Didanosine Polymorphism in a Supercritical

Antisolvent Process

R. BETTINI,1 R. MENABENI,1 R. TOZZI,1 M.B. PRANZO,1 I. PASQUALI,1 M. R. CHIEROTTI,2

R. GOBETTO,2 L. PELLEGRINO2
1
Department of Pharmacy, University of Parma, Parma, Italy
2
Department of Chemistry I.F.M., University of Turin, Turin, Italy

Received 7 July 2009; revised 20 August 2009; accepted 26 August 2009

Published online 13 October 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21962

ABSTRACT: Solid-state properties of active ingredients are crucial in pharmaceutical

development owing to their significant clinical and economical implications. In the
present work we investigated the solid-state properties and the solubility in water
of didanosine, DDI, re-crystallized from a dimethylsulfoxide solution using supercritical
CO2 as an antisolvent (SAS process) for comparison with the commercially available
drug product. We also applied modern solid-state NMR (SS NMR) techniques, namely
2D 1H DQ CRAMPS (Combined Rotation And Multiple Pulse Spectroscopy) and
1
H–13C on- and off-resonance CP (cross polarization) FSLG-HETCOR experiments,
known for providing reliable information about 1H–1H and 1H–13C intra- and inter-
molecular proximities, in order to address polymorphism issues arising from the crystal-
lization of a new form in the supercritical process. A new polymorph of didanosine was
obtained from the supercritical antisolvent process and characterized by means of 1D
and 2D multinuclear (1H, 13C, 15N) SS NMR. The particle size of the new crystal phase
was reduced by varying the antisolvent density through a pressure increase. The
structural differences between the commercial product and the SAS re-crystallized
DDI are highlighted by X-ray diffractometry and well described by solid-state NMR.
The carbon C6 13C chemical shift suggests that both commercial and re-crystallized
didanosine samples are in the enol form. The analysis of homo- and heteronuclear
proximities obtained by means of 2D NMR experiments shows that commercial and
SAS re-crystallized DDI possess very similar molecular conformation and hydrogen
bond network, but different packing. The new polymorph proved to be a metastable form
at ambient conditions, showing higher solubility in water and lower stability to
mechanical stress. ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm
Sci 99:1855–1870, 2010
Keywords: supercritical antisolvent crystallization; solid-state NMR; polymorphism;
didanosine

INTRODUCTION

Solid-state properties of active ingredients are

Additional Supporting Information may be found in the
online version of this article.
Correspondence to: R. Bettini (Telephone: 39-0521905089; Fax: 39-
0521905006; E-mail: bettini@unipr.it)
Journal of Pharmaceutical Sciences, Vol. 99, 1855–1870 (2010)
ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association
crucial in pharmaceutical development owing to
their significant clinical and economical implica-
tions.1–4 The discovery and isolation of different
crystal forms of a new or already marketed drug
represents a remarkable issue that can dramati-
cally impact on both clinical efficacy (i.e., either by
varying the drug dissolution rate and, therefore,

