Polymorphism in anti-hyperammonemic agent N-carbamoyl-l-glutamic acid

Article abstract of DOI:10.1039/c5ce00116a

Carglumic acid (CGA) is used for the treatment of hyperammonaemia. The aim of this study was to investigate novel crystalline forms of CGA which can offer improved physicochemical properties and also to understand the stability relationships between such forms. We report two novel polymorphs I and II and a degradation derivative of the drug hydantoin-5-propionic acid (5-HPA). Crystallization conditions have been optimized to obtain each polymorph in reproducible crystallization batches. 5-HPA was obtained serendipitously upon crystallization of CGA in acidic medium (2-butanone : propionic acid). The X-ray crystal structures of CGA Form-I and Form-II and 5-HPA are sustained by carboxylic acid catemer and dimer, carboxamide catemer and dimer and an acid-amide heterosynthon. The two polymorphs are monotropically related. Thermodynamic stability, phase transformation, and Hirshfeld surface maps complement the structural analysis. Both forms are stable for two months under accelerated ICH conditions of 40 °C and 75% RH. Dynamic vapor sorption (DVS) showed that Form-I is more stable under moisture conditions than Form-II. Slurry crystallization, mechanical grinding and thermal measurements confirm the thermodynamic nature of CGA Form-I.

Full text of DOI:10.1039/c5ce00116a

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Polymorphism in anti-hyperammonemic agent
N-carbamoyl-L-glutamic acid†
Cite this: DOI: 10.1039/c5ce00116a
*
D. Maddileti and Ashwini Nangia
Carglumic acid (CGA) is used for the treatment of hyperammonaemia. The aim of this study was to
investigate novel crystalline forms of CGA which can offer improved physicochemical properties and also
to understand the stability relationships between such forms. We report two novel polymorphs I and II and
a degradation derivative of the drug hydantoin-5-propionic acid (5-HPA). Crystallization conditions have
been optimized to obtain each polymorph in reproducible crystallization batches. 5-HPA was obtained ser-
endipitously upon crystallization of CGA in acidic medium IJ2-butanone : propionic acid). The X-ray crystal
structures of CGA Form-I and Form-II and 5-HPA are sustained by carboxylic acid catemer and dimer,
carboxamide catemer and dimer and an acid–amide heterosynthon. The two polymorphs are mono-
tropically related. Thermodynamic stability, phase transformation, and Hirshfeld surface maps complement
the structural analysis. Both forms are stable for two months under accelerated ICH conditions of 40 °C
and 75% RH. Dynamic vapor sorption (DVS) showed that Form-I is more stable under moisture conditions
than Form-II. Slurry crystallization, mechanical grinding and thermal measurements confirm the thermody-
namic nature of CGA Form-I.
Received 18th January 2015,
Accepted 20th February 2015
DOI: 10.1039/c5ce00116a
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in organic solvents, perhaps due to the high density of
the functional groups (dicarboxylic acid and urea) in a small
Introduction
Carglumic acid is an orphan drug used for the treatment
of hyperammonaemia in patients with N-acetylglutamate
synthase (NAGS) deficiency.1 NAGS deficiency is one the most
severe and rarest of the hereditary urea cycle disorders
(UCDs). This rare genetic disorder results in elevated levels of
ammonia in the blood, which can eventually cross the blood–
brain barrier and cause neurologic problems, cerebral edema,
coma, and eventually death. CGA is a structural analogue
of N-acetylglutamate, the naturally occurring activator of
carbamoyl phosphate synthetase (CPS), and helps to break
down ammonia to reduce its concentration in the blood and
its toxic effects.2 CGA was approved by the U.S. Food and
Drug Administration (FDA) in 2010 to treat hyperammonemia
because it can act as a replacement for NAG in NAGS-
deficient patients by activating CPS.3 It is sold under the
brand name Carbaglu® which is formulated as a water-
dispersible tablet and each tablet contains 200 mg of
carglumic acid and excipients. CGA is an acidic drug mole-
cule in the form of white crystalline powder having aqueous
solubility of 19.1 mg mL−1 (ref. 4) and is practically insoluble
molecule. Studies on hyperammonemia are limited due to the
rarity of the disease. The condition is particularly toxic to the
central nervous system (CNS).5 The X-ray crystal structure
of the analog N-acetylglutamate is reported6 (CSD Refcodes
TERRUD and TERRUD01, multiple determinations of the
same structure), but no crystal structure of CGA was found in
the Cambridge Structural Database (ver. 5.36, November 2014
update).7 In view of the importance of polymorphism in
pharmaceuticals8 to control physicochemical properties, such
as solubility, stability, dissolution rate, melting point, bio-
availability,9 etc., CGA was screened by different techniques
such as liquid-assisted grinding (LAG), slurry crystallization,
solvent removal in a rotavap, freeze drying, spray drying, and
solution crystallization.10 Two novel polymorphs named
Form-I and Form-II and a degraded derivative of CGA,
hydantoin-5-propionic acid, are reported (5-HPA, chemical
name 3-IJ2,5-dioxoimidazolidin-4-yl)propanoic acid).
