THE RIGAKU JOURNAL

VOL. 22 / NO. 1 / 2005, 215

Invited Papers

APPLICATIONS OF X-RAY POWDER DIFFRACTION IN THE

PHARMACEUTICAL INDUSTRY

GREGORY A. STEPHENSON
Eli Lilly and Company, Lilly Research Laboratories, Indianapolis, Indiana 46285. E-mail: gas@lilly.com

The objective of the following article is to present an overview of the application of X-ray
powder diffraction in the pharmaceutical industry, with discussion covering the stages of dis-
covery, development, and post product launch. Such applications range from lead optimization
where many molecules are examined for their solid-state characteristic, that is developability
assessment, in parallel with studies assessing toxicological effects and pharmacological activ-
ity. Other applications relate to development stages where the technique is used primarily for
form identification and monitoring of form during pharmaceutical processing and later quan-
tification activities aimed at product control. The role of additional applications such as struc-
ture solution using the powder method is discussed.

Introduction niques for development. Researchers have ac-

X-ray powder diffraction assumes many roles
in the analysis of pharmaceuticals. Pharmaceu-
ticals, typically organic solids, exist in numer-
ous solid forms that feature different physical
and chemical properties. The vast majority of
pharmaceuticals are administered as oral tablet
dosage forms. In order for a drug molecule to
be absorbed by the body, it must first dissolve.
One of the most effective means of modifying
the physical properties of an active pharmaceu-
tical ingredient (API) is through formation of a
salt, if the molecule possesses a basic or acidic
functional group. In general, salt forms of drugs
are more soluble in aqueous environments, po-
tentially leading to improved bioavailability
when compared to administration of a drug in
its neutral form. Furthermore, there are oppor-
tunities to modify a pharmaceuticals physical
properties by isolation of different crystallo-
graphic forms of the same chemical substance
in different polymorphic, solvated, desolvated
and amorphous solids. This phenomenon pre-
sents an opportunity for pharmaceutical scien-
tists who are interested in understanding the
structure-property relationships in organic
solids. Thus the importance of polymorphism
has prompted much interest in the characteriza-
tion of pharmaceutical solids. Different poly-
morphs require different strategies and tech-
cess to many different analytical techniques
that allow them to study many aspects of poly-
morphism. Single crystal provides the best
structural evidence for polymorphism, however
the stringent sample requirements limit its ap-
plications to high-quality, single crystals. X-ray
powder diffraction allows characterization of
materials that do not meet these criteria. This
technique has been improved through the ad-
vances in beam intensity using rotating anodes
or synchrotron radiation sources, advances in
parallel beam optics, and advances in detector
technology with solid-state detectors, linear and
area position sensitive detectors. These ad-
vances allow for rapid data collection times and
time-resolved studies of phase transformations.
Controlled environment stages allow studies of
polymorphic conversions as a function of tem-
perature and humidity. Analysis of powder dif-
fraction has become more sophisticated, thus
enabling extraction of greater amounts of infor-
mation from a diffraction pattern than ever be-
fore. Crystal indexation packages now offer a
high success rate even for low symmetry mate-
rials [1]. Powder diffraction data are widely
used for quantitative polymorphic mixtures. Ri-
etveld and other whole-powder pattern meth-
ods enable superior accuracy than single peak
methods. The powder diffraction file, the PDF,

