American Journal of Anal yt ical Chemistry, 2011, 2, 164-173
doi:10.4236/ajac.2011.22019 Published Online May 2011 (
Copyright © 2011 SciRes. AJAC
Solid State Characterization of Sodium Eritadenate
Josefine Enman1, Anuttam Patra1, Kerstin Ramser2, Ulrika Rova1, Kris Arvid Berglund1,3
1Department of Chemical Engineering and Geosciences, Luleå University of Technology, Luleå, Sweden
2Department of Computer Science and Electrical Engineering, Luleå University of Technology, Luleå, Sweden
3Departments of Forestry and Chemical Engineering & Materials Science, Michigan State University,
East Lansing, USA
Received November 22, 2010; revised January 29, 2011; accepted February 10, 2011
Knowledge of the solid state is of great importance in the development of a new active pharmaceutical in-
gredient, since the solid form often dictates the properties and performance of the drug. In the present study,
solid state characteristics of the sodium salt of the candidate cholesterol reducing compound eritadenine,
2(R), 3(R)-dihydroxy-4-(9-adenyl)-butanoic acid, were investigated. The compound was crystallized by slow
cooling from water and various aqueous ethanol solutions, at different temperatures. Further, the compound
solution was subjected to lyophilization and to high vacuum drying. The resulting solids were screened for
polymorphism by micro Raman spectroscopy (λex = 830 nm) and the crystallinity was investigated by X-ray
powder diffraction. Further, thermal analysis was applied to study possible occurrence of solvates or hy-
drates. Solids obtained from slow cooling showed crystallinity, whereas rapid cooling gave rise to more
amorphous solids. Analysis of difference spectra of the Raman data for solids obtained from slow cooling of
solution revealed subtle differences in the structures between crystals derived from pure water and crystals
derived from aqueous ethanol solutions. Finally, from the thermal analysis it was deduced that crystals ob-
tained from pure water were stoichiometrically dihydrates whereas crystals obtained from aqueous ethanol
solutions were 2.5 hydrates; this formation of different hydrates were supported by the Raman difference
Keywords: Sodium Eritadenate, Solid state Chemistry, Raman Spectroscopy, Thermal Analysis,
Crystal Hydrates, Polymorphism
1. Introduction
Cardiovascular disease is one of the most serious health
concerns in Western society and hypercholesterolemia is
a well established risk factor for the development of such
disease. To counteract hypercholesterolemia, various
statins, some of which were originally isolated from fun-
gi such as Aspergillus terreus [1], are frequently used.
Another hypocholesterolemic compound of fungal origin
is D-eritadenine, 2(R), 3(R)-dihydroxy-4-(9-adenyl)-but-
anoic acid (Figure 1), found in the shiitake mushroom,
Lentinus edodes[2,3]. The shiitake mushroom has been
shown to lower the blood cholesterol in both animals and
humans[4-6], and the cholesterol reducing mechanism of
eritadenine has been investigated in several studies on
rats [2,3,7-10]. The complete hypocholesterolemic me-
chanism of eritadenine remains to be clarified, but it has
been shown that eritadenine accelerates the removal of
blood cholesterol, with no indications of the compound
inhibiting the biosynthesis of cholesterol [10], unlike the
statins [1]. In search for new cholesterol re- ducing drugs,
eritadenine might have a potential as an
Figure 1. Chemical structure of D-Eritadenine.
active pharmaceutical ingredient, conceivably as a com-
plement to the statins.
Since active pharmaceutical ingredients are commonly
delivered as solids, one key aspect in their development
is research comprising the solid state chemistry of the
compound. The solid form of a pharmaceutical substance
greatly influences its properties and performance, such as
stability and bioavailability, and an understanding of the
solid state in relation to its functional properties is fun-
damental when developing a new drug [11,12]. A given
drug substance can be present in several different solid
forms, commonly as crystalline polymorphs, i.e. solids
with the same elemental composition but different crystal
structures; as solvates, in which solvent molecules are
present in the crystal structure; as desolvated solvates for
which the crystal structure is principally retained upon
solvent loss, or as amorphous solids, which show no or
only partial crystallinity [11]. In the pharmaceutical in-
dustry a variety of methods are applied for production of
solids and which crystal form is obtained depends on the
crystallization conditions applied, e.g. solvent composi-
tion [13] and temperature [14].
