 10ЊC/min to
recently been suggested that lyophilized proteins exhibited “pH 95ЊC, in order to determine the onset Tg, the point at which a
memory” of the aqueous solution from which they were lyophi- change in heat capacity first occurs. This will be referred to as
lized and its initial pH conditions (12). A study was also carried the first scan. Generally, this heating and cooling cycle was
out using a 1:5 molar weight ratio of citric acid to sucrose repeated once more to give what will be referred to later as a
where the solution was adjusted to pH 1.87.
second scan.
Kinetic studies of chemical reactivity were carried out in
open 2 ml vials in desiccators over phosphorus pentoxide under
vacuum at 50Њ Ϯ 1ЊC using a laboratory oven. Vials were
MATERIALS AND METHODS
Sucrose (Mallinckrodt, AR grade) and citric acid monohy- removed periodically and subjected to DSC and XRD analysis,
drate (Mallinkrodt, USP grade) were used as received. Water as described above, and to polarimetric analysis in solution as
used for lyophilization and polarimetric analysis was deionized described below. The possible effects of any phosphoric acid
using a Barnstead water purification system. Two solid citric formed during storage in causing chemical changes of sucrose
acid:sucrose weight ratios of 1:10 and 1:5 were prepared by were addressed by periodically changing the P O during long-
2
5
lyophilization of aqueous solutions of citric acid and sucrose. term storage. Also we found no decomposition of pure sucrose
One group of solutions containing 31.6% and 40.1% w/w of over the entire storage period and no solution pH change before
total solids, representing 1:10 and 1:5 ratios, respectively, were and after freeze drying, thus supporting the conclusion that no
adjusted to pH 1.87. In addition, two other solutions at a 1:10 such effects occurred.
ratio were prepared with 19.4% and 5.5% total solids, to give
The inversion of sucrose to glucose and fructose was fol-
initial solution pH values of 2.05 and 2.43, respectively. Freeze- lowed with time using a Perkin-Elmer model 121 polarimeter.
drying was performed in a Dura-Stop DC freeze-dryer (FTS It had been shown previously that results using polarimetry to
Systems, Stone Ridge, NY). Solutions were transferred into follow this reaction in solution and with solid materials con-
petri dishes and frozen with liquid nitrogen immediately after taining residual water were consistent with those obtained using
the solutions were made to minimize sucrose inversion in solu- other methods (13). Weighed amounts of sample were dissolved
tion. The frozen solutions were placed in the freeze-drier at in water to obtain about a 3% w/w solution. The extent of
Ϫ40ЊC, immediately after which vacuum was applied. At the sucrose inversion, x, was calculated using the following
residual pressure of 50–70 mT, the temperature was maintained equation:
at Ϫ40ЊC for 4 days and then increased to 25ЊC at about 5Њ/
␣s Ϫ ␣
␣s Ϫ ␣i
day for at least 5 more days. Analysis of samples for any
chemical conversion of sucrose during freeze drying indicated
no significant degradation and there was no collapse of the
freeze-dried cake. The lyophilized material was gently ground
with a mortar and pestle, placed into 2 ml vials, and dried over
phosphorus pentoxide at room temperature in a desiccator under
vacuum for at least 6 days. The material was then placed in a
desiccator over phosphorus pentoxide under vacuum for 1 day
at 50ЊC to completely dry the samples. All operations with
freeze-dried materials were performed in a glove box under
nitrogen flow. Residual water content for all solid samples
was determined by the Karl Fischer method using an Aquastar
C2000 coulometric titrator (EM Science, Cherry Hill, NJ). It
was shown that all samples treated in the manner described
above contained no more than 0.1% water, the lower limit of
sensitivity of this technique.
x ϭ
(1)
where ␣ is the specific rotation of the 100% sucrose sample,
equal to 66.4 (14); ␣ is the specific rotation of the sample under
consideration, and ␣ is the specific rotation of the pure invert
sugar, equal to Ϫ19.7 (15). Specific rotation of the sample was
s
i
calculated as
1
00 ␣c
␣
ϭ
(2)
LCS
where ␣ is the instrument reading, C is the weight percent
c
S
of solute in the solution, and L is the length of the cuvette.
In general the optical rotation of the solution samples,
representing various extents of chemical reaction, decreased
with time and asymptotically approached a constant value after
about 2–3 hours at room temperature. These changes are due
to the mutorotation of the fructose and glucose produced as
the result of sucrose inversion (13). The extent of sucrose
inversion, therefore, was calculated using solutions maintained
for two or more hours after dissolution of the solid sample in
the deionized water, to correct for the mutorotation lag. The
relative uncertainty in the determination of the extent of chemi-
cal changes is estimated to be within one percent.