2 The Rigaku Journal

can be useful for identification of unknowns and
for unraveling the crystalline composition of
formulated products. More recently, structure
solution from powder diffraction data has en-
abled structure determination of organic mole-
cules whose structures were previously unable
to be solved. Each of these advances has ex-
tended the application of X-ray powder diffrac-
tion in the pharmaceutical industry. The follow-
ing paper will attempt to provide a better under-
standing of the many roles that X-ray diffraction
assumes in the pharmaceutical laboratory as
one progresses a molecule from discovery
through development and into the post mar-
keted phase of a drug products life cycle.
1. Applications in Discovery
During the discovery phase, X-ray powder dif-
fraction plays little role, whereas both large and
small molecule crystallography play a signifi-
cant role. Large molecule techniques provide in-
formation about the three dimensional structure
of receptor sites and target enzymes, often with
potential drug candidates as bound ligands.
These studies enable rational drug design such
that structural changes can be made that opti-
mize a drug molecules interaction with the tar-
get structure.
1.1. X-ray Crystallography of Small Molecules
X-ray crystallography is used through out the
discovery and development process. It serves
the primary role of structural elucidation of mol-
ecular conformation. It is used in the confirma-
tion of structure (COS) reports that are pre-
sented in the investigational new drug applica-
tion (IND) and new drug application (NDA) that
prove the identity of the molecule to be tested
in clinical trials. The technique serves other
niche roles in the pharmaceutical industry. It
can elucidate the absolute configuration of chi-
ral molecules, and aids in structural determina-
tion of systems that contain heterocyclic sys-
tems that are not readily amenable to magneti-
zation transfer used in two-dimensional NMR
techniques. Furthermore, heterocyclic systems
give rise to complex fragmentation patterns in
their mass spectra making their interpretation
more complex. Despite advances in the deter-
mination of structures from X-ray powder dif-
fraction data, the importance of such a defini-
tive, high-resolution technique as single crystal
X-ray diffraction remains as the favorite of syn-
thetic organic chemists.
Vol. 22 No. 1 2005
1.2. High Throughput Salt and Polymorph
Screening
After tens of thousands of molecules have
been screened for activity against a particular
target for disease intervention, a subset of ac-
tive molecules advance into lead optimization
where they undergo toxicological testing. Dur-
ing toxicological testing the primary objective is
to establish the blood levels that the compound
can safely be dosed, using animal models, and
determine if any short-term reaction to the po-
tential therapeutic agent is detected. Prior to the
investment of large amounts of money and
time into a given compound, it becomes impor-
tant to assess whether or not the drug candi-
date can be produced in a form that might be
developable as a product. Material is committed
to salt screening activities to produce materials
that can be tested to see if they are crystalline,
chemically stable, and bioavailable. At this
point, the purity of the material produced be-
gins to approach that acceptable for a final
product, hence crystallizations using this mater-
ial becomes more relevant. Purity is not the
only factor involved in crystallization, the re-
lated substance profile also plays a major role
in determining the crystallization outcome. Al-
teration of the synthetic route will alter the re-
lated substance profile and potentially the re-
sults observed from a crystallization screen. As
a consequence, studying the crystallization
characteristics of a molecule at the very early
stages does not supplant the further investiga-
tion of its crystallization properties using repre-
sentative material later in development. Salt
screening is attempted when sufficient material
becomes available and one becomes concerned
about achieving sufficient blood levels to con-
duct meaningful toxicological testing. Consider-
able challenges can result when trying to relate
the observations from one toxicological study
to the next if the solid form is not well charac-
terized. Furthermore, often it is necessary to
achieve much greater levels of bioavailability
during toxicological studies aimed at establish-
ing the margin of safety of a drug. In such stud-
ies, one often attempts to reach blood levels far
greater than the anticipated therapeutic level.
The enhanced bioavailability achieved by salt
formation can enable such studies to be more
effectively conducted. Figure 1, provides an ex-
ample of a drug and its active metabolites
bioavailability when administered as a mesylate
salt versus being dosed as an HCl salt [2]. In this
study, a 2.6 fold increase was observed by ad-
ministration of the drug in the mesylate form.
3
 3500

3000
LY333531(HCl)
2500 LY333531(M esylate)

ng/mL Plasma
LY338522 (HCl)
2000
LY338522 (M esylate)
1500

1000

500

0
0 8 16 24 32 40 48 56 64 72 80 88 96
Hours

Fig. 1. Mean plasma concentrations in male beagle dogs orally administered. The AUC val-
ues in each dog were approximately 2.6 times higher following administration of mesylate
salts compared to HCl.

Fig. 2. High throughput salt crystallization and characterization. The design of the chemistry
to be conducted is dispensed and crystallized by the three most common industrial proce-
dures, cooling, evaporation, and antisolvent addition.

The combination of the large number of mole-
cules to evaluate, the large array of potential
crystallization conditions and methods to be ex-
amined, and the need for assessment of the
physical properties of the crystallization hits
makes this a problem ideally suited for applica-
tion of a combinatorial chemistry approach. The
chemistry is conducted in an arrayed fashion,
typically a 96-well titer plate format, see Figure
2, so that the samples may be collectively trans-

4 The Rigaku Journal

 Fig. 3. Plate views of X-ray powder diffraction patterns collected on crystals obtained by
HTS.
ferred from where the chemistry occurs to
where the samples are characterized. In the salt
screen, the amount of sample available for
analysis in our process is between one half to
two milligrams per well. As for characterization
of the results from the screen we use birefrin-
gence microscopy, Raman microscopy, X-ray
powder diffraction and solubility analysis by
high performance liquid chromatography
(HPLC). X-ray diffraction remains the gold stan-
dard technique for distinguishing crystals forms
of a drug substance, as shown in Figure 3, how-
ever there are numerous challenges one faces
in designing a system capable of conducting
diffraction analysis from such a sample format.
Moreover, the sample deviates from the ideal-
ized perfectly flat, infinitely thick, compact of
fine particles, having dimensions of less than
ten microns. In such instances, an area detec-
tor is best suited for a variety of reasons. The
speed of data collection is considerably faster,
the collection of a significant portion of the dif-
fraction cone provides better averaged intensity
than would be obtained using a conventional
point detector. When one characterizes a large
number of samples using very small sample
sizes of particles of variable size, one is provid-
ing sub-optimal data for the task of phase iden-
tification. Poor particle statistics and preferred
orientation effects are at their worst. The task of
sorting through thousands of patterns collected
on a material presents itself as a new challenge
to the diffractionist and is greatly facilitated by
application of chemometric approaches such as
hierarchical cluster or principle component
analysis [3].
There is a choice to be made between trans-
mission and reflection geometry. While reflec-
Vol. 22 No. 1 2005
tion geometry may afford one the ability of in-
troducing the sample into the diffraction beam
without transferring the sample from the crys-
tallizer to the diffractometer, pharmaceuticals
typically have large unit cells having diffraction
peaks that fall within four and forty degrees
two-theta, using a Copper Ka radiation source.
In order to collect data at low angles, the foot-
print of the incident beam impinging upon the
sample is elongated and is elliptical in shape
when using a pin-hole collimator. This large
footprint limits how close the samples may be
positioned with respect to one another if the
samples are introduced in an arrayed format.
Consideration must be made as to the over-
spray into an adjacent sample. The ideal
geometry for diffraction by high throughput
screening (HTS) would most likely be transmis-
sion geometry. In such a scenario, the incident
beam passes through the entire organic sam-
ple. Unfortunately, the sample must be crystal-
lized or moved to a highly localized position or
spot. This requires transfer or manipulation of
the sample so that it is presented to the incident
beam. This is not easily automated and many
crystallization attempts result in gels or oils
such that transfer becomes problematic. In our
laboratory we have found both reflection and
transmission approaches yield suitable diffrac-
tion data.
One additional benefit of the HTS is that
many times the crystals obtained are large
enough to isolate and determine their crystallo-
graphic structure. Figure 4 provides photomi-
crographs and unit cell packing diagrams of the
single crystal structures determined on five dif-
ferent polymorphs that were isolated from a
single plate during a HTS run. Thanks in part to
5