The objectives of the present study were to investigate
the effects of solvent composition and temperature on the
crystal structure of the cholesterol reducing compound
eritadenine. For acidic drug products the sodium salts are
commonly the chosen derivatives, because of higher so-
lubility and generally increased biocompatibility [15].
Consequently the sodium salt of eritadenine, sodium
eritadenate, was investigated in the present study. As
solvent systems, pure water and various aqueous ethanol
solutions were used since these are frequently applied in
the pharmaceutical industry. Sodium eritadenate was
slowly crystallized from water and different aqueous
ethanol solutions, at different temperatures, by means of
cooling, and subjected to rapid cooling from water solu-
tions by either lyophilization or drying under high vac-
uum. The resulting solids were collected and analyzed by
X-ray powder diffraction (XRPD), micro Raman spec-
troscopy and thermal analysis, to investigate for crystal-
linity and for the occurrence of possible polymorphs,
solvates or hydrates.
2. Materials and Methods
2.1. Synthesis of Sodium Eritadenate
Sodium eritadenate was synthesized as previously de-
scribed [16]. In summary, methyl 2,3-O-isopropylidene-
-D-ribofuranoside was first synthesized [17] and proc-
essed to methyl 2,3-O-isopropylidene-5-O-p-toluene-su-
-D-ribofuranoside [18]. The latter reacted with
the sodium salt of adenine to give the compound methyl
-D-ribofuranoside, which underwent hydrolysis to 5-
(6-aminopurin-9H-9-yl)-5-deoxy-D-ribofuranose. In the
final step, oxidation of the latter in alkaline media re-
sulted in sodium eritadenate [19].
2.2. Solubility Studies
The solubility of sodium eritadenate in pure water and in
15, 30 and 50% (v/v) aqueous ethanol solutions was stu-
died. The concentrations of sodium eritadenate used
were in the ranges 50 - 300 mg/mL and 5 - 150 mg/mL,
for pure water and aqueous ethanol solutions, respec-
tively. All solubility studies took place in microtiter
wells, which were tightly covered and placed in a Ther-
mo forma orbital shaker, at 200 rpm. The solubility was
observed after 24 hours at 20, 30, 40 and 50˚C for all
solvent systems, and additionally at 60˚C for pure water.
2.3. Crystallization Procedure
Based on the solubility studies, 15 - 60 mg of sodium eri-
tadenate in 200 μl of water, 6 - 30, 3 - 14 and 1 - 12 mg of
sodium eritadenate in 200 μl of 15, 30 and 50% aqueous
ethanol solutions, respectively, were added to microtiter
wells. The wells were tightly covered and the samples
heated and slowly cooled down, using a Thermo forma
orbital shaker. Solid forms obtained at 20, 30, 40 and
50˚C were collected and dried. Eritadenine in pure water
was also subjected to high vacuum drying and to lyophi-
2.4. Characterization of Solids
2.4.1. X-Ray Powder Diffraction Measurements
X-ray powder diffraction data of selected solid samples
were recorded with a Siemens D5000 diffractometer,
using CuKα radiation and variable slits. The samples
were investigated in the 2-theta range 7˚ - 90˚, in Bragg-
Brentano geometry, with a step size of 0.01, for 15
2.4.2. Raman Measurements
The solid state Raman spectra of all solids were recorded
with a Renishaw 2000 micro Raman spectrometer and
approximately 20 mg of sodium eritadenate, on a glass