Powder X-ray diffraction patterns were obtained with a
PadV, Scintag Powder X-ray Diffractometer (Scintag, Inc.,
Santa Clara, CA) controlled by a computer (Model B10610,
Tetronix, Inc., Wilsonville, OR). The radiation used was gener-
˚
ated by a copper K␣ filter with a wavelength of 1.5418 A at
5 kV and 40 mA. Samples were scanned from 5Њ to 40Њ 2␪
4
at a scanning rate of 5Њ per minute. In all cases initial and
reacted samples exhibited no diffraction peaks and had the halo
characteristic of a completely amorphous phase.
Differential Scanning Calorimetry (DSC) measurements
were carried out on all amorphous samples to measure the glass
RESULTS
Table I lists the various samples used in this study, includ-
transition temperature, Tg, using a Seiko SSC/5200 instrument ing the citric acid:sucrose weight ratio, initial solution pH, the

3
68
Shalaev, Lu, Shalaeva, and Zografi
Table I. Sample Composition and Tg in Colyophilized Sucrose:Citric
Acid Mixtures
Tg,a Tg, Tg calculated from (Eq. 3)
Citric acid:
Initial 1st
2nd using second scan data for
sucrose wt ratio
pH
scan scan
sucrose Tg (ЊC)
0
1
1
1
1
:1
5.87
1.87
2.05
2.43
1.87
80
68
68
69
69
72
57
57
58
52
—
66
66
66
61
:10
:10
:10
:5
a
Annealed at 50ЊC for 24 hours prior to measurement.
glass transition temperature measured from the first DSC scans
after annealing samples at 50ЊC for one day and a Tg obtained
from a second scan. Usually, a second scan is used to report
Tg since the first scan eliminates the previous history of the
sample and allows uniformity in reported values. However,
in this study, chemical reactivity occurs in samples having a
particular thermal history, i.e., in samples annealed at 50ЊC,
which is below Tg. Hence, it is believed to be important to
report values of Tg for both annealed and unannealed samples.
The Tg for pure amorphous sucrose, treated in this manner was
in good agreement with previous results (9). The Tg of pure
Fig. 1. Kinetic curves for sucrose:citric acid freeze-dried mixtures.
citric acid has been reported to be 11ЊC (16). As can be seen Results for pure sucrose (⌬) are also given for comparison. ▫:
in Table 1, the two citric acid:sucrose mixtures had lower values sucrose:citric acid ϭ 10:1, freeze-dried from solution with pH 2.4; ●:
of Tg than sucrose, indicating the plasticizing effects of citric sucrose:citric acid ϭ 10:1, freeze-dried from solution with pH 2.05;
⅙
: sucrose:citric acid ϭ 10:1, freeze-dried from solution with pH 1.87;
acid. Also listed in Table 1 are estimates of Tg for these mixtures
assuming ideal mixing behavior, as reflected in the Gordon
Taylor equation (17) where
ࡗ: sucrose:citric acid ϭ 5:1; freeze-dried from solution with pH 1.87.
Lines present fitting of the experimental data using Eq. 5.
w Tg ϩ Kw Tg
2
1
1
2
Tg ϭ
(3)
w ϩ Kw2
1
DISCUSSION
and
The results of these experiments would indicate that citric
(4) acid has a major effect on this reaction in the absence of a
Tg1␳1
Tg2␳2
K Ϸ
significant amount of water. The influence of the acid on this
where w and w are weight fractions and ␳ and ␳ are densities reaction is consistent with conclusions made from studies in
1
2
1
2
of each component. The density of amorphous sucrose is 1.43 solution and the melt (1,11,19) that acidic species are directly
Ϫ3
Ϫ3
gcm (9) and that of amorphous citric acid is 1.61 gcm (16). involved in the inversion of sucrose. The prevailing view, as
It can be seen that the experimental values of Tg are lower seen in Scheme 1, therefore, would suggest that in solution
than those predicted by equation 3, indicative of the non-ideal a rapid pre-equilibration protonation (step 1) followed by a
mixing of the two components in the molecular dispersion unimolecular rate-determining splitting of the glycosidic bond
formed by lyophilization (18).
(step 2) and conversion to glucose and fructose (step 3) occurs:
The kinetic curves for the loss of sucrose with time, taken
as an average of at least two independent samples, are shown
in Fig. 1. Here, it can be seen that significant degradation occurs
at 50ЊC, for the 1:10 citric acid:sucrose samples at 3 initial
solution pH values; the rates increase as the initial solution pH
decreases. Also included in Fig. 1 are the results obtained for
amorphous sucrose, prepared in the same manner, but without
citric acid present. In this case no chemical change occurred
over comparable time periods. In Fig. 1 we also present a
comparison of the changes occurring with the 1:10 and 1:5
citric acid-sucrose samples, lyophilized from a pH 1.87 solution.
Here it can be seen that the rate of sucrose reactivity is greater
the higher the citric acid concentration. It is also apparent from
Scheme 1
+
+
1) H ϩ S s SH
+
+
2
) SH → GH ϩ F
or
+
+
SH → FH ϩ G
+
+
3
) GH ϩ H O → G ϩ H
2
or
+
+
FH ϩ H O → F ϩ H
2
Fig. 1 that the maximum extent of reaction was greater in the where S, G, and F represent sucrose, glucose, and fructose,
mixture with the higher citric acid level. respectively.