Fig. 4. Photomicrograph and unit cell diagrams of sin-
gle crystal structures solved from crystals obtained from
different wells using HTS.
HTS, it is rare that a drug will achieve the IND
stage without its single crystal structure having
been determined, thus reducing the need for
manual attempts at crystal growth for the sole
purpose of determination of structure.
2. Applications in Development
The development cycle for a drug candidate
typically takes approximately 13 years and
nearly one billion dollars. One in 100,000 com-
pounds make it through the toxicological test-
ing, and clinical trials where the safety and effi-
cacy of the drug is established. During this time
frame, a large amount of effort is placed on the
development of robust processes whereby the
drug product can be produced in high yield and
purity. Furthermore, analytical tests are devel-
oped that enable assessment of a drug API and
formulated products quality and performance.
X-ray powder diffraction assumes many differ-
ent roles during the development process and
extends throughout the life of the product.
6 The Rigaku Journal
2.1. Phase Identification
The role of X-ray powder diffraction for iden-
tification of unknown chemicals is limited in the
pharmaceutical industry, since there are numer-
ous techniques available for determining mole-
cular structure of organic molecules. The tech-
niques of mass spectrometry and nuclear mag-
netic resonance are dominant in this role. On
the other hand, due to its speed of data acquisi-
tion and sensitivity, X-ray powder diffraction ex-
ists as the primary tool for phase identification,
that is identifying the unique crystallographic
form of a given substance.
With the intent and objective to assist in the
establishment of a single set of global specifica-
tions for new drug substances and products,
guidelines were written that provide guidance
on setting and justifying acceptance criteria and
the selection of test procedures for new drug
substances of synthetic chemical origin which
have not been previously registered in the U.S.,
European Union or Japan. In particular, a guide-
line was written that provides a rationale for ap-
proaching the development of pharmaceuticals
that are polymorphic. In the guideline, the
broad sense of the word polymorphism is used
such that it encompasses true polymorphs, hy-
drates, solvates, and amorphous forms. Differ-
ences in forms could, in some cases, affect
quality or performance of new drug products. In
cases where differences exist which have been
shown to affect drug product performance,
bioavailability or stability, then the appropriate
solid state should be specified. The polymor-
phic form of a drug product is monitored during
stability testing. If a change of form that could
affect safety or efficacy of the product does
occur then acceptance criteria are established
for the product.
2.2. Diffraction Analysis of Effects of Process-
ing on Crystal Form
Polymorphic mixtures can result from a range
of pharmaceutical processes, each step may be
responsible for generation of a different form.
One example is the drying process of the API.
2.2.1. Monitoring Crystallization Processes
It is also important to ensure that a salt is ac-
tually formed during recrystallization. When
forming the salt of a relatively weak acid or
base, it is possible for them to disproportionate
upon recrystallization or upon suspension in an
aqueous vehicle. Suspensions are commonly
used for toxicological studies as well as in pedi-
atric or gerontology samples. If disproportiona-
tion occurs, the characteristics of the solid will
 Acid + Free Base (Salt not Formed)
2000
Free Base
Acid
0
5 10
2-Theta - Scale
Fig. 5. The X-ray powder diffraction pattern showing that disproportionation of the salt oc-
curred during recrystallization.
no longer be that of the salt form, but rather of
the individual components. For this reason it is
important to study the stability of an API in toxi-
cological formulations prior to administration.
Sometimes disproportionation occurs during
the actual recrystallization steps, particularly
common with polar solvents and weakly basic
compounds. Rather than observing a unique
pattern for the salt form, the pattern observed is
of the two individual components that were re-
acted to give the desired salt form. By analysis
of the diffraction pattern it is easy to establish
that the salt was not formed and different crys-
tallization conditions should be explored to pro-
vide the desired salt. The sample shown in Fig-
ure 5 was prepared for toxicological testing and
was analyzed by diffraction prior to testing. If it
had been tested, one might anticipate the sam-
ple to have lower bioavailability compared to
the desired salt form and potentially hinder es-
tablishment of appropriate dosing levels for
clinical investigations.
2.2.2. Monitoring Drying of Active Pharmaceu-
tical Ingredient
After the drug is recrystallized and is filtered
from its mother liquor, it is typically dried by
one of a variety of processes. If the drug is ini-
tially isolated as a hydrated form, it can un-
dergo dehydration and conversion to an anhy-
drous form or perhaps remain in the same crys-
tallographic form, but with altered physical
properties. Figure 6 shows a study conducted
Vol. 22 No. 1 2005
20
NIST 675
011
hydrated
200
004
1 day
7 days
32 days
8 10 12 14 16 18 20
degrees 2Q
Fig. 6. X-ray powder diffraction patterns of erythro-
mycin A dihydrate before and after dehydration. The
shifting of unit cell parameters toward higher angle indi-
cated a reduction of unit cell volume during relaxation of
the desolvated lattice.
using an environmental chamber where the
drug, erythromycin A dihydrate, was dehy-
drated at low humidity and allowed to exist in a
moisture free environment for an extended pe-
riod of time. Though the crystal form does not
collapse to an amorphous state nor does it
transform to a crystallographically distinct
phase, it shows relaxation behavior over time
that relates to a reduction in unit cell volume.
The lattice energy is expressed as a function of
the van der Waals, coulombic, and hydrogen
bonding energies, see equation 1. While the van
der Waals contribution is not as well described
as hydrogen bonding interactions for organic
molecules, the relatively large number of van
der Waals contacts make its contribution to the
7
lattice energy very important in determining the
stability of an organic substance. The van der
Waals term is often expressed as the Lennard
Jones functional form where r represents the
interatomic separation and A and B are
LennardJones coefficients, equation 2. As the
crystal lattice of the sample relaxes as its cell
volume reduces, heat is given off as the lattice
achieves a more favorable close packing of its
desolvated structure, see Figure 7.
Elattice
Evan der WaalsEcoulombicEhydrogen bonding
(1)
Evan der Waals(r )
Ar 12Br 6
Similarly, such studies involving humidity-
controlled experiments have resulted in better
understanding of cromolyn and cefazolin
sodium non-stoichiometric hydrate drying
processes, whose unit cell parameters vary con-
30
Hydrated
Dried
25
20
15
mW/g
10
5 entation is reduced, whereas significant degree
0
0 72 144 216 288
time hrs.
Fig. 7. Isothermal calorimetry, the change in
power output over time was monitored that corre-
lated with the reduction in unit cell volume of the ery-
thromycin A dehydrate.
Fig. 8. X-ray powder diffraction pattern of olanzapine during milling process.
8 The Rigaku Journal
tinuously depending upon the humidity of their
environment [4].
2.2.3. Monitoring the Influence of Milling of
Active Pharmaceutical Ingredient
After the API is dried, it is typically milled to a
specific particle size by pin milling, ball milling,
slurry milling or jet milling operations, depen-
dent upon material properties and the desired
particle size for the API. The milling process can
generate significant amounts of heat and shear
stress. As a consequence, phase transforma-
tions and amorphous components can be gen-
erated. X-ray diffraction in combination with
(2) solid-state NMR is used to understand the im-
pact of milling processes. In Figure 8, the
changes that occurred as the result of milling of
olanzapine crystals are demonstrated. As the
particle size is reduced, the intensity of reflec-
tions systematically decreased as preferred ori-
entation effects decreased, furthermore broad-
ening of the peaks is observed as the result of
particle size reduction.
One can gain better understanding by model-
ing such effects by Rietveld refinement. Figure 9
demonstrates the influence of micronization on
the X-ray powder diffraction pattern of olanzap-
ine. There is considerable reduction in the peak
intensities of the series of reflections relating to
the crystal morphology. The most developed
faces are those whose relative intensity reduce
most dramatically upon milling as preferred ori-
of particle size broadening is observed with
longer milling times and finer particles.
360
2.2.4. Monitoring Wet Granulation Processes
One often will use X-ray powder diffraction to
assess the processing properties of wet granu-
lation processes or freeze drying processes. Wet
granulation involves mixing the active ingredi-
ent and possibly some excipients in a mixer. A
 Fig. 9. Demonstration of the influence of particle size reduction on the intensity of the 1 0 0
reflection of olanzapine.