Results and discussion
Since the API is routinely exposed to various conditions rang-
ing from production to tableting to transportation before
consumption, the risk of process-induced transformations
and stability of polymorphs must be properly understood for
phramaceuticals.11 Thus, a sound understanding of a poly-
morphic system is a prerequisite for product quality and per-
formance. Even though strong hydrogen bonding functional
School of Chemistry, University of Hyderabad, Prof. C. R. Rao Road, Central
University PO, Gachibowli, Hyderabad 500 046, India.
E-mail: ashwini.nangia@gmail.com
† Electronic supplementary information (ESI) available: Molecular torsion
angles, IR and NMR spectra shifts, and PXRD overlay plots. CCDC 1043890–
1043892. For ESI and crystallographic data in CIF or other electronic format see
DOI: 10.1039/c5ce00116a
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groups COOH and urea are present in the CGA molecule,
which generally promote crystallization, we had to try a range
of solvent systems and mixtures to obtain diffraction quality
crystals. A majority of crystallization experiments gave micro-
crystalline powders which matched with the PXRD pattern of
the starting material (Form-I). The solubility of CGA in aque-
ous medium is very high, but in organic solvents, it is practi-
cally insoluble. This limitation narrowed down the variety of
solvents which could be used for crystallization screen, and
hence, mostly Form-I was observed. Form-I was crystallized
by slurry grinding the starting compound in EtOH, freeze dry-
ing in water (lyophilization), and spray drying from water.
Novel Form-II was produced upon rotovaporization of CGA
for molecule A and 178.7°IJ1) and −57.6°IJ2) for molecule B in
Form-II, respectively. The main difference in the torsion
angles of both Form-I and Form-II was observed for τ1 in the
alkyl chain portion (C2–C3–C4–C5 Δτ about 100°, see Table S1,
ESI†) for these conformational polymorphs.13 The overlay dia-
gram of the CGA molecules from Form-I and II shows confor-
mational changes (Fig. 1). From the molecular overlay and
structural analysis, we can classify both forms as conforma-
tional and synthon polymorphs. Reflection data were col-
lected using Mo-Kα X-radiation, and the S-configuration at
the chiral center of the molecule was assigned based on the
natural L-glutamic acid derived starting material.
in water : acetone (1 : 5 v/v solvent) (see the experimental Crystal structure analysis
section). The above procedures were optimized to obtain
reproducibly pure polymorphs I and II. A degradation deriva-
Form-I
Upon crystallizing commercial pharmaceutical grade CGA from
tive of CGA, 5-HPA, was serendipitously obtained upon solu-
tion crystallization of CGA from 2-butanone : propionic acid
(1 : 1 v/v) (Scheme 1). The three crystal structures illustrate
acid–acid catemer and dimer, amide–amide homodimer, and
acid–amide heterosynthons12 (Scheme 2). In this paper, we
describe X-ray crystal structures of two novel forms and one
degraded form of CGA, which are fully characterized by
spectroscopic (FT-IR, 13C ss-NMR), thermal (DSC), field emis-
sion scanning electron microscopy (FESEM), and powder
X-ray diffraction (PXRD) analyses, and their structures were
confirmed by single crystal X-ray diffraction. Crystallographic
parameters of the novel forms and their hydrogen bonds are
listed in Tables 1 and 2.
The chemical structure of CGA (Scheme 1) indicates that
the conformationally flexible molecule will exhibit different
torsion angles. The two symmetry-independent molecules
in the asymmetric unit of Form-II are labeled A and B.
The values of τ1 (C2–C3–C4–C5) and τ2 (C6–N1–C2–C1) are
79.7°IJ2) and 81.26°IJ16) in Form-I and 177.4°IJ1) and −55.8°IJ2)
MeOH : 2-butanone (1 : 1 v/v), needle-shaped crystals were
obtained which were solved and refined in the orthorhombic
space group P212121. CGA molecules are connected by the acid
catemer synthon of C(4) graph set notation,14 which is stabi-
lized by C–H⋯O (2.58 Å, 144°) interaction resulting in a 1D
tape along [100] (Fig. 2a). The second carboxylic acid and
N-carbamoyl groups are connected through N–H⋯OC (acid)
(1.90 Å, 167°) and N–H⋯OC (carbamoyl) (2.09 Å, 147°)
H-bonds resulting in an R22(11) motif.14 Such tapes are inter-
connected through an acid–amide R22(8) heterosynthon in a
corrugated sheet structure (Fig. 2b).
Form-II
Crystallization of CGA from CH3CN : m-cresol (1 : 1 v/v) mix-
ture afforded block crystals which were solved and refined in
the monoclinic space group P21 with two molecules of CGA
in the asymmetric unit. One of the carboxylic acid and the
N-carbamoyl group of CGA forms an acid–amide R22(8) motif
Scheme 1 The transformation of CGA to 5-HPA during crystallization. The natural L-glutamic acid S-configuration is shown.
Scheme 2 Hydrogen bonded synthons in crystal structures.