slide, were used for each measurement. The excitation
wavelength was 830 nm, giving excellent signal strength
while minimizing photo-induced damage. The power
onto the sample was 50 mW and the integration time was
10 sec. For all measurements a 20 long working dis-
tance (LWD) microscope objective was used. Correction
for the energy sensitivity of the spectrometer was per-
formed by measuring the spectrum of a calibrated light
Copyright © 2011 SciRes. AJAC
Copyright © 2011 SciRes. AJAC
source and calculating the intensity wave number re-
sponse curve. Each Raman spectrum was filtered by the
noise-reduction algorithm according to Eilers [20]. The
background was automatically subtracted using the algo-
rithm by Cao et al. [21], which fits a piecewise modified
polynomial to the spectrum, and spectra were vector
normalized to get equal integrated areas. The preproc-
essing algorithms, except the one by Eilers, were written
in-house and implemented in Matlab (version R2007b
including Statistics Toolbox version 6.1). The occurrence
of possible polymorphs of sodium eritadenate was inves-
tigated by examining the ratio of the Raman intensity
lines and by plotting difference spectra between crystals
obtained from water, between crystals obtained from
aqueous ethanol, and by investigating the difference
spectrum between crystals derived from water and crys-
tals derived from ethanol solutions. The Full Width at
Half Maximum (FWHD) of the Raman line that exposed
the most prominent difference, i.e. at 742 cm1, was cal-
culated for several spectra.
2.4.3. Thermal Analysis
Differential scanning calorimetry (DSC).The thermal
behavior of selected solid samples was studied using a
Thermal Advantage DSC Q1000 (TA instrument). The
samples were scanned from 20 to 260˚C, at a heating rate
of 10˚C/min, under nitrogen purge.
Thermogravimetric analysis (TGA). The changes in
sample mass with temperature were measured by ther-
mogravimetric analysis (TGA) using the Thermal Ad-
vantage TGA Q5000 (TA instrument-Waters, LLC) mod-
ule. The samples were heated from 20 to 260˚C, at a
heating rate of 10˚C/min, under nitrogen purge.
Evolved gas analysis-mass spectrometry (EGA-MS).
For identification of the evolved gas on heating, a
Netzsch STA 409 instrument equipped with simultane-
ous thermo-gravimetric (TG), differential scanning calo-
rimetric (DSC) and quadropole mass spectrometric (QMS)
analysis was used. The experiments were con- ducted in
flowing argon while heating from 20 to 260˚C, at a heat-
ing rate of 10˚C/min.
3. Results and Discussion
The solubility of sodium eritadenate was comparatively
high in pure water and virtually insoluble in absolute
ethanol (Figure 2); hence the highest ethanol concentra-
tion used for crystallization in this study was 50% (v/v).
Sodium eritadenate was crystallized from water and
aqueous ethanol solutions, by controlled cooling, and
additionally subjected to rapid cooling from water solu-
tions. The resulting solids were then investigated by
means of X-ray powder diffraction, Raman spectroscopy
and thermal analysis.
X-ray powder diffraction is a powerful technique for
identifying crystalline phases [22] and was employed to
Figure 2. Solubility of sodium eritadenate in pure water (), 15% aqueous ethanol (), 30% aqueous ethanol () and 50% aqueous
ethanol ().