Fig. 10. Characterization of changes occurring during drying of a wet granulated formula-
tion. The top trace is of lactose monohydrate. The middle three are of the formulation com-
posed of a mixture of hydrated and anhydrous lactose, as well as the drug (D) at 15, 60, and
120 minutes (top to bottom). The lower trace shows a diffraction pattern of anhydrous lactose.

binder is typically added in the dry mix state or
dissolved in the fluid used for granulating. The
granulating solution or suspension is added to
the dry powders in the mixer and mixed until
the desired characteristics are achieved. This
usually produces a granule that is of suitable
characteristics for producing tablets with ade-
quate hardness, dissolution, content uniformity,
and other physical characteristics. After the wet
granulation step, the product is most often
dried and then milled after drying to get a major
percentage of the product within a desired size
range. The dry granulation is then processed to
get an acceptable size range by first screening
with a sieving device, and then milling the over-
sized particles. For normal compressed tablets,
the broad particle size range produced by this
method is usually satisfactory. It is important to
understand the phase conversions that occur
during processing. By profiling the process, one
can determine which phases dissolve and then
recrystallized upon drying. If the API dissolves,
it is important to understand whether it recrys-
tallizes or remains amorphous, or if it forms a
hydrate or different polymorph. Certainly if it
dissolves and recrystallizes, its particle size will
differ from that of the API introduced into the
dry blend of the formulation. All of these factors
can impact the chemical stability of the drug
product, and potentially its bioavailability or
tablet disintegration. Figure 10 shows a profil-
ing of a formulation that is predominantly com-
posed of lactose monohydrate, a smaller
amount of anhydrous lactose and a small con-