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 4, APRIL 2010 1855

1856 BETTINI ET AL.
the bioavailability or by allowing the drug
administration through a new route) and indus-
trial production of the dosage forms. Sometimes
even small changes in the crystal structure afford
significant variation in drug dissolution rate or
stability.5
Modern technologies based on supercritical
fluids offer new perspectives for the design and
production of innovative materials6 as they afford
peculiar process conditions and offer the possi-
bility of avoiding or minimizing the use of organic
solvents.7
Supercritical fluids are nowadays proposed in
the pharmaceutical field as alternative ‘‘solvent’’
for a series of important operations such as
micron- and sub micron-sized particle production,
residual solvents removal and drug loading of
polymers.7 In all these cases, a great innovation
potential exists for the production of new crystal-
line or amorphous phases with improved and
advantageous technological and biopharmaceuti-
cal characteristics and features.6 As opposed
to traditional techniques for micron-sized
particle production (crystallization followed by
comminution), that may lead to cohesive, elec-
trically charged and poorly crystalline powders,
the supercritical fluid based processes have the
potential for affording in one production step
noncohesive, free flowing and highly crystalline
powders.8
The production of microparticles by super-
critical fluids can be achieved mainly by means
of three possible alternatives: (i) rapid expansion
of a drug solution in a supercritical fluid,9 (ii) drug
re-crystallization by adding supercritical CO2 as
antisolvent,10–13 or (iii) drug precipitation in an
amorphous state or as nano-sized crystals on
hydrophilic polymers.7 The crystallization of a
drug from solutions in supercritical CO2 can
exploit peculiar process conditions (e.g., pressure
and/or temperature variation, rate of solvent
evaporation) and operation parameters (fine
tuning of the density and the solvent power of
the crystallization medium by modulating tem-
perature and pressure) that cannot be obtained
with traditional crystallization processes from
liquid solvents, because of the obvious operational
constraints (e.g., temperature range, cooling rate,
solute concentration variation due to the progres-
sive separation of solid phases).
Great attention has been recently devoted to
drug re-crystallization by means of processes that
use supercritical CO2 as an antisolvent (SAS, or
Supercritical AntiSolvent).10–13
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 4, APRIL 2010
Figure 1. Chemical structure of DDI.
Didanosine (20 ,30 -dideoxyinosine), DDI (Fig. 1)
is a reverse transcriptase inhibitor, effective
against HIV and used in combination with
other antiretroviral drugs as part of highly active
antiretroviral therapy.
Nevertheless, it presents some drawbacks such
as tendency to hydrolyze at the acid pH of
the stomach, low oral bioavailability, short
biological half-life. These issues can be addressed
by site specific drug delivery systems14–17 or by
exploiting possible alternatives to oral route.18–20
This approach may require the need for accurate
control of the physico-chemical properties of
the drug particles such as size, shape, crystalline
purity and dissolution rate. No reports are
presently available on the solid-state chemistry
(X-ray structure, polymorphism studies, etc.) of
this active principle.
The aim of this work was to investigate the
solid-state properties and the solubility in water of
didanosine re-crystallized from a dimethylsulf-
oxide (DMSO) solution using supercritical CO2
as an antisolvent (SAS process) for comparison
with the commercially available drug product.
We also applied modern solid-state NMR (SS
NMR) techniques, namely 2D 1H DQ CRAMPS
(Combined Rotation And Multiple Pulse Spectro-
scopy) and 1H–13C on- and off-resonance CP
(cross polarization) FSLG-HETCOR experiments,
known for providing reliable information about
1
H–1H21,22 and 1H–13C23–25 intra- and intermole-
cular proximities, in order to address polymorph-
ism issues arising from the crystallization of a new
DOI 10.1002/jps
 DIDANOSINE POLYMORPHISM IN A SUPERCRITICAL ANTISOLVENT PROCESS
form in the supercritical solvent process. These
pulse sequences allowed comparisons and con-
siderations about the crystal packing and hydro-
gen bonds since X-ray structures are not available
because of the unsuitable size of DDI specimens
for single crystal diffraction.26
EXPERIMENTAL
Chemicals
DDI was a kind gift of Bristol-Myers Squibb
Pharmaceutical Research Institute; CO2 (99.9%
grade, Sapio, Italy); DMSO, methanol lecithin,
cyclohexane ammonium acetate and concentrated
ammonia were of analytical grade.
Solubility of DDI in SC CO2
The solubility measurements of DDI in super-
critical CO2 were performed by means of a
laboratory scale apparatus (SPE-ED SFE, Applied
Separation, Allentown, PA), operating under
dynamic conditions at low flux according to the
method described by Bettini et al.27 Experiments
were carried out at 40–458C and 90–100 bar.
SAS Re-Crystallization
The apparatus for the SAS crystallization was set
up by modifying the apparatus used for solubility
measurement (Fig. 2). Here the saturation cell
was substituted with a precipitation vessel.
In a typical SAS experiment the precipitation
vessel (32 cm3 i.v., 20 cm length, 1.42 cm i.d.) was
connected through a low volume T valve with the
lines of the pressurized CO2 and the DDI solution
in DMSO. The drug solution was pumped at
0.01 mL min1 by an HPLC pump (LKB-2248,
Pharmacia, Uppsala, Sweden). A sintered glass
frit was positioned at the bottom of the precipita-
tion vessel to collect the re-crystallized DDI. The
temperature of the thermostatic chamber was set
at 458C while the CO2 pressure was 100, 150, or
200 bar.
A second HPLC pump (Waters-510 Millipore,
Milford, MA) pumped methanol (1 mL min1)
through the tubing below the precipitation vessel
in order to avoid possible obstruction stemming
from downstream precipitation of the drug. The
circuit ended with a chilled bath for solvent
condensation.
The CO2 was made to flow through the
precipitation vessel at a flow rate of about
DOI 10.1002/jps
1857
Figure 2. Schematic representation of the apparatus
used for SAS experiments: 1) CO2 reservoir; 2) High
pressure pump; 3) Chilled bath; 4) CO2 inlet valve; 5) T
connector; 6) Precipitation vessel; 7) Three ways valve;
8) Temperature controller; 9) Outlet valve; 10) Chilled
bath; 11) DDI in DMSO solution reservoir; 12) HPLC
pump; 13) Methanol reservoir; 14) HPLC pump.
4.5 mmol min1. The CO2 flux was manually