Copyright © 2011 SciRes. AJAC
establish if the solids of sodium eritadenate were crystal-
line or amorphous. The XRPD data presented are in the
2-theta range 10˚ - 50˚, since all distinctive peaks were
found in this range (Figures 3(a)-(c)). These data indi-
cated that solids resulting from slow cooling were crys-
talline and their patterns similar, irrespective of the sol-
vent used (Figures 3(a)-(b)), whereas solids obtained
from rapid cooling gave rise to more amorphous patterns
(Figure 3(c)). However, some crystalline pattern could
be observed for the lyophilized solid and this could be
attributed to small crystals in the amorphous phase. By
optical microscopy studies (data not shown) it was veri-
fied that there were small crystals within the amorphous
Micro Raman spectroscopy is a highly valuable tech-
nique for investigating structural properties of molecules,
such as polymorphism [23,24] and all solid forms of so-
dium eritadenate obtained from the crystallization pro-
cedure in this study were screened for the occurrence of
polymorphism, by Raman spectroscopy. Three different
Raman spectra were recorded for each solid form and the
spectra were background corrected and normalized in
order to optimize comparison. In accordance with the
XRPD measurements (Figure 3), the solids obtained
from slow cooling showed more crystallinity in the Ra-
man spectra (Figures 4(a)-(b)) and the solid forms ob-
tained from fast cooling (Figures 4(c)-(d)) gave rise
more amorphous patterns. The latter is plausible since
amorphous forms are readily obtained by lyophilization
or other means of rapid cooling [25-27]. In some cases,
the amorphous forms of pharmaceuticals are used as
products because of enhanced solubility [28] and occa-
sionally increased bioavailability [29], but generally this
form is not marketed due to lower chemical stability than
the crystalline counterpart [27]. The Raman spectra of all
solids resulting from slow cooling of solution had a sim-
ilar appearance, irrespective of temperature and sol- vent
composition (Figures 4(a)-(b)). Further, they were
highly reproducible and correlated well with the earlier
assignments of the eritadenine Raman spectrum [30].
Based on the Raman intensity ratio, there was no sig-
nificant difference between the different solid forms. In
order to study more subtle differences in the crystal
structures, difference spectra were plotted (Figure 5).
The analysis of the difference spectra revealed small
changes at several Raman lines, i.e. at 742, 1320/1354
Figure 3. X-ray powder diffraction pattern of sodium eritadenate solids obtained by slow cooling from water (a) and by slow
cooling from aqueous ethanol (b).
Figure 4. Raman spectra of sodium eritadenate solids obtained by slow cooling from water (a), by slow cooling from aqueous
ethanol (b), by lyophilization (c), by high vacuum drying (d), by dehydration of A (e) and by dehydration of B (f) . The inset
shows the region between 1000 and 1150 cm–1, where the Raman lines arise from vibrations in the carbon chain moiety. The
upper graph in the inset is from (c), while the lower graph is from (b).
and 1583 cm–1 (breathing and deformation of the ring
structure), as well as at 880, 926 and at 1365 cm–1
(stretching vibrations of the carbon chain). These differ-
ences were negligible when comparing spectra for crys-
tals obtained from water at different temperatures (Fig-
ure 5(a)), and likewise for crystals obtained from differ-
ent aqueous ethanol concentrations (Figure 5(b)). How-
ever, the differences increased significantly when spectra
of crystals derived from water were subtracted from
spectra of crystals derived from aqueous ethanol solu-
tions (Figure 5(c)). This indicates that these two types of
crystals might form slightly different hydrates or solvates
influencing the crystal structure. The Raman line that re-
vealed the most prominent difference was found at 742 cm–1.
When comparing the FWHD of this Raman line for the
spectra of crystals derived from water with the spectra of
crystals derived from aqueous ethanol solutions, a
broadening by 2 cm–1 was found for sodium eritadenate
crystallized from water solutions (Inset of Figure 5).
Narrow bands indicate a rigid structure, hence, it can be
argued that crystals derived from aqueous ethanol solu-
tions are more crystalline than those obtained from pure
Copyright © 2011 SciRes. AJAC
water. A preliminary study with solid state NMR spec-
troscopy supports these results. Analyzing both 13C and
15N SS-NMR spectra for crystals derived from water and
aqueous ethanol solutions, it was found that there are
slight, but noticeable differences in some of the peak
positions, indicating the presence of two different types
of crystals. A more detailed discussion will be published
To investigate the thermal behavior and possible exis-
tence of solvates or hydrates, crystalline solids were stu-
died by thermal analysis. The TGA curve (Figure 6(a))
of sodium eritadenate crystallized from water showed a
mass loss in the temperature range 70˚C - 130˚C, which
was estimated to 12%. The corresponding temperature
Figure 5. Raman difference spectrum for sodium eritadenate crystals obtained from water solutions (a), for sodium eritade-
nate crystals obtained from aqueous ethanol solutions (b) and for crystals of sodium eritadenate obtained from water and
aqueous ethanol solutions (c). A broadening of the peak at 742 cm–1 of 2 cm–1 for water derived crystals is indicated and was
verified by the FWHD, as shown in the inset from (a).