Vol. 22 No. 1 2005 9

centration of a water-insoluble drug when the
dry blend is formed. After adding the granulat-
ing solution the lactose is exclusively converted
to the monohydrate form, as the drying process
proceeds, most of the lactose monohydrate is
converted to anhydrous lactose after two hours
of drying. Throughout this process, due to the
drugs low solubility, the API remains in the
same crystalline state as it was first introduced
into the formulation. It is not completely un-
common for acid-base reactions to occur during
wet granulation. In fact, many of the an-
giotensin converting enzyme (ACE) inhibitor
formulations, such as that of enalipril maleate,
have used basic excipients to neutralize the
acidic API and render it more chemically stable
in the tablet.
2.2.5. X-ray Powder Diffraction of Freeze Dry-
ing Processes
When one produces a freeze-dried formula-
tion such as one for parenteral administration
of a drug substance, a bulking agent is often
added to make a uniform plug that rapidly
dissolves upon reconstitution. One of the most
common bulking agents is mannitol. In one
low-temperature study, X-ray powder diffrac-
tion was used to identify a new form of manni-
tol, a hemi-hydrate that is stable only at low
temperature, see Figure 11 [5]. This crystalline
form is thought to be involved in a well known
vial breakage problem that causes a significant
percentage of vials to break as they are brought
back to room temperature while under vacuum.
The mechanical stress afforded by the phase
transition combined with defects in the glass
cause vials to break as they are heated, under
vacuum, to room temperature.
2.3. X-ray Powder Diffraction of Forensic Sam-
ples
X-ray powder diffraction is not the primary
means for determination of chemical identifica-
tion of organic molecules, lagging well behind
nuclear magnetic resonance and mass spec-
trometry. As the proprietor of relatively sophis-
ticated instrumentation, and perhaps the most
straightforward method for determining the
structural identity of inorganic materials, X-ray
powder diffraction is commonly called upon to
identify inorganic unknowns from manufactur-
ing processes. Whether the investigation in-
volves analysis of metals when mechanical fail-
ure occurs, gasket materials, or common inor-
ganic salts that are formed as byproducts of re-
actions, X-ray diffraction is the tool of choice.
One of the more interesting identifications
10
Fig. 11. Evidence of hydrated form of mannitol at
initial, low-temperature (1) conditions prior to heating
to room temperature (2) versus stick diagrams of
known crystal forms of mannitol.
problems was when a protein product was crys-
tallizing to a size that was 100 times the product
specification. The only thing unique about this
recrystallization was that a new supplier of a
buffer ingredient, sodium phosphate, had been
used. When the sample was examined more
closely by X-ray powder diffraction, it was evi-
dent that the supplier had formed a significant
amount, 18 percent, sodium pyrophosphate as
the result of an errant drying process. Spiking
of crystallizations with pyrophosphate resulted
in reproduction of the larger crystal size and
demonstrated the role of pyrophosphate in en-
hancing crystal growth.
2.4. Reverse Engineering of Drug Products
It is often desirable, particularly if one is in
the generic industry, to reverse engineer a com-
petitors formulation. This is particularly useful,
since the innovator company has already estab-
lished sufficient compatibility of the API with
the excipient ingredients. As a consequence, it
can be quite useful to determine the composi-
tion of a competitors product, since it will ad-
vance ones formulation development consider-
ably. Many of the pharmaceutical excipients are
amorphous, whereas others such as mannitol,
lactose monohydrate, and anhydrate are crys-
talline. A combination of IR, X-ray powder dif-
fraction, and SSNMR provide a great under-
standing of the composition and solid state
forms of the components that make up the for-
mulation.
The Rigaku Journal
2.5. Quantitative Phase Analysis Using X-ray
Powder Diffraction
Because the physical form of a drug can im-
pact pharmaceutical drug product performance,
there are occasions when it is not sufficient to
merely qualitatively identify the forms present
in the API or final product. In such cases, one
may need to develop a quantitative method to
monitor the production process and ensure that
the active pharmaceutical ingredient remains
within manufacturing control limits and the
drug product performance is not compromised.
To meet regulatory requirements for drug prod-
uct registration, flow charts were constructed
for investigators to use as guidelines for charac-
terizing compounds under development [6]. Ac-
cording to Byrn et al., quantitative methods are
called for only in cases where mixtures of poly-
morphs or hydrates, that are known to have dif-
ferent physical properties that are relevant to
dosage form performance (bioavailability or
chemical stability) or manufacturing repro-
ducibility, cannot be avoided. In such cases, val-
idated methods would be needed to ensure that
the ratio of forms is reproducible and the pro-
duction process is controlled. A variety of physi-
cal techniques (crystallography, spectroscopy,
thermal analysis, and microscopy) are useful for
characterizing the solid forms of pharmaceuti-
cals and have recently been reviewed [7].
X-ray powder diffraction has been used ex-
tensively for quantitative analysis of mixtures of
crystal forms and to a lesser extent the determi-
nation of the degree of crystallinity. There are
two primary methods for quantification; using
either individual peaks or the whole patterns to
establish the relationship between phase com-
position and the intensity of individual peaks or
of patterns of the phases being quantified. Klug
and Alexander first outlined the basic mathe-
matical relationships for quantitative analysis of
powder mixtures in 1948 [8]. The primary as-
sumption of the diffraction method relies on the
particle size to be sufficiently small that extinc-
tion and micro-absorption effects are negligible.
Furthermore, accurate quantification relies
heavily on our ability to minimize the effects of
preferred orientation. With inorganic samples,
this is typically accomplished by grinding of the
