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

2 The Rigaku Journal


can be useful for identification of unknowns and 1.2. High Throughput Salt and Polymorph
for unraveling the crystalline composition of Screening
formulated products. More recently, structure After tens of thousands of molecules have
solution from powder diffraction data has en- been screened for activity against a particular
abled structure determination of organic mole- target for disease intervention, a subset of ac-
cules whose structures were previously unable tive molecules advance into lead optimization
to be solved. Each of these advances has ex- where they undergo toxicological testing. Dur-
tended the application of X-ray powder diffrac- ing toxicological testing the primary objective is
tion in the pharmaceutical industry. The follow- to establish the blood levels that the compound
ing paper will attempt to provide a better under- can safely be dosed, using animal models, and
standing of the many roles that X-ray diffraction determine if any short-term reaction to the po-
assumes in the pharmaceutical laboratory as tential therapeutic agent is detected. Prior to the
one progresses a molecule from discovery investment of large amounts of money and
through development and into the post mar- time into a given compound, it becomes impor-
keted phase of a drug products life cycle. tant to assess whether or not the drug candi-
date can be produced in a form that might be
1. Applications in Discovery developable as a product. Material is committed
During the discovery phase, X-ray powder dif- to salt screening activities to produce materials
fraction plays little role, whereas both large and that can be tested to see if they are crystalline,
small molecule crystallography play a signifi- chemically stable, and bioavailable. At this
cant role. Large molecule techniques provide in- point, the purity of the material produced be-
formation about the three dimensional structure gins to approach that acceptable for a final
of receptor sites and target enzymes, often with product, hence crystallizations using this mater-
potential drug candidates as bound ligands. ial becomes more relevant. Purity is not the
These studies enable rational drug design such only factor involved in crystallization, the re-
that structural changes can be made that opti- lated substance profile also plays a major role
mize a drug molecules interaction with the tar- in determining the crystallization outcome. Al-
get structure. teration of the synthetic route will alter the re-
lated substance profile and potentially the re-
1.1. X-ray Crystallography of Small Molecules
sults observed from a crystallization screen. As
X-ray crystallography is used through out the
a consequence, studying the crystallization
discovery and development process. It serves
characteristics of a molecule at the very early
the primary role of structural elucidation of mol-
stages does not supplant the further investiga-
ecular conformation. It is used in the confirma-
tion of its crystallization properties using repre-
tion of structure (COS) reports that are pre-
sentative material later in development. Salt
sented in the investigational new drug applica-
screening is attempted when sufficient material
tion (IND) and new drug application (NDA) that
becomes available and one becomes concerned
prove the identity of the molecule to be tested
about achieving sufficient blood levels to con-
in clinical trials. The technique serves other
duct meaningful toxicological testing. Consider-
niche roles in the pharmaceutical industry. It
able challenges can result when trying to relate
can elucidate the absolute configuration of chi-
the observations from one toxicological study
ral molecules, and aids in structural determina-
to the next if the solid form is not well charac-
tion of systems that contain heterocyclic sys-
terized. Furthermore, often it is necessary to
tems that are not readily amenable to magneti-
achieve much greater levels of bioavailability
zation transfer used in two-dimensional NMR
during toxicological studies aimed at establish-
techniques. Furthermore, heterocyclic systems
ing the margin of safety of a drug. In such stud-
give rise to complex fragmentation patterns in
ies, one often attempts to reach blood levels far
their mass spectra making their interpretation
greater than the anticipated therapeutic level.
more complex. Despite advances in the deter-
The enhanced bioavailability achieved by salt
mination of structures from X-ray powder dif-
formation can enable such studies to be more
fraction data, the importance of such a defini-
effectively conducted. Figure 1, provides an ex-
tive, high-resolution technique as single crystal
ample of a drug and its active metabolites
X-ray diffraction remains as the favorite of syn-
bioavailability when administered as a mesylate
thetic organic chemists.
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.