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Table 1 Crystallographic parameters of CGA polymorphs and 5-HPA
CGA-Form-I
CGA-Form-II
Hydantoin-5-propionic acid (5-HPA)
Chemical formula
Formula weight
Crystal system
Space group
T [K]
a [Å]
b [Å]
c [Å]
α [°]
C6H10N2O5
190.16
Orthorhombic
P212121
100(2)
5.1179(8)
12.2079IJ18)
12.7508IJ19)
90
C6H10N2O5
190.16
Monoclinic
P21
C6H8N2O4
172.14
Monoclinic
P21
298(2)
6.2475(4)
7.1111(6)
17.3692IJ11)
90
100(2)
5.3615(5)
15.8123IJ15)
9.9631(9)
90
β [°]
γ [°]
90
90
90.899(2)
90
96.792(5)
90
Z
4
4
4
V [Å3]
796.7(2)
1.585
0.139
844.54(14)
1.496
0.131
766.24(10)
1.492
0.127
Dcalc [g cm−3
]
M [mm−1
]
Reflns. collected
Unique reflns.
Observed reflns.
R1 [I > 2IJI)]
wR2 (all)
Goodness-of-fit
Diffractometer
8323
1576
1532
0.0264
0.0632
1.089
8820
3335
3159
0.0323
0.0777
1.049
3079
2191
1973
0.0362
0.0804
1.068
Bruker SMART Apex
Bruker SMART Apex
Oxford CCD
(N–H⋯O: 1.90 Å, 167°; 1.94 Å, 160°; O–H⋯O: 1.63 Å, 172°;
1.66 Å, 170°) resulting in an infinite linear chain along the
[001] direction (Fig. 3a). These chains extend in a 2D sheet
parallel to the (020) plane, being connected to the other car-
boxylic acid and carbamoyl group through N–H⋯OC (acid)
(1.82 Å, 173°; 1.86 Å, 156°) and N–H⋯OC (carbamoyl) (2.21 Å,
147°; 2.25 Å, 146°) bonds (Fig. 3b). Adjacent sheets are
connected through O–H⋯O (1.73 Å, 166°; 1.73 Å, 166°)
hydrogen bonds resulting in a ladder-like structure, and
C–H⋯C short contacts connect these ladders (Fig. 3c).
Table 2 Hydrogen bond metrics in crystal structures
H⋯A D⋯A
D–H⋯A
(°)
Interaction
(Å)
(Å)
Symmetry code
CGA-Form-I
O1–H1⋯O5
1.63
1.72
2.548(1) 167
2.660(2) 176
−x + 1, +y + 1/2, −z +
1/2 + 1
O4–H5⋯O3
x − 1/2, −y + 1/2 + 2,
−z + 1
N1–H6⋯O2
N2–H2A⋯O5
N2–H2B⋯O2
1.90
2.09
2.09
2.914(2) 167
3.009(2) 147
3.084(2) 161
x + 1, +y, +z
x + 1, +y, +z
−x + 1, +y − 1/2, −z +
1/2 + 1
Hydantoin-5-propionic acid (5-HPA)
C3–H3A⋯O4
CGA-Form-II
O1–H1⋯O8
O4–H5⋯O5
O6–H7⋯O3
O9–H11⋯O10
N1–H6⋯O2
N2–H2A⋯O5
N2–H2B⋯O3
N3–H12⋯O7
N4–H13A⋯O8
2.58
3.515(2) 144
x + 1, +y, +z
Hydantoin-5-propionic acid (5-HPA, chemical name 3-IJ2,5-
dioxoimidazolidin-4-yl)propanoic acid) was obtained as a
1.73
1.66
1.73
1.63
1.82
2.25
1.90
1.86
1.94
2.662(2) 171
2.587(2) 170
2.664(2) 166
2.558(2) 172
2.841(2) 173
3.159(2) 146
2.915(2) 167
2.832(2) 156
2.931(2) 160
3.119(2) 147
3.299(2) 141
3.505(2) 138
3.381(2) 145
x, y, z
x, +y, +z − 1
x − 1, +y, +z
x, +y, +z + 1
x + 1, +y, +z
x + 1, +y, +z
x, +y, +z + 1
x + 1, +y, +z
x, +y, +z − 1
x + 1, +y, +z
x, y, z
N4–H13B⋯O10 2.21
C4–H4A⋯O6
C9–H9B⋯O2
2.39
2.63
x, y, z
x − 1, +y, +z
C10–H10A⋯O1 2.44
Hydantoin-5-propionic acid (5-HPA)
O1–H1⋯O6
O5–H7⋯O2
N1–H5⋯O4
N2–H6⋯O7
N3–H11⋯O8
N4–H12⋯O3
C2–H2A⋯O1
C2–H2B⋯O4
C3–H3A⋯O5
C8–H8A⋯O8
C10–H10⋯O3
1.72
1.74
1.89
1.79
1.88
1.81
2.29
2.58
2.53
2.62
2.48
2.646(3) 171
2.670(3) 172
2.914(2) 176
2.820(2) 171
2.900(2) 168
2.801(2) 161
3.239(4) 145
3.618(3) 161
3.397(3) 137
3.551(3) 144
3.365(3) 139
x, y, z
x, y, z
x − 1, +y, +z
x + 1, +y, +z + 1
x + 1, +y, +z
x − 1, +y, +z − 1
−x + 1, +y − 1/2, −z + 1
x − 1, +y, +z
Fig. 1 Overlay diagram of CGA molecules in Form-I and Form-II.