Acid-Catalyzed Inversion of Sucrose in the Amorphous State
369
The species formed in step 2 can be either glucose or
It should be pointed out that this kinetic analysis is over-
fructose depending on the site of bond cleavage. Glycosyl- simplified, since while it allowed us to explain the greater extent
oxygen cleavage will produce fructose and glucose ion, whereas of reaction in the mixture with a higher citric acid (and higher
fructosyl-oxygen cleavage will produce glucose and fructose proton) level, and the similar values of Xm for all 1:10 samples,
ion. Both processes have been shown to occur in solution (20). it does not explain why the initial rate of reactivity was higher
According to this scheme, therefore, water does not participate in the citric acid:sucrose 1:5 mixture. Consequently, our results
as a direct reactant in sucrose cleavage, but it does participate can only be used to compare rates and maximum extents of
in the later conversion of the carbonium ions into neutral reactivity and we must be cautious about speculations concern-
monosaccharides.
ing exact mechanisms. From this analysis, we can conclude
To compare the rates of reactivity in samples with different that the maximum extent of the reaction is directly related to
sets of conditions, we have carried out the following kinetic the amount of citric acid present, presumably a limitation on
analysis. In solution, sucrose inversion is generally described the amount of protonated sucrose that can form. The generally
by a first-order equation where the experimental rate constant enhanced rate of reactivity likewise at the same “pH,” but with
+
is equal to a constant k times [H ] (1). It is usually assumed a different ratio of citric acid present, again, most likely reflects
+
that in solution [H ] is constant during the reaction, but this the role of citric acid in protonating sucrose.
undoubtably is not the case in the present study where citric
From Table I we note that the citric acid-sucrose samples
acid is most likely the major and limited source of protons. In all have about the same first-scan Tg values and, therefore, on
Scheme 1, we see the importance of citric acid in producing this basis alone they would be expected to have similar rates of
protonated sucrose and the importance of water in facilitating solid-state reactivity if molecular mobility was the predominant
the final conversion of monosaccharide carbonium ions. Conse- factor. However, from Fig. 1 and Table II it is apparent that
quently, the amount of citric acid present in the sample initially initial rates as reflected in the constant k are quite different,
in the presence of negligible amounts of water should limit the which we believe illustrates the importance of medium effects
extent to which this reaction can proceed and, indeed, from the on chemical reactions in the amorphous state, in general.
curves in Fig. 1 it can be seen that the extent of reaction over
the entire time frame is greater the higher the level of citric
acid at the same initial solution pH before freeze drying.
The effect of citric acid as a limiting factor for the maxi-
mum extent of reaction can be expressed in a more quantitative
basis by considering the overall reaction to consist of several
parallel and consecutive elementary reactions. To simplify the
analysis, we assume that unimolecular cleavage of sucrose is
the rate-determining step as has been known to be true in
solution (19). Furthermore, we assume that sucrose cleavage
is an irreversible reaction and that the maximum extent of
conversion is determined by the extent of sucrose protonation
possible. In other words, we assume that there are two groups
of sucrose molecules present, the protonated species capable of
reacting by first-order kinetics, and a nonreactive nonprotonated
species. This allows us to express the extent of reaction, ␣ at
any time, t, as
CONCLUSIONS
This study has shown the significant sensitivity of amor-
phous sucrose to degradation in the presence of a molecularly
dispersed acidic solid at temperatures as low as 50ЊC and in
the presence of negligible amounts of residual water. It is further
shown that the initial rate of reactivity depends on the pH of
the solution used to prepare the lyophilized sample and that
the maximum extent of reaction is limited by the amount of
acid present and, most likely, its role in the formation of an
important intermediate, the protonated sucrose molecule. Con-
sequently, even in the relatively dry state, lyophilized samples
containing sucrose and some acidic ingredients are susceptible
to chemical degradation under conditions of storage that could
easily be encountered during stability assessment and the shelf-
life of the product.
␣
(t) ϭ X exp (Ϫ kt) ϩ (1 Ϫ X )
(5)
m
m
where Xm is the weight fraction of protonated sucrose (SH+)
representing the maximum extent of reaction, and k is a rate
constant characterizing the rate of reactivity. Table II presents
ACKNOWLEDGMENTS
the values of k and X estimated for data presented in Fig. 1.
m
The authors acknowledge the financial support of the
Purdue/Wisconsin Program on Physical and Chemical Stability
of Pharmaceutical Solids and the technical assistance of David
Albers and Ping Tong.
Fits to equation 5 were obtained with k and X as adjustable
m
parameters using a Microcal Origin software package.
Table II. Rate Constants (k) and Maximum Extents of Reaction (Xm)
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