sample. With organic compounds, this may not
be so readily accomplished. The potential of
phase inter-conversion while reducing particle
size is of major concern. Oftentimes crystalline
samples can be made amorphous, solvates des-
olvate, and metastable phases convert to more
thermodynamically stable forms. This problem
Vol. 22 No. 1 2005
is particularly troublesome during the earliest
stages of development when only limited
amounts of unmilled material are available
and lot sizes are smaller. As the process is
scaled up and controls are made on the particle
size of the API, the precision and accuracy of
the method tends to improve. There are numer-
ous considerations that must be made when
considering exactly what method is best to de-
velop [9]. Most whole pattern methods require
a greater level of knowledge about the phases
to be analyzed than the single peak methods.
Two exceptions would be the whole pattern ap-
proaches such as factor-based Partial Least
Squares (PLS) or the whole pattern method de-
scribed by Smith et al. Factor-based PLS is a
multivariate method that has found widespread
analytical application [10]. Such methods in-
volve establishing a calibration set that is used
to derive a predictive model for analysis of fu-
ture data sets. The calibration set should con-
tain as many sources of sample variation as
possible. In doing so, one might expect to be
able to empirically correct for (or at least par-
tially compensate for) the influence of preferred
orientation [11]. Such methods require no more
information than single peak methods because
they rely on empirically derived correlation of
intensity/composition through training sets.
The program GMQUANT, developed by
Smith et al., uses a whole powder pattern ap-
proach that does not require indexation of the
individual components of the mixture [12]. In-
dexation of pharmaceuticals can be highly
problematic due to their low symmetry and
commonly large unit cell axes. This require-
ment is perhaps the greatest limitation to the
utility of the whole powder pattern approaches
described later. GMQUANT uses least squares
minimization of the difference between the digi-
tized experimental pattern of a mixture and that
of a convolution of the digitized pattern of the
individual phases related by weighting factors.
This approach represents perhaps the most eas-
ily applied whole pattern method and is suitable
for quality control applications, since it requires
minimal interaction of the analyst.
Whole Powder-pattern Decomposition Meth-
ods (WPPD)
In WPPD the integrated intensity parameters,
unit-cell parameters, and the peak profile para-
meters are refined by least squares fitting pro-
cedures along with an overall scale-factor relat-
ing the individual phases (or even amorphous
background). intensity of the diffraction pattern
11
profile intensity, Y, at individual steps, i, of 2q is
decomposed
N cation of approach.
Y (2i )
B (2i )  S I
k
1
k
j
jk P (2 i ) jk
where B(2q i ) is the background function, P(2q i )
is the profile function and Sk is the scale factor.
There are a number of different background
functions and profile functions that describe the
diffraction profile [13].
In the WPPD as implemented by Toraya et al.
[14], the peak profiles and background functions
are decomposed to obtain the best fit to the ex-
perimental powder pattern of the individual
pure phase data by least-squares refinement.
The integrated intensities of the pure phases
are then stored with the other refined parame-
ters, such as the profile parameters and the
unit-cell parameters of the phases to be quanti-
fied. During quantification step, the integrated
intensity of the phase being quantified is scaled,
as defined in equation 3, such that the total of
the scale factors for the component phases sum
to unity. The scale factors of the individual com-
ponents are then refined by least-squares meth-
ods until a best fit is observed with respect to
the pattern of unknown composition. In the Ri-
etveld method, essentially the same approach is
used as in Torayas method except a structural
model (typically from a crystallographic deter-
mination) is used to calculate the intensities of
the individual phases.
There seems to be an endless number of ap-
proaches to quantification by X-ray powder dif-
fraction, some of which have been briefly dis-
cussed herein. When deciding what approach to
use there are many considerations one must
take into account; are you developing a method
to guide the development of a process, are you
developing a method that will ultimately serve
as a quality control application. Many factors in-
fluence the lab-to-lab or instrument-to-instru-
ment transferability of a diffraction method. For
instance, it might not be advisable for one to
transfer a Rietveld method to a quality control
laboratory since the success of quantification
relies heavily on obtaining a global minimum
from a non-linear least squares refinement
process. The robustness of such a methodology
is highly dependent upon the skill level of the
analyst and may not be readily automated.
Since organic compounds may decompose with
time, consideration must also be made about
the long-term availability of standards if stan-
dard curves are to be used. Development of a
good quantitative method requires careful con-
12
sideration of many factors and oftentimes de-
pends on trade-off between ease and sophisti-
(3)
2.5.1. Applications of Quantitative Analysis to
Pharmaceuticals in the Solid-state
Quantification of mixtures of crystalline forms
Christ et al. reported the first application of
quantitative X-ray powder diffraction to a phar-
maceutical system in 1948 [15]. In this work, X-
ray powder diffraction was used to quantify the
amount of crystalline sodium penicillin G in
samples containing a mixture of five other re-
lated substances from the fermentation
process. Measuring the intensity of a single re-
flection against an external standard and plot-
ting its ratio versus concentration determined
sodium penicillin G content. A straight-line
working curve was obtained. They found that
addition of carbon black to the sample reduced
the effect of preferred orientation. We have
found this approach effective for minimizing
preferred orientation of a number of other phar-
maceuticals. The advantage of carbon black is
that its color provides a visible indication of the