Color code: green = Form-I, red = CGA-Form-IIA, blue = CGA-Form-IIB.
Note that A and B are the two symmetry-independent molecules in
the crystal structure of Form-II.
−x + 2, +y − 1/2, −z + 1
x + 1, +y, +z
−x + 1, +y + 1/2, −z+1
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Fig.
2 (a) 1D tape formed through acid–acid C(4) catemer and
supporting C–H⋯O and N–H⋯O interactions. (b) An acid–acid C(4)
chain and an acid–amide R22(8) heterosynthon result in a corrugated
structure.
Fig. 3 (a) An infinite linear chain formed through an acid–amide R22(8)
heterosynthon. (b) Hydrogen bonded 2D sheet structure formed
through N–H⋯O interactions. (c) O–H⋯O and C–H⋯π interactions in
the inter-sheet region viewed down the a-axis.
by-product of crystallization of CGA from 2-butanone :
propionic acid (1 : 1 v/v) solvent mixture. The structure was
solved and refined in the monoclinic space group P21 with
two molecules of 5-HPA in the asymmetric unit. The mole-
cules form an infinite linear chain through acid–acid R22(8)
(O–H⋯O: 1.72 Å, 171°; 1.74 Å, 172°) and amide–amide R22(8)
(N–H⋯O: 1.79 Å, 171°; 1.81 Å, 161°) homosynthons. Such
chains are inter-connected through N–H⋯O (1.88 Å, 168°;
1.89 Å, 176°) and C–H⋯O (2.58 Å, 161°; 2.62 Å, 144°)
H-bonds in a corrugated 2D sheet, with weak C–H⋯O (2.48 Å,
139°; 2.53 Å, 137°; 2.29 Å, 145°) interactions to complete the
structure (Fig. 4).
Spectroscopic characterization
IR spectroscopic analysis of CGA forms showed significant
differences in the vibrational patterns of their functional
groups.15 The NH2 asymmetric stretch of CIJO)NH2 in
Form-I gives a strong band at 3439.9 cm−1 and symmetric
NH2 stretch at 3344.3 cm−1 with a somewhat weaker inten-
sity, whereas for secondary NH stretch it showed peaks at
3295.4 cm−1. The asymmetric and symmetric stretching fre-
quencies of CIJO)NH2 in Form-II appeared at 3454.3 cm−1
and 3315.3 cm−1 and for secondary NH at 3247.3 cm−1. The
Fig. 4 (a) Infinite linear chain formed through acid–acid R22(8) and
amide–amide R22(8) homosynthons. (b) A 2D corrugated sheet structure
assembled through N–H⋯O and C–H⋯O interactions.
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CO stretching vibration of the carboxylic acid gave a strong
absorption at 1728.5 cm−1 in Form-I and 1709.9 cm−1 in
Form-II. The stretching frequencies for amide CO reso-
nated at 1697.3 cm−1 in Form-I and 1666.9 cm−1 in Form-II.
The signature frequencies are listed in Table S2 (see ESI†),
and the spectra are presented in Fig. 5.
calculated lines of CGA-Form-II crystal structure are due to
temperature effects; the experimental PXRD was recorded at
room temperature and X-ray crystal structure reflections were
collected at 100 K (Fig. 7). The two polymorphs did not show
any phase transformation up to heating at 160 °C in indepen-
dent experiments as monitored by PXRD.
We also characterized CGA forms through solid-state NMR
because this technique provides information about differ-
ences in hydrogen bonding, molecular environment, and
local short range differences.16 13C solid-state NMR spectra
are presented in Fig. 6. The chemical shift values of the alkyl
chain of CGA did not exhibit significant differences for
C2, C3, and C4 and also for amide C6 atoms (Fig. 6 and
Table S3, ESI†). The carboxylic acid C1 and C5 atoms showed
peak values at 175.4 and 179.4 ppm in Form-I, whereas in
Form-II in addition to the C1 and C5 (175.3, 179.4 ppm)
peak positions two extra peaks were observed at 177.33 and
181.7 ppm, which is due to the two molecules in the asym-
metric unit of Form-II (from X-ray analysis).
Thermal and FESEM analyses
After confirming the bulk purity of the polymorphic mate-
rials, DSC analysis18 was carried out to understand their
thermodynamic relationship. No phase transformation was
observed prior to melting at 167.9 °C for Form-I and 160.6 °C
for Form-II (Fig. 8 and Table 3). The high melting polymorph
Form-I has a higher heat of fusion (48.4 kJ mol−1), while the
lower melting polymorph Form-II has a lower heat of fusion
(36.5 kJ mol−1), and hence both polymorphs are mono-
tropically related.19 Both forms were kept at 160 °C for
40 min in a programmable oven to see the thermal effect;
they were stable and no phase transformations were observed
confirming the DSC measurements. Thus Form-I is the stable
polymorph with higher enthalpy of fusion and higher melting
point. The broad endotherm for Form-II was observed in
multiple batches. Normally a broad endotherm in DSC
implies that there is some other event such as phase transfor-
mation or water evolution occurring along with the melting
process or due to low thermal conductivity of the sample,
kinetic effects, etc. The exact reason for a broad endotherm
for Form-II but a sharp peak for Form-I is not clear at the
moment. What we did observe for both Form-I and Form-II is
that heating the sample just beyond the melting point,
Powder X-ray diffraction
CGA Form-I and Form-II are readily distinguishable from
their unique powder XRD line patterns17 (blue lines in Fig. 7).