set by means of a micrometric valve positioned
at the end of the pressurized circuit. After
15 min equilibration 1 mL of the DDI solution
(100 mg mL1) was pumped into the precipitation
vessel through the T valve. After complete DDI
precipitation, the vessel was submitted to a 2 h
final washing with supercritical CO2 alone to
ensure complete solvent removal from the pre-
cipitated solid particles and to prevent DMSO
condensation during depressurization.
Thermal Analysis
Differential scanning calorimetry was performed
on an indium calibrated Mettler DSC 821e
instrument (Mettler Toledo, Columbus, OH)
driven by STARe software (Mettler Toledo).
DSC traces were recorded by placing accurately
weighed amounts (5–6 mg) of powder sample in a
40 mL aluminum pan which was sealed and doubly
pierced. Scans were performed between 25 and
2008C at 10 K min1 under a flux of dry nitrogen
(100 mL min1). Each powder sample was ana-
lyzed at least in triplicate.
Powder X-Ray Diffractometry
X-ray diffraction patterns on powder were record-
ed on a Miniflex (Rigaku, Tokyo, Japan) diffract-
ometer using Cu Ka radiation (l ¼ 1.5418 Å)
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 4, APRIL 2010
1858 BETTINI ET AL.
generated with 30 kV. The goniometer was set at a
scan rate of 0.58 min1 over the 2u interval 2–358.
Elemental Analysis
Elemental analysis for C, H and N was carried out
with a FlashEA 1112 (ThermoQuest, Milan, Italy)
running at 9508C on powder samples of about 1 mg
accurately weighed in Sn crucibles. The accuracy
of the analysis ranges between 0.01% and 0.3% of
the absolute value of element composition.
Particle Size Analysis
Particle size analysis was done with a Malvern
Mastersizer X (Malvern Instruments LTD,
Malvern, UK) laser light diffractometer. Lenses
of 45 and 100 mm ranges were used according to
the sample needs and the laser beam length was
set at 2.40 mm. The samples were suspended in a
1% solution of lecithin in cyclohexane, mixed in an
ultrasound bath and introduced into the light
scattering cell by a Malvern stirring pump.
NMR Experiments
Solid-state NMR measurements were run on a
Bruker AVANCE II 400 instrument operating at
400.23, 100.65, and 40.55 MHz for 1H, 13C, and
15
N, respectively. 13C, 15N spectra were recorded
at room temperature at the spinning speed of
12 kHz. Cylindrical 4 mm o.d. zirconia rotors with
sample volume of 120 mL were employed. For
CPMAS experiments, a ramp cross-polarization
pulse sequence was used with contact times of
3.5 ms (13C) or 4 ms (15N), a 1H 908 pulse of 3.30 ms,
recycle delays of 10–15 s, and 128 (13C) or about
1000 (15N) transients. The two pulse phase-
modulation (TPPM) decoupling scheme was used
with a frequency field of 75 kHz.
For the spectral editing experiment a CPPISPI
sequence28 was used with a polarization-inversion
period of 50 ms.
2D 13C–1H HETCOR spectra were measured
according to the method of van Rossum et al.29,30
The MAS rate was set to 12 kHz. The proton rf
field strength used during the t1 delay for FSLG
decoupling and during the acquisition for TPPM
decoupling was 82 kHz. Two off-resonance pulses
with opposite phases (i.e., þx, x or þy, y) during
the FSLG decoupling were set to 9.96 ms. The
contact time was 100 ms. The magic angle (54.78)
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 4, APRIL 2010
pulse length for protons was set at 2.0 ms while
recycle delays used were 10–15 s. Quadrature
detection was achieved by using the states-TPPI
method. All the data for 40t1 increments with
240 scans were collected. For the off-resonance CP
(LG-CP) FSLG-HETCOR NMR the intensity of
the B1(1H) field for the CP was 75 kHz with a
mixing period of 2.0 ms. The 1H chemical shift
scale in the HETCOR spectra was corrected by a
scaling factor of 1/H3 since the 1H chemical-shift
dispersion is scaled by a factor of 1/H3 during
FSLG decoupling.
1D 1H CRAMPS and 2D 1H DQ CRAMPS
experiments were performed with a 2.5 mm
Bruker probe. 1H CRAMPS spectra were acquired
using a windowed-PMLG pulse sequence of
dipolar decoupling at the spinning speed of
13 kHz. 2D 1H–1H DQ CRAMPS were acquired
at 12.5 kHz of spinning speed with the window-
less-eDUMBO-131 and the windowed-PMLG32
pulse sequences for homonuclear dipolar decou-
pling during t1 and t2, respectively. For all
samples, 1H 908 pulse lengths of 1.9 ms and recycle
delays of 10–15 s were used. For each of 64
increments of t1, 96 transients were averaged. The
pulse width and the RF power were finely
adjusted for best resolution and a minimal zero-
frequency line in the spectra. The q1 and q2
prepulses were both of duration 0.5 ms (commer-
cial) or 0.8 ms (SAS 200 bar). In t2, one complex
data point was acquired in each acquisition
window (2.2 ms). Including the prepulses, the
effective dwell time in t2 was 16.1 ms (maximum
t2 ¼ 11 ms). The DUMBO blocks were 25.6 ms long
(corresponding to one basic DUMBO cycle). DQ
excitation and reconversion was achieved using
two elements of POST-C7,33 corresponding to a
recoupling time of 45.7 ms. A 16-step nested phase
cycle was used to select Dp ¼ 2 on the DQ
excitation pulses (four steps), and Dp ¼ 1 on the
z-filter 908 pulse (four steps). The States-TPPI
method was used to achieve sign discrimination in
the F1 dimension.
1
H, 13C, and 15N scales were calibrated
with adamantate (1H signal at 1.87 ppm), glycine
(13C methylene signal at 43.86 ppm) and
(NH4)2SO4 (15N signal at 355.8 ppm with respect
to CH3NO2) as external standards.
Optical Microscopy
Optical microscopy with polarized light was per-
formed with an Optiphot2-Pol microscope (Nikon,
Tokyo, Japan) at 10 and 20 magnification.
DOI 10.1002/jps
 DIDANOSINE POLYMORPHISM IN A SUPERCRITICAL ANTISOLVENT PROCESS
DDI Solubility in Water
Weighed amounts of DDI (50 mg), in excess
with respect to saturation, were added to 1 mL of
double distilled water and magnetically stirred in
EppendorfTM vials at 25  0.58C for 48 h. There-
after, DDI concentration in solution was deter-
mined by HPLC after filtering the suspensions
(0.45 mm, regenerated cellulose) and diluting the
filtrate with a solvent mixture constituted by
8 volumes of mobile phase B and 92 volumes
of mobile phase A (see below HPLC analysis).34
Each measurement was replicated at least three
times.
DDI Dissolution Rate
Drug dissolution experiments were carried out at
258C on accurately weighed amount of DDI
(50 mg) using a USP 32 flow through dissolution
apparatus (Dissotest CE6, Sotax, Basel, CH).
Double distilled water flowing at 5 mL min1
was used as dissolution medium. Aliquots corre-
sponding to 1 mL of the flowing fluid were
collected at the cell exit at fixed time (within