homogeneity of mixing. Being relatively inert
and amorphous, it is non-reactive and disfavors
successive layering of platy crystals, as was the
case with penicillin G samples. In 1963, Shell
studied the application of quantitative XRPD to
pamoic acid, sulfonamide, tetracycline, and
novobiocin. He further surveyed the use of di-
rect simple calibration curves and the use of in-
ternal standards. In this work, Shell demon-
strated the use of quantitative XRPD for the
completion of a salt forming reaction. Duddu
and coworkers studied the reaction between
two enantiomers to form a racemic crystal [16].
In the method, physical mixtures were made of
the racemic crystalline form and one of the
enantiomers. A linear relationship was ob-
served in a plot of the peak area versus the
weight fraction. The standard curve was used to
determine the amount of crystalline racemate
formed during the solid-state reaction.
More recently, the development of quantita-
tive methods by the group of Suryarananan to
investigate carbamazepine has occurred. A sin-
gle-peak powder diffraction method was devel-
oped to quantify the relative amounts of anhy-
drous carbamazepine and carbamezapine dihy-
drate to study the kinetics of transformation
upon suspending the anhydrous form in water
[17]. Although the study used a single-peak
method, which may be most affected by pre-
ferred orientation, its influence was minimized
The Rigaku Journal
by selection of lines least influenced by pre- considerable advancement in the utility of the
ferred orientation. This was accomplished by technique beyond mere phase identification.
systematic evaluation of the standard deviation
3.1. Calculated Powder Diffraction Patterns
of the individual peak areas as the result of re-
Preferred orientation can cause problems in
peat sample preparation. Another important as-
interpretation of powder diffraction patterns, it
pect of the study was that it demonstrated the
provides uncertainty in the intensity that peaks
minimal influence of a change of hydration
are to be observed. Consequently the appear-
state on the mass absorption coefficient of the
ance of peaks at locations where there previ-
compound, changing from 5.21 cm2 g1 to
ously might not have been is a cause for alarm
5.87 cm2 g1 in the anhydrous versus hydrated
for the diffractionist. The use of calculated pow-
phase. In most of the pharmaceutical literature,
der diffraction patterns has become an indis-
this difference is not considered. The lack of
pensable tool for the diffraction laboratory,
correction for differences in mass absorption of
where the presence of peaks that are not ac-
different states of hydration of pharmaceuticals
counted for in the calculated powder diffraction
being quantified can be expected to introduce a
pattern or indexed pattern provides unambigu-
relatively small, albeit unnecessary error in
ous evidence for phase impurity.
such analyses.
It has become common practice to calculate
In another study by Suryanarayanan, a pow-
powder diffraction patterns from single crystal
der diffraction method was developed to quan-
data to aid in the establishment of phase purity.
tify the active ingredient in intact tablets [18].
The amount of information afforded by such
Two model drugs were examined, lithium car-
comparison is many-fold. First of all, the estab-
bonate and carbamazepine. The drugs of vari-
lishment of the relationship between structure
ous weight fractions were mixed with micro-
and experimental observation of the pattern en-
crystalline cellulose, as well as starch in the
ables one to understand the variances of a dif-
case of carbamazepine, and then compressed
fraction pattern due to preferred orientation,
into tablets. Though use was not made of the
poor particle size statistics, or even the alter-
entire diffraction pattern, the intensities of sev-
ation of unit cell parameters due to changes in
eral different reflections were used for quantifi-
environment, most commonly with respect to
cation, thereby resulting in a more robust
relative humidity. Such comparisons also pro-
method. The objective was to monitor the drug
vide assurance of the chemical composition of a
content in individual tablets during accelerated
given form by definitively establishing the drug
stability studies. The method was carried out on
to counter ion and or solvent stoichiometry.
intact tablets and resulted in a simple procedure
With the relatively common occurrence of for-
that could readily be automated.
mation of concomitant polymorphs in organic
There are numerous opportunities for further
solids [19], the comparison of calculated and
development of quantitative methods for phar-
experimental patterns is an indispensable tool
maceuticals. One can expect to see more fre-
helping us ensure the phase purity of reference
quent application of whole pattern ap-
patterns. Furthermore, the comparison provides
proaches to quantitative X-ray powder diffrac-
an added level of confidence in the quality of a
tion analysis of pharmaceuticals. Because of the
sample used to generate data for publication or
ever-increasing need to reduce particle size to
patent purposes. Currently the International
enhance bioavailability of highly permeable-
Centre for Diffraction Data (ICDD) has included
water insoluble compounds, there continues to
calculated powder diffraction patterns in the
be a largely unmet need for the detection of low
powder diffraction file (PDF) [20]. While the in-
levels of an amorphous component present in
clusion of such data greatly expands the num-
crystalline pharmaceuticals as well as a lack of a
ber of organic phases available in the database,
good internal standard for quantification of or-
most of which were collected at room tempera-
ganic phases.
ture, the potential effect of temperature of data
collection should be considered during phase
3. Interplay of Single Crystal and Pow-
identification. Considerable differences may be
der Diffraction
observed between a calculated powder diffrac-
Because of the commonness of programs
tion pattern obtained from a low temperature
that are useful for calculation of X-ray powder
data set versus an experimentally determined
diffraction data from single crystal structures
diffraction pattern collected at Room tempera-
and our ability to make comparisons with ex-
ture, see Figure 12.
perimental powder patterns, there has been