Copyright © 2011 SciRes. AJAC
Copyright © 2011 SciRes. AJAC
range in the DTG curve showed that the mass loss oc-
curred in two sequential steps, and the EGA-MS further
identified it as water loss. The total mass loss calculated
amid the two peaks shown in the DTG curve, suggest
that two water equivalents departed the crystal structure,
one after another. Hence, the water molecules are differ-
ently bound in this type of hydrate, which could be sup-
ported by the crystal structure of sodium eritade- nate
determined previously [31]. Approximately one third of
all active pharmaceutical ingredients are able to form
crystal hydrates [12] and sodium salts of all differ- ent
types of drugs are particularly apt to form crystal hy-
drates [15]. In the DSC curve, the water loss was rep-
resented by an endothermic peak, which was followed by
a sharp endothermic peak starting at about 135˚C. It is
plausible that the latter peak corresponds either to melt-
ing of the compound or to a phase transition, since no
mass change occurred.
In a separate experiment, upon heating the crystals to
160˚C under nitrogen atmosphere, no melting could be
observed. However, by investigating this anhydrous form
by Raman spectroscopy (Figure 4(e)), it could be seen
that there was a transition to amorphous phase upon de-
hydration. Solvent molecules commonly stabilize the
crystal structure and desolvation can thus cause amor-
phous materials [32]. In this case the water molecules act
as stabilizers, due to their incorporation in the crystal
structure [31], and hence dehydration would reasonably
disrupt the crystal structure. Since water is a small mo-
lecule it is apt to fill vacancies and its exceptional hy-
drogen bonding capacity, to other water molecules or-
functional groups, enables the formation of stable crystal
structures [12]. Starting at about 225˚C, an endothermic
peak was observed in the DSC curve (Figure 6(a));
however, at the same temperature a mass change was
seen in the TGA curve. This, combined with carbon di-
oxide formation as shown by EGA-MS, indicated that
decomposition of the compound took place.
Figure 6. Thermal analysis for sodium eritadenate crystals obtained from water solutions (a) and from aqueous ethanol solu-
tions (b).
Copyright © 2011 SciRes. AJAC
For the crystals forming from aqueous ethanol, a mass
loss was detected between 55 - 125˚C in the TGA curve
(Figure 6(b)) and estimated to 14%. The mass loss was
further observed by one broad peak in the DTG curve
and a broad endothermic peak in the DSC curve. The
EGA-MS showed that water was departing the crystals,
whereas no ethanol could be detected. Hence, in these
types of crystals there are reasonably 2.5 molecules of
water per molecule sodium eritadenate, according to the
mass loss. As stated above, sodium salts of drug mole-
cules are prone to form hydrates. It should also be em-
phasized that, due to a low ability of forming multi-point
hydrogen bonding, ethanol is distinguished by a low li-
kelihood to be incorporated into solvates [33]. Accom-
panying the water loss, a diffuse endothermic peak could
be observed in the DSC curve. Again, upon heating the
crystals to 160˚C, under nitrogen atmosphere, and inves-
tigating the anhydrous form by micro Raman spectros-
copy (Figure 4(f)) it could be seen that there was a tran-
sition to amorphous phase upon dehydration. Starting
around 235˚C, a sharp endothermic peak was detected in
the DSC curve (Figure 6(b)). Since there was no mass
loss at this temperature, the compound was plausibly
The thermal analysis of crystals derived from water
and from aqueous ethanol solutions indicated the former
as dihydrates and the latter as 2.5 hydrates. These two
types of hydrates showed a subtle difference in crystal
structure, as indicated by micro Raman spectroscopy and
clearly displayed different thermal behavior. A higher
temperature was required for loss of water from the di-
hydrates, and the loss was more stepwise. Based on these
observations it is plausible that one or more water equi-
valents are attached differently and/or more loosely to
the crystal lattice of the 2.5 hydrates, as compared to the
dihydrates. However, since there were no major dis- si-
milarities between the crystal structures of the two types
of hydrates, the difference in water binding is plau- sibly
small and tentatively pertains to secondary water mole-
cules more outside the lattice. Further, for both types of
crystals the water loss resulted in more amor- phous ma-
terials, verifying that water has a stabilizing effect on
both types of hydrates.