The experimental PXRD pattern of commercial CGA matches
with the calculated pattern of CGA Form-I (Fig. S1, ESI†). Bulk
material of novel Form-II prepared by rotovaporization
exhibited a unique powder diffraction line pattern that
matches with the calculated pattern of the single crystal X-ray
crystal structure of Form-II (Fig. 7). The slight variations in
the overlay of peaks of the experimental PXRD and the
Fig. 5 A FT-IR spectral comparison of CGA forms indicates that there are significant differences in stretching frequencies between the two
polymorphs.
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Fig. 6 13C ss-NMR spectrum of CGA polymorphs shows that there is a significant difference in chemical shifts. Multiple peaks for carboxylic
C atoms in Form-II are for the two molecules in the asymmetric unit (of the crystal structure).
Fig. 7 The PXRD overlay of the experimental patterns of the CGA forms exhibits unique powder lines and matches with the calculated lines from
the respective crystal structures.
cooling to room temperature and then re-heating did not
show any DSC peak at the same temperature (Fig. S2†). This
implies some kind of chemical transformation of the sample
on heating.
needles and Form-II was found to have a block morphology
at 20 μm magnification scale, and these observed morphol-
ogies were in excellent match with those of the single crystals
mounted for X-ray diffraction.
Field emission scanning electron microscopy is extremely
informative for pharmaceutical solids to understand their
morphology at the nm scale.20 The individual polymorphs
were spread uniformly on a carbon-coated copper grid and
then FESEM was recorded (Fig. 9). Form-I appeared as
Hirshfeld surface and 2D fingerprint plots
Hirshfeld molecular surfaces21 and the associated fingerprint
plots generated using CrystalExplorer 3.0 on the basis of
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Fig. 8 DSC thermograms of CGA Form-I and Form-II to show direct melting without any phase transition.
Table 3 Melting point and enthalpy values of CGA forms
CGA forms
M.p. (°C) IJTonset/Tpeak
)
ΔHfus (kJ mol−1
)
Packing fraction (%)
Stability relation
Monotropic, Form-I is thermodynamic
CGA-Form-I
CGA-Form-II
167.98/169.82
160.61/165.79
48.4
36.5
73.8
69.4
Fig. 9 SEM images of CGA polymorphs (a) Form-I and (b) Form-II to show the differences in morphology and bulk particles.
X-ray diffraction provide quantitative differences between
the polymorphs. The dnorm Hirshfeld surfaces are displayed
using a red–white–blue color scheme, where red highlights
shorter contacts, white is used for contacts around the van
der Waals separation and blue is for longer contacts. The
surfaces are shown as transparent to allow visualization of
the orientation of the molecule and to show the conforma-
tional differences of the CGA molecules in both Form-I and
Form-II. Fig. 10 presents Hirshfeld surfaces of the mole-
cules in both polymorphs. In Form-I, three large circular
depressions (deep red) visible on the top left of the surface
are indicative of hydrogen-bonding (O–H⋯O) contacts, and
additional faint red circles, one on the top left and another
on the right, are from the N–H⋯O hydrogen bonds
(Fig. 10a). In general, the color intensity is high for stron-
ger interactions, and this was observed for Form-I with
shorter H-bonds (O–H⋯O: 1.63 Å, 167°, 1.72 Å, 176°;
N–H⋯O: 1.90 Å, 167°, 2.09 Å, 147°). For Form-II (Fig. 10b),
there are three red regions on the left of the surface and
two intense large circles due to the strong O–H⋯O (1.73 Å,
171°, 1.63 Å, 172°) and also from N–H⋯O (1.94 Å, 160°)
bonds. Additional red regions at the left and right of the
surface arise from the N–H⋯O (1.86 Å, 156°) and O–H⋯O
(1.73 Å, 166°) contacts.
Hirshfeld surfaces and fingerprint plots proved to be partic-
ularly suited for comparing environments of structures with
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complementary surface regions. In Fig. 11, di represents the
distance from the surface to the nearest atom in the
molecule itself, and de represents the distance to the nearest
atom outside the molecule. In Form-I, the presence of long
sharp spikes towards the lower left corner near de + di ≈ 1.6 Å
of the plot indicates O⋯H interactions and it is a characteris-
tic for strong hydrogen bonds resulting from N–H⋯O and
O–H⋯O and C–H⋯O hydrogen bonds (Fig. 11). The contri-
bution of these interactions to the Hirshfeld surfaces is
53.2% in Form-I (Fig. 12). Two sharp spikes are present for
Form-II as well, but because of the two molecules in the
asymmetric unit of the Form-II structure, the contribution
of O⋯H interactions increases (57.8% for molecule A and
56.6% for molecule B) (Fig. 12). Overall there are more
numerous short O⋯H interactions (from N–H⋯O, O–H⋯O,
C–H⋯O) in Form-II. The wings for C–H⋯C interactions are
slightly different for the two forms due to the different meth-
ylene CH atoms involved in C⋯H interactions, but their con-
tribution is very low (2–3%) (Fig. 12). The H⋯H interaction
values are in a similar range (33% for Form-I and 31%
and 32% for Form-II). The calculated density and packing
Fig. 10 Hirshfeld surfaces with mapped dnorm property and
neighboring molecules for (a) Form-I and (b) Form-II projected and
transparency to show the conformation of the molecules in the forms.