30 min) filtered (0.45 mm, regenerated cellulose)
and analyzed by HPLC for DDI quantification.
High Performance Liquid Chromatography (HPLC)
DDI quantification was carried out according to
the method reported in the European Pharmaco-
poeia34 using a high performance liquid chroma-
tographer constituted by 2 LC-10 ATvp pump, and
a SPD 10Avp detector set at 254 nm (Shimadzu
Tokyo Japan) equipped with a Sperisorb1 5 mm
ODS2 column (Waters Corporation, Milford, MA).
The volume of injection was 20 mL (Rheodyne
injector 7725i). The mobile phase was constituted
by different volume ratios of mobile phase A
(8 volumes of methanol and 92 volumes of a
3.86 g L1 solution of ammonium acetate adjusted
to pH 8.0 with concentrated ammonia) and mobile
Table 1. Particle Size Distribution of Commercial DDI and DDI Re-Crystallized at Three Different Antisolvent
Pressures
Powder Dv, 0.1
Commercial DDI 1.86
SAS DDI obtained at 100 bar 1.99
SAS DDI obtained at 150 bar 1.25
SAS DDI obtained at 200 bar 1.01
DOI 10.1002/jps
1859
phase B (30 volumes of methanol and 70 volumes
of a 3.86 g L1 solution of ammonium acetate
adjusted to pH 8.0 with concentrated ammonia).
Powder Milling
Weighed amounts (about 300 mg) of either com-
mercial or re-crystallized DDI were ground for 2 or
4 h, in a vibration mill (Mk 11, RIIC, Glenrothes,
Fife, Scotland, UK) equipped with an agate
cylindrical cell (33 mm height, 9.5 mm i.d.) con-
taining 2 agate balls (6.0 mm diameter).
RESULTS AND DISCUSSION
The selection of the crystallization method as well
as the operating condition in terms of temperature
and pressure range was based on preliminary
solubility measurements and literature data
relevant to CO2–DMSO phase behavior.35,36
Preliminary solubility measurements of DDI in
pure SC CO2 indicated the unsuitability of a
process based on the rapid expansion of a super-
critical solution for the drug re-crystallization. In
fact, the DDI solubility values in supercritical
CO2, that is, at 40–458C and 90–100 bar, resulted
in the range 1  109–2  107 mole fraction,
values that are too low to afford a suitable yield.
On the other hand, DDI is very soluble in
DMSO34 which is among the most frequently used
organic solvents in the SAS process, owing to its
good miscibility with SC CO2. The temperature
and the pressure range used in the present work
were based on the work of Gonzalez et al.35 and
Kordikowski et al.36 who reported the tempera-
ture and pressure conditions for complete mis-
cibility of DMSO and CO2 in the supercritical
phase.
SAS re-crystallization was carried out at 458C
with a CO2 pressure ranging from 100 to 200 bar.
The yield of the re-crystallization process ranged
between 22% and 26%.
(mm) Dv, 0.5 (mm) Dv, 0.9 (mm) Span
4.98 20.35 3.71
6.30 30.05 4.45
5.68 17.16 2.80
3.66 11.25 2.79
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 4, APRIL 2010
1860 BETTINI ET AL.
Figure 3. Cumulative undersize distribution of DDI
particles obtained by SAS re-crystallization at three
different antisolvent pressures.
The particle size distribution of the SAS
re-crystallized DDI is reported in Table 1 as a
function of the CO2 pressure in comparison with
the commercially available drug.
The particle size and the particle size distribu-
tion (the Span value is defined as the ratio
Dv, 0.9  Dv, 0.1/Dv, 0.5) of the powder obtained at
100 bar were higher compared to the commercial
DDI powder. On the other hand, for the re-
crystallized powders, a significant and progres-
sive decrease of the particle size and particle size
distribution can be observed with the increased
pressure (Fig. 3). In agreement with the findings
of other authors,13 the geometric mean diameter
of the SAS processed particles decreases with the
increase of the CO2 density (Fig. 4).
The phenomenon can be mainly ascribed to an
effect of the CO2 pressure variation on the fluid
Figure 4. Geometric mean diameter of DDI particles
obtained by SAS re-crystallization as a function of CO2
density.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 4, APRIL 2010
dynamics: the pressure increase implies an
increase of the fluid density and velocity and,
therefore, of the Reynolds number which, in turn,
results in a better mixing efficiency between the
organic solution and the supercritical fluid.6
Another parameter that should be taken into
account is the supercritical fluid to drug solution
volume ratio. The fluxes of the organic solution of
the drug and the flux of the CO2, measured after
the final expansion valve (at ambient conditions),
were kept constant at 0.01 and 100 mL min1,
respectively. The density values of CO2 at given
pressures and temperatures were calculated
by means of the Refprop software (NIST,
Gaithersburg, MD). Based on these values, the
ratios of the volume of the organic solution and the
supercritical CO2 into the precipitation vessel
were calculated as 39.7, 26.6, and 24.3 at 100, 150,
and 200 bar, respectively. The progressive reduc-
tion of this ratio with the pressure (and therefore
with the density) resulted in an increase of the
concentration of the drug solution at the super-
saturation point when the antisolvent effect took
place.37
Beside the particle size, also the crystal
morphology was affected by the SAS process.
Commercial DDI (Fig. 5A) appeared as aggregates
of irregular shaped crystals, while SAS re-crystal-
lization afforded needle shaped and more regular
crystals (Fig. 5B and C). No effect of pressure can
be evidenced on the crystal habit of the DDI re-
crystallized under different conditions.
As far as the purity of the crystallized compound
is concerned, in particular with respect to possible
traces of residual DMSO, elemental analysis was
carried out on the SAS re-crystallized DDI as well
as on the commercially available product. Accord-
ing to the pharmacopoeial classification DMSO
belongs to Class 3 which comprises the solvents of
low toxic potential and whose daily exposure
should not exceed 50 mg.38 In this respect, for a
DDI daily dose of 400 mg39 the maximum accep-
table DMSO concentration in the active ingredi-
ent would be 12.5% (w/w). Nevertheless, the
analysis carried out on DDI samples re-crystal-
lized by SAS at 100 and 150 bar (Tab. 2) indicated
a C, H, and N concentration very close to the
values measured for the commercial product and
practically superimposed to the theoretical ones
calculated on the base of the C10H12N4O3 formula.
Table 2 reports also the C, H, and N percentages
relevant to the hypothetical case of a DMSO
content of 0.5%, which should be considered as a
very low value in relation to the toxicity of the
DOI 10.1002/jps
 DIDANOSINE POLYMORPHISM IN A SUPERCRITICAL ANTISOLVENT PROCESS 1861