The decrease of structural order for the amorphous
materials was especially apparent in the region between
1000 and 1150 cm–1, where the Raman lines are due to
vibrations of the carbon chain moiety (Inset of Figure 4).
Previously carried out polarization measurements show-
ed that a change of the polarization direction by 90˚ re-
sulted in an increase of the lines at 1066 and 1085 cm–1
ascribed to the
(C - O) and
(C - C)[30], indicating an
increase in structural order. The carbon chain moiety has
structural properties that enable certain orientations,
which are stabilized by adjacent water molecules. For
solid states of sodium eritadenate achieved through ly-
ophilization, high vacuum drying or dehydration by
heating above 120˚C, these bands more or less disap-
peared (Figures 4(c)-(f)). Hence, by removing the stabi-
lizing water molecules, the order of the solid pertaining
to the carbon chain moiety is lost and a more amorphous
structure is achieved.
Conclusively, for both types of crystals, water mole-
cules are presumably both highly incorporated into the
crystal lattice and more loosely bound to the crystals.
Further, the additional water molecule for every two
molecules of sodium eritadenate in crystals derived from
aqueous ethanol solutions, as compared to water derived
crystals, is plausibly attached to the crystal lattice, but
not as a part of the crystal structure. The thermal behav-
ior as described above was shown to be highly repro-
ducible for both types of hydrates.
4. Conclusions
The solid forms of sodium eritadenate, as crystallized
from water or aqueous ethanol solutions, at various tem-
peratures and cooling methods, were investigated. Solids
forming from slow cooling from either water or aqueous
ethanol showed crystallinity and similar Raman and
XRPD patterns, irrespective of temperature of formation,
whereas rapid cooling resulted in more amorphous solids.
However, a slight difference in the structure between
crystals obtained from water and crystals obtained from
aqueous ethanol could be observed by analyzing the dif-
ference spectrum of the Raman data. Thermal analysis
showed that the former were stoichiometrically dihydrates
whereas the latter were 2.5 hydrates. The difference in
water equivalents had a minor effect on the crystal
structures, as observed by Raman spectroscopy but gave
rise to clearly dissimilar thermal behaviors. Finally, both
anhydrous forms were more amorphous than their hy-
drated counterparts, verifying the stabilizing effect of
water on the crystal structures.
Future studies have to reveal whether there are any
differences between the two hydrates with respect to
stability and bioactivity and hence which solvent system
to use for crystallization. Further, if the amorphous ma-
terial of sodium eritadenate will be shown to exhibit
equal biocompatibility and fulfil stability requirements,
this form might also be considered as an active pharma-
ceutical ingredient. In this case solidification can be
achieved significantly faster.
5. Acknowledgements
We wish to acknowledge Stefan Candefjord for preproc-
essing of the Raman data, Linda Sandström for assis-
tance with XRPD measurements and Amjad Alhalaweh
and Ryan Robinson for assistance with thermal analysis.
JE, UR and KAB acknowledge the financial support of
Innovationsbron, the Research Council of Norrbotten,
and Working Bugs AB. AP acknowledges financial sup-
port from the foundation to the memory of J. C. and Seth
M. Kempe (research grants JCK-2701 and JCK-2905).
KR acknowledges financial support from the Swedish
research council.
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