Z′ > 1 where the complex interplay between crystallographically
unique molecules could be rationalized in terms of the
Fig. 11 2D Hirshfeld fingerprint plots for Form-I and Form-II. Note that the A and B represent the two crystallographically independent molecules
in Form-II.
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converted into Form-I in 10–12 h in water and ethanol, but in
pentane, it took 24 h for conversion to Form-I (Fig. S3, ESI†).
Interestingly, slurry experiments on Form-I at alkaline pH 9.0
and acetic acid pH 2.4 solutions were found to convert to
Form-II after 24 h, whereas Form-II under similar experimen-
tal conditions was found to be stable for 24 h. We did not
observe trends such as non-polar solvents preferentially
giving metastable forms and polar solvents giving stable
forms;22 however, in our study alkaline pH 9.0 and acetic
acid pH 2.4 solutions resulted in metastable Form-II. In
competitive slurry experiments, wherein a mixture of the two
forms was taken in a 1 : 1 ratio, the product was pure Form-I
(in water, ethanol, and pentane) after 8–10 h of slurry grind-
ing (Fig. 13 and S4, ESI†).
Fig. 12 Contribution of major interactions to the Hirshfeld surface for
Form-I and Form-II of CGA.
Processing of solids can have a major impact on solid–
solid phase transformations which consequently affects the
dissolution of the crystal forms. Neat grinding/liquid assisted
grinding (NG/LAG) at room temperature was carried out on
CGA forms up to 1 h to gain an insight into mechanically
activated polymorph conversion. Grinding is one process that
has been shown to cause changes in polymorphs, and such
a polymorphic transformation is often detrimental to the
efficacy of the formulation, for example, chloramphenicol
palmitate23 and ritonavir24 cases. NG/LAG of pure Form-II was
converted into Form-I upon grinding for 60 min (Fig. 14).
However, extended NG/LAG on Form-I for 2 h did not show
any effect. In addition, competitive NG/LAG experiments on a
mixture of forms gave the stable Form-I after 20 min (Fig. 15).
efficiency of the thermodynamic Form-I is higher (1.585 g cm−3,
73.8% vs. 1.496 g cm−3, 69.4%).
Grinding and solvent-mediated transformations
The most suitable form for drug formulation is the one
which exhibits an appropriate balance between solubility
and stability. Therefore, it is important to establish the stabil-
ity of polymorphic systems. Solution-mediated polymorphic
transformation is a fast, easy, and reliable method to deter-
mine the stable polymorph under conditions relevant to
pharmaceutical tablets. Hence, slurry grinding experiments
were performed on CGA polymorphs in a range of solvents,
e.g. water, ethanol, pentane, alkaline pH 9.0 solution, and
acetic acid pH 2.4 solution. Both polymorphs were subjected
to slurry grinding separately in these solvents to understand
the effect of solvent on the stability outcome of solvent-
mediated processes. Upon slurry grinding in water, ethanol,
and pentane, pure Form-I was stable for 48 h. Pure Form-II
Effect of humidity on CGA forms
Storage conditions of temperature and humidity affect the
stability of pharmaceuticals.25 In particular, hygroscopicity
can have a significant impact on the physical and chemical
stability of active pharmaceutical ingredients (APIs). Therefore,
Fig. 13 The mixture of Form-I and Form-II (blue) was found to convert into Form-I (magenta) after slurry grinding in EtOH solvent. The calculated
PXRD pattern of Form-I (red) is shown for comparison.
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Fig. 14 Overlay of the experimental PXRD pattern of CGA-Form-II (green) with the ground material PXRD pattern of the same form, and Form-II
was found to convert into Form-I after 1 h grinding. The calculated pattern of Form-I is shown for comparison (red).
Fig. 15 Overlay of experimental PXRD patterns of the mixture of Form-I and Form-II (blue), and the mixture was found to convert into Form-I
after 20 min grinding (green). The calculated pattern of Form-I is shown for comparison (red).
it is important to know the physicochemical stabilities of
different forms and the kinetics of their transformation at
different humidity levels. A stability study for CGA poly-
morphs was carried out under accelerated ICH conditions
of 40 °C and 75% RH. Both forms were found to be stable
(no phase transformation or hydrate formation) for the test
period of 2 months based on PXRD analysis (Fig. 16).
Whereas Form-I is thermodynamically stable, the fact that
there was no change in Form-II also came as a surprise.
Under ambient conditions of Hyderabad (35 °C and 40%
RH), the polymorphs are stable for more than six months.
The study of hydration and dehydration of drug substances
quantitatively is an important part of the investigations on
polymorphs. This is achieved by measuring the water sorption–
desorption isotherms on CGA polymorphs by dynamic vapor
sorption (DVS). Samples were subjected to a RH cycle of 10–
90–10% at 40 °C (detailed in the experimental section) and
their water adsorption/desorption behavior was analyzed.