Many literature reports underscored the effect

Figure 5. Pictures taken at the polarized light
microscope of (A) commercial DDI crystals and SAS
re-crystallized DDI B) 100 bar, C) 200 bar. Original
magnification 20X.
solvent. It is noteworthy that the relevant figures
are far away from the measured one. This allows
one to conclude that the re-crystallized products
present a negligible residual solvent content.
of the supercritical process on the solid-state
characteristics of the treated drug in particular
with respect to the degree of crystallinity, crystal
phase and polymorph generation.11,13,40,41
DSC analysis carried out on commercial
DDI evidenced melting with decomposition at
186  0.18C. Traces that displayed no significant
differences were obtained from the re-crystallized
powders, that showed melting point temperatures
of about 18C around the melting peak temperature
of the commercial DDI. However, these variations
could not be correlated to the experimental con-
ditions (pressure value or CO2 density) adopted
during the SAS process. The composite nature of
the phenomenon (endothermic melting with con-
comitant exothermic decomposition) makes the
DSC unsuitable for detecting possible melting
point variations stemming from different crystal
phases.
In this respect powder X-ray diffraction proved
to be a more appropriate tool.
Figure 6 reports the diffraction patterns
obtained from commercial and SAS re-crystallized
(200 bar) DDI. Nonsuperimposable traces were
obtained. Main differences are in the 8–118,
12–138, 16–208, and 26–288 2u regions. This
observation strongly suggested that a new poly-
morph was isolated upon SAS re-crystallization.
To our best knowledge, no data relevant to
DDI polymorphism are presently available in
literature nor on the Cambridge Structural
Database (http://www.ccdc.cam.ac.uk/products/
csd/).
The powders obtained by SAS at different
pressures did not show any difference in PXRD
patterns, thus suggesting that the formation of
a new crystal phase might be ascribed to a
combination of both the organic solvent charac-
teristics and the crystallization conditions im-
posed by the supercritical milieu.
In fact, considering that DMSO shows quite a
low vapor tension (boiling point 1898C at ambient
pressure) the supercritical fluid played a crucial

Table 2. Composition Percent of the Commercial DDI and DDI Obtained after SAS Re-Crystallization at 100 and
150 bar along With the Theoretical Values Calculated for Pure DDI or DDI Containing 0.5% (w/w) of DMSO

Theoretical Measured

Element % Pure DDI DDI þ 0.5% DMSO SAS DDI (100 bar) SAS DDI (150 bar) Commercial DDI
C 50.84 44.79 50.21 50.04 50.10
H 5.12 4.54 5.16 5.14 5.08
N 23.71 20.81 23.72 23.77 23.91