Vol. 22 No. 1 2005 13

 Fig. 12. Comparison of simulated powder diffraction pattern of olanzapine based on struc-
tures collected at 100C (lower) and 23C (upper).
3.2. Structures Solution from Powder Diffrac-
tion Data
While single crystal X-ray diffraction will con-
tinue to be the sole provider of absolute config-
uration data for organic molecules [21], X-ray
powder diffraction continues to advance to a
state where it can provide reasonably accurate
information about molecular conformation,
three dimensional packing arrangement, and
hydrogen bonding patterns in organic molecu-
lar solids. With the increasing performance and
availability of programs that apply Monte
Carlo/Simulated Annealing (MC/SA) or molecu-
lar searching methods to powder diffraction
data, the solution of structures that were previ-
ously inaccessible to single crystal methods are
now possible [19, 2224]. The use of powder
diffraction for the solution of structures has re-
cently been reviewed [25]. Certain types of crys-
talline substances are difficult or beyond the ca-
pability of single crystal techniques, even when
performing micro-crystal diffraction using a
synchrotron radiation source. In particular,
metastable polymorphic forms that are isolated
by rapid crystallization from the melt or that
rapidly grow from solution are typically ex-
tremely small or highly flawed crystals. Desol-
vation processes commonly result in crystals
that appear to have the same particle size as the
crystals from which they were formed; how-
ever, upon examination by polarized light mi-
croscopy they are usually composed of micro-
crystalline aggregates [26]. As in single crystal
diffraction, the first step, indexing, presents per-
haps the biggest obstacle. Typically if one can
successfully index a crystal, its structure can be
determined. This is apparently true for the pow-
der method also. The number of variables of
the problem can be limited by restraining bond
distances and angles of the molecule to known
values. Portions of molecules may be consid-
ered rigid bodies based on chemical intuition
14
and when similar fragments are found to have
consistent conformations in different structures
that are reported in the Cambridge Structural
Database (CSD) [27], or by searching ones own
library of molecules of a particular therapeutic
category. Alternatively, conformations can be
calculated using molecular mechanics, semi-
empirical, or ab initio methods. As a result,
seemingly complex molecules can be reduced
to merely a few torsion variables describing the
molecules internal degrees of freedom, three
Cartesian coordinates describing the position of
the molecule in the unit cell, and three Eulerian
angles describing molecular orientation.
As described in section 2.2.5, a previously un-
known hydrated crystalline phase was identified
using low temperature X-ray powder diffrac-
tion. That crystalline phase had been implicated
in the common vial breakage problem of
freeze-drying mannitol containing parenteral
formulations. Despite the sample consisting of
a mixture of crystalline phases of mannitol, the
structure was solved on the phase-mixture pat-
tern, having the peaks associated with the
phases whose structures had previously been
determined subtracted from the pattern [28]. The
intensity and resolution afforded by the synchro-
tron radiation source resulted in the reasonably
low R factor. The information gained from the
studies of the freeze drying process using low
temperature X-ray diffraction combined with
the subsequent structure solution by the pow-
der method highlights the important role that X-
ray diffraction can play in the pharmaceutical
industry as it helped understand a problem that
has plagued parenteral product manufacturing
for more than one half of a century.
4. Conclusions
X-ray powder diffraction continues to be the
gold standard technique for identification of
different crystalline forms of a given substance
The Rigaku Journal
in the pharmaceutical industry. While it does
serve as a means for identification of primarily
inorganic unknowns, it remains the dominant
technique for studies of polymorphism, solva-
tion, and salt form identification. We have seen
advances of the application to high throughput
screening for crystal form in the discovery
phases of pharmaceutical research. The tech-
nique continues to play a primary role in exami-
nation of lots of active pharmaceutical ingredi-
ent produced and the influence of pharmaceuti-
cal process on crystal form and crystallinity. The
increased availability of single crystal data on
discovery compounds and programs for com-
parison of calculated and experimental diffrac-
tion patterns has had significant impact on our
ability to quantify phase purity and to solve
structures of materials whose structures have
eluded us for many years.
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15