CGA-Form-II was found to adsorb 100.5% water at 90% RH
and there was no significant percentage of water retention
upon bringing down to 10% RH (Fig. 17b). CGA-Form-I
was found to be the least hygroscopic compared to Form-II
(Fig. 17a). Interestingly, moisture sorption profiles of both
forms showed that somewhat different trends may be due to
differences of the structure in the solid state. Water adsorp-
tion and desorption character of the solid materials depends
on the physical characteristics of the material, such as parti-
cle size, surface area, and pore size. From crystal structure
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Fig. 16 The overlay of PXRD patterns of (a) Form-I and (b) Form-II after the stability study at 40 °C and 75% RH shows no form change/hydrate
formation up to 2 months.
Fig. 17 DVS isotherms of the CGA polymorphs show the stability towards moisture uptake for (a) Form-I, whereas (b) Form-II shows negligible
(0.5%) water uptake at 90% RH, but there is no retention of water after desorption.
analysis, both polymorphs have strong O–H⋯O and N–H⋯O
hydrogen bonds, but the packing density of Form-II is lower,
thereby leaving pores for water to enter uniformly during
the sorption cycle (and leave during desorption), whereas the
tighter packing of Form-I means that the behavior of water
sorption/desorption is erratic due to lattice reorganizations
as water molecules interact with the crystal surface. Hence
the DVS plots of polymorphs are different (Fig. 17), but
there is very small net gain/loss (<0.5%) of moisture by the
sample.
we found two polymorphs Form-I and Form-II and a
degraded form hydantoin-5-propionic acid (5-HPA). We were
successful in obtaining diffraction quality single crystals for
all the forms and determined their X-ray crystal structures.
The procedures were optimized to obtain each polymorph in
bulk quantity for PXRD and DSC/DVS analysis. The main dif-
ference in the synthons of these polymorphs is the acid–acid
catemer synthon in Form-I and the acid–amide hetero-
synthon in Form-II. There are significant conformational dif-
ferences between these synthon polymorphs. The higher den-
sity Form-I is thermodynamically stable and the dimorphs
are monotropically related. The polymorphs were fully char-
acterized by FT-IR, 13C ss-NMR, DSC, FESEM, PXRD, and
DVS techniques. Thermodynamic stability and phase trans-
formations were established, and furthermore, the major
Conclusions
Polymorph screening was carried out on the anti-
hyperammonemic drug carglumic acid (CGA). In our search,
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intermolecular interactions in the two crystal structures
were quantified through Hirshfeld surface analysis. Both
polymorphs were stable for 2 months under ICH conditions
of 40 °C and 75% RH, and DVS analysis showed that Form-I
is more stable to moisture than Form-II.
Vibrational spectroscopy
A Thermo-Nicolet 6700 Fourier transform infrared spectro-
photometer with an NXR-Fourier transform Raman module
(Thermo Fisher Scientific, Waltham, Massachusetts) was
used to record IR spectra. IR spectra were recorded on sam-
ples dispersed in KBr pellets. The data were analyzed using
the Omnic software (Thermo Fisher Scientific, Waltham,
Massachusetts).
Experimental section
Materials and methods
Carglumic acid was purchased from Sigma-Aldrich (Hydera-
bad, India) and used without further purification. All other
chemicals were of analytical or chromatographic grade. Water
purified from a deionizer-cum-mixed-bed purification system
(AquaDM, Bhanu, Hyderabad, India) was used in the
experiments.
Solid-state NMR spectroscopy
Solid-state 13C NMR (ss-NMR) spectroscopy provides structural
information about differences in hydrogen bonding, molecu-
lar conformations, and molecular mobility in the solid state.16
The solid-state 13C NMR spectra were obtained using a Bruker
Ultrashield 400 spectrometer (Bruker BioSpin, Karlsruhe,
Germany) utilizing a 13C resonant frequency of 100 MHz
(magnetic field strength of 9.39 T). Approximately 100 mg of
the crystalline sample was packed into a zirconium rotor with
a Kel-F cap. The cross polarization, magic angle spinning
(CP-MAS) pulse sequence was used for spectral acquisition.
Each sample was spun at a frequency of 5.0 0.01 kHz and
the magic angle setting was calibrated by the KBr method.
Each data set was subjected to a 5.0 Hz line broadening
factor and subsequently Fourier transformed and phase
corrected to produce a frequency domain spectrum. The chemi-
Preparation of CGA forms
CGA-Form-I
The commercial material obtained from Sigma-Aldrich
matches with Form-I in our study which was confirmed by
PXRD overlay. The bulk material in pure Form-I can also be
obtained by slurry grinding in EtOH and freeze drying in
water. The bulk material was characterized by FT-IR, 13C
ss-NMR, PXRD and DSC. Single crystals suitable for X-ray
diffraction were obtained upon dissolving 30 mg of CGA in
10 mL of hot MeOH : 2-butanone (1 : 1 v/v) solvent mixture
and left for slow evaporation under ambient conditions.
Colorless needle crystals suitable for X-ray diffraction were
obtained after 3–4 days upon solvent evaporation (M.p. 170–
172 °C).
cal shifts were referenced to TMS using glycine (δglycine
43.3 ppm) as an external secondary standard.