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1862 BETTINI ET AL.
Figure 6. X-Ray diffraction patterns of commercial DDI powder and SAS re-crystal-
lized DDI (200 bar) powder.
role in the crystal formation in a very short
time. In this respect, the otherwise difficult to
realize rapid solvent evaporation determined by
the antisolvent effect exerted by the SC CO2, made
affordable the crystallization process.
Solid-State NMR Analysis
It is well known that advanced solid-state NMR
techniques provide several structural and
dynamic data useful in many areas, in particular
those concerning polymorphism in pharma-
ceutical,4,42 organic,43–45 and in crystal engineer-
ing46,47 fields. Due to recently developed
techniques based on the dipole–dipole interaction
between homo- and heteronuclei,23 solid-state
NMR is becoming essential where X-ray struc-
tures are not available.48,49 These experiments
are nowadays able to give sufficient structural
information to elucidate conformation and aggre-
gation even for unlabelled systems.23 Recent
improvements in probe technology with spinning
speed up to 70 kHz and in homodecoupling
techniques employing the Lee–Goldburg (LG)
approach (e.g., FSLG, frequency switched, or
PMLG, phase modulated)32,50 allow the acquisi-
tion of high resolution 1H spectra in both 1D and
2D modes. Several CRAMPS schemes have been
used in order to elucidate hydrogen bond net-
works in many systems while the DQ spectra
provide reliable information about 1H–1H proxi-
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 4, APRIL 2010
mities.51,52 Furthermore, unambiguous 1H signal
assignments can be achieved by on-resonance CP
1
H–13C HETCOR in which polarization transfer
occurs during a very short time, namely the
contact time (CT). For longer CT, the detection of
additional 1H–13C coherences provides further
geometrical constraints if analyzed with respect to
the formation of 1H–1H polarization coherences.25
For a complete analysis, the use of an off-
resonance CP achieved by LG spin-lock (LG–
CP) efficiently suppresses unwanted 1H–1H spin
exchange leading to the observation of long-range
1
H–13C contacts.29
For these reasons the solid-state characteristics
of the SAS crystallized DDI in comparison to those
of the commercially available product were
further investigated by 1H CRAMPS, 13C, and
15
N CPMAS, spectral editing 13C CPPISPI (Cross
Polarization Polarization Inversion Simultaneous
Phase Inversion), 1H DQ CRAMPS and 1H–13C
on- and off-resonance CP (LG-CP) FSLG-HET-
COR NMR.
1
H and 13C chemical shifts with assignments
are listed in Table 3 while 15N chemical shifts are
reported in Table 4.
The 13C solution spectrum of the DDl53 shows
many differences with respect to 13C CPMAS
spectra of commercial and SAS crystallized DDI
(Fig. 7) related to significant conformational
changes and/or geometrical constraints due to
crystallization forces such as weak interactions or
hydrogen bonds.
DOI 10.1002/jps
 DIDANOSINE POLYMORPHISM IN A SUPERCRITICAL ANTISOLVENT PROCESS 1863

1 13
Table 3. H and C Chemical Shifts With Assignment for the Commercial and SAS 200 bar DDI Samples

Solution-State NMRa Solid-State NMR

Commercial, Commercial, 200 bar, 200 bar,

1 13 1 13 1 13
Atom Note H (ppm) C (ppm) H (ppm) C (ppm) H (ppm) C (ppm)
C6 C–
–O — 157.5 156.9 156.6
155.4 155.2
C2 (H)NC(H)N 8.34 (s, 1H) 146.5 8.0 149.3 (2) 8.5 148.3 (2)
C4 C(q) — 148.4 147.8 148.3 (1)
145.5 145.5
C8 (R)NC(H)N 8.05 (s, 1H) 139.1 7.7 138.5 8.0 137.3
7.3 136.0 7.6 135.3
C5 C(q) — 125.2 123.8 123.7
122.0 121.9
C10 , C40 CH 6.20 (dd, 1H) 85.3 4.7 85.3 (2) 5.7 85.0 (3)
4.10–4.13(m, 2H) 82.9 5.3 84.7 (2) 5.1 83.9
C50 CH2OH 3.53–3.62(m, 2H) 63.5 3.8 61.7 (2) 4.2 61.7 (2)
C20 CH2 2.35–2.51(m, 2H) 33.0 2.3 33.9 2.6 33.2
2.3 30.9 3.0 30.2
C30 CH2 1.99–2.06 (m, 2H) 26.3 2.3 26.5 2.5 25.6
2.4 23.7 2.7 24.4
NH 12.33 (brs, 1H) 10.4/14.7 10.3/14.9
OH 5.0 (brs, 1H) 14.7/10.4 14.9/10.3
a
From Ref.,53 DMSO-d6 solvent.

The solid-state 13C assignment was obtained

15
Table 4. N Chemical Shifts With Assignment for with the help of the CPPISPI experiment (Fig. 8)
the Commercial and SAS 200 bar DDI Samples that allows the discrimination of carbon atoms
according to the number of attached protons based
Atom Note Commercial 200 bar on differences in CP dynamics. We optimized the
N(1) NH 152.8 153.6 polarization inverse period to 50 ms in order to
N(1) NH 146.3 146.0 obtain positive C and CH3 signals, negative CH2
N(3) N(q) 186.7 186.9 and suppressed CH peaks.
N(3) N(q) 178.9 179.5 Almost all carbon atoms of commercial DDI
N(7) N(q) 211.3 211.1 gave rise to two peaks, due to the presence of two
N(9) N(q) 159.5 159.7
N(9) N(q) 156.6 156.4

Figure 8. 13C CPMAS (A) and 13C CPPISPI (spectral

Figure 7. 13C CPMAS spectra of the commercial and
SAS 100, 150, and 200 bar DDI recorded at 100.63 MHz
with a spinning speed of 12 kHz.
editing) (B) spectra of the commercial sample of the
DDI. In the CPPISPI spectrum the polarization-inver-
sion period was set to 50 ms in order to obtain
positive Cq, negative CH2 and null CH (marked with
circles).

DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 4, APRIL 2010
1864 BETTINI ET AL.

Figure 9. 1H CRAMPS spectra of the commercial and

Figure 10. Comparison among 15N CPMAS spectra
SAS 200 bar DDI, recorded at 400.23 MHz with a spin-
of commercial DDI and SAS re-crystallized didanosine
ning speed of 13 kHz.
at 100, 150, and 200 bar of CO2.

independent molecules in the crystallographic C5 would be strongly influenced, leaving all other
asymmetric unit. This aspect might also be a resonances almost unchanged, whereas this
consequence of a keto-enol tautomeric equili- was not observed. The signal C6 (C –– O) at
brium, but in this case signals C6, C2, C4, and 156.6 ppm might indicate that both molecules

Figure 11. 2D 1H–13C FSLG-HETCOR (A) and off-resonance CP 1H–13C FSLG-HET-

COR (B) spectrum of commercial DDI, recorded with a contact time of 100 and
2000 ms, respectively.

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 4, APRIL 2010 DOI 10.1002/jps
 DIDANOSINE POLYMORPHISM IN A SUPERCRITICAL ANTISOLVENT PROCESS 1865

exist in the enol form: indeed, it has been reported simple acquisition of 1H MAS spectra at 35 kHz
for similar systems that the C – O group did not provide enough resolution, so we decided
resonates in the solid state at about 180 ppm, to use a CRAMPS approach by means of the
while C–OH groups give rise to peaks around windowed-PMLG scheme.32 The 1H CRAMPS
167 ppm,54 though they may shift to 167–166 and spectra of the commercial and SAS crystallized
154–155 ppm respectively, depending on ring DDI are reported in Figure 9. They are quite
substituents.55 similar and characterized by five resonances, two
As far as the SAS re-crystallized DDI was of which (at 10.5 and 14.8 ppm) are clearly related
concerned, spectra of powders obtained at 100, to hydrogen bonded atoms involved in a weak and
150, and 200 bar were identical (Fig. 7). Therefore, in a strong interaction, respectively.
only the sample prepared at 200 bar will be The 15N spectra are characterized by seven
discussed. signals (Fig. 10): the comparison of the com-
The 13C CPMAS spectrum of SAS re-crystal- mercial and the SAS crystallized samples shows a
lized DDI was quite similar to that of the shift in the range of 0.2–0.5 ppm for all reso-
commercial form. However, small but significant nances. Main differences involve (a) the N1 atom
differences confirmed the isolation of a new that shifts from 152.8 to 153.6 ppm with increas-
crystalline product. The main changes involve ing of the line width at half height (from 48 to
resonances C30 and C10 : the former in SAS re- 57 Hz) and (b) the sharpening of N7 atom from
crystallized samples gave rise to two signals at 100 to 40 Hz.
25.6 and 24.4 ppm, closer than those of the After the 13C assignment, 1H resonances were
commercial product. In SAS DDI the C10 signal detected from one-bond correlation signals high-
is split into two peaks (83.9 and 85.0 ppm), one of lighted by the on-resonance CP 1H–13C FSLG-
which is overlapped with C40 , while in the HETCOR spectrum (Fig. 11A) acquired with a
commercial sample C10 atoms gives rise to a
single peak (84.7 ppm). Furthermore, in the SAS
DDI, C8 resonances are shifted to lower frequen-
cies of about 1.2 and 0.7 ppm, respectively,
whereas the high frequency C4 peak is completely
overlapped with C2. The remaining signals did
not show any significant variations compared to
the commercial product.
By comparing solution chemical shifts (which
refer to monomeric or dissolved structures) with
solid-state chemical shifts (related to aggregated
or crystallized structures) we observed the highest
shifts (dliq–d) for carbon atoms C4, C8, and C5 with
a Dd of 2.9–2.9, 3.1–3.8, and 3.2–3.3 ppm, respec-
tively (reported values refer to the most shifted
peak only of the two independent molecules; first
value, commercial DDI, second value SAS DDI).
These carbon atoms experience the strongest
packing forces, probably because involved in
noncovalent bonding. In the case of C8, this is
in agreement with its ability to correlate
through the space with many other hydrogen
atoms (see below). The presence of a single signal
at 157.5 ppm for C6 in solution suggests the
existence of a tautomeric equilibrium probably
shifted to the enol form.
Figure 12. 2D 1H DQ CRAMPS spectrum of the com-
Usually, the easiest technique for averaging mercial DDI together with the single-quantum projec-
the strong 1H–1H homonuclear dipolar interac- tions. The pair of DQ signals in both spectra
tion present in organic solids is to spin the corresponding to the intramolecular proximity of hydro-
sample in small rotors (1.3–2.5 mm o.d.) up to gen-bonded protons with high-ppm resonance signals to
30–70 kHz.56,57 Unfortunately, in this case, the the same nearby proton are highlighted.

DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 4, APRIL 2010
1866 BETTINI ET AL.
very short CT (100 ms). Indeed, cross peaks
relating to long-range correlations or due to
1
H spin diffusion are almost completely sup-
pressed. On the other hand, the off-resonance CP
1
H–13C FSLG-HETCOR experiment (Fig. 11B),
acquired with a CT of 2000 ms, allowed una-
mbiguous assignments of long-range spatial
1
H–13C correlations since the LG 1H spin-lock
avoids 1H–1H spin exchange during the CP. In
this way, structural information such as confor-
mations, angles and distances becomes poten-
tially available allowing an NMR crystallography
analysis that in some case provides accurate
short-range structures.58,59 Unfortunately, the
presence of two molecules in the asymmetric unit
dramatically complicates the analysis: indeed,
while two 13C signal sets are clearly visible (for
almost all carbon atoms), it is not the same for
1
H resonances. However, even if it is difficult to
distinguish between inter- and intramolecular
correlations, several conclusions can be drawn.
All long-range correlations are highlighted in
Figure 11B. Polarization transfer is observed
Figure 13. 2D 1H–13C FSLG-HETCOR (A) and off-resonance CP 1H–13C FSLG-HET-
COR (B) spectrum of SAS 200 bar DDI, recorded with a contact time of 100 and
2000 ms, respectively.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 4, APRIL 2010
among all proton and carbon atoms of the pyrrole
moiety. We were able to trace correlations from
the proton H8 to carbon atoms C10 , C40 , C4, C5,
and C6. All these proximities, indicating a
particular conformation of the pyrrole moiety
around the C8–H8 group, explain its large shift
with respect to the solution state. We also
observed polarization transfers from H20 and
H30 to C8 and from H10 and H40 to C4. Further-
more, both hydrogen bonded signals show correla-
tions with the carbon C6 confirming the presence
of an enol (C–OH) rather than a carbonyl (C – O)
group. Interestingly they transfer polarization
also to carbon atoms C2 and C5 in agreement
with the presence of N1  H–O or N3  H–O
interactions.
These results suggest that the two molecules in
the asymmetric unit possess very similar con-
formations, while the main difference stems in the
hydrogen bond strength (signals at 10.4 and
14.7 ppm which refer to the same C6–O–
H1  N interaction in the two molecules both
correlating with carbon atoms C6). They differ for
DOI 10.1002/jps
 DIDANOSINE POLYMORPHISM IN A SUPERCRITICAL ANTISOLVENT PROCESS
Figure 14. 2D 1H DQ CRAMPS spectrum of the SAS
200 bar DDI, together with the single-quantum projec-
tions. The pair of DQ signals in both spectra correspond-
ing to the intramolecular proximity of hydrogen-bonded
protons with high-ppm resonance signals to the same
nearby proton are highlighted.
the correlation with C5 only present for the signal
at 14.7 ppm.
Also the hydrogen bond network seems to be
very similar, as observed in the 1H DQ CRAMPS
(Fig. 12) where both hydrogen-bonded atoms show
proximity to H8.
The same analysis has been performed on the
SAS DDI 200 bar (Figs. 13 and 14). Interestingly,
the LG-CP HETCOR spectrum highlights the
same correlations observed in the commercial
sample, somewhat more intense. Furthermore,
by comparing 1H DQ CRAMPS spectra (Figs. 12
and 14), we can assert that hydrogen bond
correlation patterns for the two forms are equal
as well as all other hydrogen proximities, of course
by taking into account small differences in the
chemical shifts. Then, the two forms possess
very similar structural conformation but at
least differences in the molecular packing. If we
consider that also the interaction networks are
similar we can surmise that they probably differ
mainly in the cell volume and density, that is, in
the interaction lengths and in the intermolecular
distances.
DOI 10.1002/jps
1867
The solubility in water was determined at 258C
for investigating possible differences between
commercial and re-crystallized DDI, as well as,
as a coarse indicator of the relative thermody-
namic stability of the two crystal forms.
The values obtained from the commercial DDI
and the DDI SAS re-crystallized at 200 bar were
25.4  0.7 and 26.9  0.5 mg mL1, respectively.
The difference was very low but statistically
significant ( p < 0.05 by Student’s t-test) and
suggested that the small variation observed in
the crystal conformation played a role on the
energetic content of the molecule. Therefore it
could be affirmed that the SAS re-crystallized DDI
represents a metastable polymorph of the com-
mercial DDI.60,61
It is worth underscoring that the accurate
determination of equilibrium solubility of two
polymorphs is often a difficult task. In fact,
solution mediated conversion of the less stable
form to the more stable one may prevent dif-
ferences in solubility from being detected.
Nevertheless, this small difference in solubility
observed resulted in a difference, although not
statistically significant, in dissolution rate bet-
ween the re-crystallized and commercial DDI
(Fig. 15).
Finally, a further indication of the lower
thermodynamic stability of the re-crystallized
DDI with respect to the commercial product
stemming from the different energy content of
the crystal lattice was obtained by submitting
the two powders to prolonged milling using a
vibration mill.
The decrease of the intensity of the X-ray
diffraction peaks indicated that the commercial
product underwent a reduction of the degree
of crystallinity after 2 h milling Figure 16A. No
Figure 15. Dissolution profile of commercial DDI and
DDI re-crystallized at 200 bar.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 4, APRIL 2010
1868 BETTINI ET AL.
Figure 16. Powder X-ray diffraction patterns of
(A) commercial DDI and (B) SAS re-crystallized DDI
at 200 bar recorded upon different milling time.
further decrease was observed upon 4 h milling.
On the contrary, the SAS re-crystallized powder
(200 bar) showed a progressive decrease of the
peak intensity with the milling time, accompanied
by an elevation of the baseline, that indicated a
reduction of the degree of crystallinity with the
formation of an amorphous phase (Fig. 16B).
CONCLUSIONS
A new polymorph of DDI was produced by a
supercritical antisolvent process and charac-
terized by means of 1D and 2D multinuclear
(1H, 13C, 15N) SS NMR. The structural differences
between the commercial product and the SAS re-
crystallized DDI are underlined by powder X-ray
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 4, APRIL 2010
diffractometry and well described by solid-state
NMR. The carbon C6 13C chemical shift suggests
that both DDI samples are in the enol form. The
analysis of homo- and heteronuclear proximities
obtained by means of 2D NMR experiments shows
that commercial and SAS 200 bar DDI possess
very similar conformation and hydrogen bond
network, but different packing.
The new polymorph proved to be a metastable
form showing higher solubility in water and lower
stability to mechanical stress.
The particle size of the new crystal phase can
be reduced by varying the antisolvent density
through a pressure increase.
ACKNOWLEDGMENTS
This work was supported by a grant from Italian
Ministry for University and Research through the
PRIN 2006 program. The authors would like to
thank Prof. Mino R. Caira (University of Cape
Town, South Africa) for the helpful discussion.
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