=
Differential scanning calorimetry (DSC)
DSC was performed on a Mettler Toledo DSC 822e module.
Samples were placed in crimped but vented aluminium sam-
ple pans. The typical sample size is 3–5 mg, and the tempera-
ture range is 30–250 °C at 5 °C min−1. Samples were purged
by a stream of dry nitrogen flowing at 80 mL min−1.
CGA-Form-II
Form-II was obtained in bulk upon dissolving 100 mg of CGA
commercial material in 20 ml of water : acetone (1 : 5 v/v) sol-
vent mixture and heated until a clear saturated solution was
obtained. This solution was subjected to rotoevaporation
until the complete removal of the solvent and the resultant
material was kept at high vacuum for 30 min to obtain a dry
powder material. This powder material was confirmed as
Form-II by FT-IR, 13C ss-NMR, PXRD and DSC. Colorless sin-
gle crystals were obtained upon dissolving 30 mg of CGA in
12 mL of hot CH3CN : m-cresol (1 : 1 v/v) solvent mixture and
left for slow evaporation under ambient conditions. Colorless
block crystals suitable for X-ray diffraction were obtained
after 3–4 days upon solvent evaporation (M.p. 166–168 °C).
Field emission scanning electron microscopy (FESEM)
The shape and morphology of the CGA forms were examined
using a Carl Zeiss model Merlin Compact 6027 FESEM with a
beam voltage of 3.0 kV. The sample was spread on a carbon-
coated copper grid. Prior to FESEM imaging, an ultrathin
layer of gold was coated in order to enhance the conductivity
of the sample.
Dynamic vapor sorption (DVS)
Dynamic vapor sorption (DVS) was used to obtain sorption–
desorption kinetic data of the CGA polymorphs. DVS mea-
surements were performed using a Q5000SA vapor sorption
analyzer (TA Instruments, Delaware, USA) at 40 °C. About 4–
10 mg of the sample was placed in a metalized quartz sample
pan and subjected to relative humidity flux from 10% RH to
90% RH and back to 10% RH with a step size of 10% change
in humidity. A dwell time of 60 min was set for a weight
change of >0.1% in the adsorption/desorption phase at a
particular RH (5 min dwell time for a weight change of
Hydantoin-5-propionic acid (5-HPA)
The degraded form (5-HPA) of CGA was obtained upon dis-
solving 30 mg of CGA commercial material in 10 ml of
2-butanone : propionic acid (1 : 1 v/v) solvent mixture and
heated until a clear solution was obtained. The solution was
filtered and left for slow evaporation under ambient condi-
tions. Colorless needle crystals suitable for X-ray diffraction
were obtained after 4–5 days upon solvent evaporation (M.p.
170–152 °C).
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<0.1%). Thus, if the weight loss/gain is >0.1% at a particular
RH, the instrument maintains the same RH for 60 min and
then automatically sets at the next higher/lower value. If the
weight gain/loss is <0.1%, the DVS cycle (10%–90%–10%)
will be completed within 2 hours; otherwise, it will take a
longer duration time.
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X-ray reflections for Form-I and Form-II were collected at
100 K using a Bruker SMART-APEX CCD diffractometer
equipped with a graphite monochromator and Mo-Kα fine-
focus sealed tube (λ = 0.71073 Å). Data reduction was performed
using Bruker SAINT software.26 Intensities were corrected for
absorption using SADABS,27 and the structure was solved and
refined using SHELX-97.28 X-ray reflections for hydantoin-5-
propionic acid (5-HPA) was collected at 298 K using an Oxford
Xcalibur Gemini Eos CCD diffractometer with Mo-Kα radiation
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solve and refine the structures. All non-hydrogen atoms were
refined anisotropically. Hydrogen atoms on heteroatoms were
located from difference electron density maps and all C–H
hydrogens were fixed geometrically. Hydrogen bond geome-
tries were determined using Platon.31 X-Seed32 was used to
prepare packing diagrams. Crystal structures are deposited as
part of the ESI† and may be accessed at www.ccdc.cam.ac.uk/
data_request/cif (CCDC no. 1043890–1043892).
Powder X-ray diffraction
Powder X-ray diffraction of all the samples were recorded on
a Bruker D8 Advance diffractometer (Bruker-AXS, Karlsruhe,
Germany) with Cu-Kα X-radiation (λ = 1.5406 Å) at 40 kV and
30 mA power. X-ray diffraction patterns were collected over
the 2θ range of 5–50° at a scan rate of 1° min−1. Powder Cell
2.4 (ref. 33) (Federal Institute of Materials Research and Test-
ing, Berlin, Germany) was used for Rietveld refinement of
experimental PXRD and calculated lines from the X-ray crys-
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Acknowledgements
D. M. thanks CSIR for fellowship. We thank the Department
of Science and Technology for J. C. Bose fellowship SR/S2/
JCB-06/2009, DST-SERB Scheme Novel solid-state forms of
API's SR/S1/OC-37/2011 for funding. DST (IRPHA, PURSE) and
University Grants Commission (UPE) are thanked for provid-
ing the instrumentation and infrastructure facilities at UOH.
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