Vol. 22 No. 1 2005 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- makes this a problem ideally suited for applica-
cules to evaluate, the large array of potential tion of a combinatorial chemistry approach. The
crystallization conditions and methods to be ex- chemistry is conducted in an arrayed fashion,
amined, and the need for assessment of the typically a 96-well titer plate format, see Figure
physical properties of the crystallization hits 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 tion geometry may afford one the ability of in-
where the samples are characterized. In the salt troducing the sample into the diffraction beam
screen, the amount of sample available for without transferring the sample from the crys-
analysis in our process is between one half to tallizer to the diffractometer, pharmaceuticals
two milligrams per well. As for characterization typically have large unit cells having diffraction
of the results from the screen we use birefrin- peaks that fall within four and forty degrees
gence microscopy, Raman microscopy, X-ray two-theta, using a Copper Ka radiation source.
powder diffraction and solubility analysis by In order to collect data at low angles, the foot-
high performance liquid chromatography print of the incident beam impinging upon the
(HPLC). X-ray diffraction remains the gold stan- sample is elongated and is elliptical in shape
dard technique for distinguishing crystals forms when using a pin-hole collimator. This large
of a drug substance, as shown in Figure 3, how- footprint limits how close the samples may be
ever there are numerous challenges one faces positioned with respect to one another if the
in designing a system capable of conducting samples are introduced in an arrayed format.
diffraction analysis from such a sample format. Consideration must be made as to the over-
Moreover, the sample deviates from the ideal- spray into an adjacent sample. The ideal
ized perfectly flat, infinitely thick, compact of geometry for diffraction by high throughput
fine particles, having dimensions of less than screening (HTS) would most likely be transmis-
ten microns. In such instances, an area detec- sion geometry. In such a scenario, the incident
tor is best suited for a variety of reasons. The beam passes through the entire organic sam-
speed of data collection is considerably faster, ple. Unfortunately, the sample must be crystal-
the collection of a significant portion of the dif- lized or moved to a highly localized position or
fraction cone provides better averaged intensity spot. This requires transfer or manipulation of
than would be obtained using a conventional the sample so that it is presented to the incident
point detector. When one characterizes a large beam. This is not easily automated and many
number of samples using very small sample crystallization attempts result in gels or oils
sizes of particles of variable size, one is provid- such that transfer becomes problematic. In our
ing sub-optimal data for the task of phase iden- laboratory we have found both reflection and
tification. Poor particle statistics and preferred transmission approaches yield suitable diffrac-
orientation effects are at their worst. The task of tion data.
sorting through thousands of patterns collected One additional benefit of the HTS is that
on a material presents itself as a new challenge many times the crystals obtained are large
to the diffractionist and is greatly facilitated by enough to isolate and determine their crystallo-
application of chemometric approaches such as graphic structure. Figure 4 provides photomi-
hierarchical cluster or principle component crographs and unit cell packing diagrams of the
analysis [3]. single crystal structures determined on five dif-
There is a choice to be made between trans- ferent polymorphs that were isolated from a
mission and reflection geometry. While reflec- single plate during a HTS run. Thanks in part to

Vol. 22 No. 1 2005 5


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
Fig. 4. Photomicrograph and unit cell diagrams of sin- quality or performance of new drug products. In
gle crystal structures solved from crystals obtained from cases where differences exist which have been
different wells using HTS.
shown to affect drug product performance,
bioavailability or stability, then the appropriate
HTS, it is rare that a drug will achieve the IND solid state should be specified. The polymor-
stage without its single crystal structure having phic form of a drug product is monitored during
been determined, thus reducing the need for stability testing. If a change of form that could
manual attempts at crystal growth for the sole affect safety or efficacy of the product does
purpose of determination of structure. occur then acceptance criteria are established
for the product.
2. Applications in Development
2.2. Diffraction Analysis of Effects of Process-
The development cycle for a drug candidate
ing on Crystal Form
typically takes approximately 13 years and
Polymorphic mixtures can result from a range
nearly one billion dollars. One in 100,000 com-
of pharmaceutical processes, each step may be
pounds make it through the toxicological test-
responsible for generation of a different form.
ing, and clinical trials where the safety and effi-
One example is the drying process of the API.
cacy of the drug is established. During this time
frame, a large amount of effort is placed on the 2.2.1. Monitoring Crystallization Processes
development of robust processes whereby the It is also important to ensure that a salt is ac-
drug product can be produced in high yield and tually formed during recrystallization. When
purity. Furthermore, analytical tests are devel- forming the salt of a relatively weak acid or
oped that enable assessment of a drug API and base, it is possible for them to disproportionate
formulated products quality and performance. upon recrystallization or upon suspension in an
X-ray powder diffraction assumes many differ- aqueous vehicle. Suspensions are commonly
ent roles during the development process and used for toxicological studies as well as in pedi-
extends throughout the life of the product. atric or gerontology samples. If disproportiona-
tion occurs, the characteristics of the solid will

6 The Rigaku Journal


Acid + Free Base (Salt not Formed)

2000

Free Base

Acid

0
5 10 20

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 NIST 675


011
the individual components. For this reason it is hydrated
200
004
important to study the stability of an API in toxi-
cological formulations prior to administration.
1 day
Sometimes disproportionation occurs during
the actual recrystallization steps, particularly
7 days
common with polar solvents and weakly basic
compounds. Rather than observing a unique
pattern for the salt form, the pattern observed is 32 days

of the two individual components that were re- 8 10 12 14 16 18 20


acted to give the desired salt form. By analysis degrees 2Q

of the diffraction pattern it is easy to establish Fig. 6. X-ray powder diffraction patterns of erythro-
that the salt was not formed and different crys- mycin A dihydrate before and after dehydration. The
tallization conditions should be explored to pro- shifting of unit cell parameters toward higher angle indi-
cated a reduction of unit cell volume during relaxation of
vide the desired salt. The sample shown in Fig-
the desolvated lattice.
ure 5 was prepared for toxicological testing and
was analyzed by diffraction prior to testing. If it
using an environmental chamber where the
had been tested, one might anticipate the sam-
drug, erythromycin A dihydrate, was dehy-
ple to have lower bioavailability compared to
drated at low humidity and allowed to exist in a
the desired salt form and potentially hinder es-
moisture free environment for an extended pe-
tablishment of appropriate dosing levels for
riod of time. Though the crystal form does not
clinical investigations.
collapse to an amorphous state nor does it
2.2.2. Monitoring Drying of Active Pharmaceu- transform to a crystallographically distinct
tical Ingredient phase, it shows relaxation behavior over time
After the drug is recrystallized and is filtered that relates to a reduction in unit cell volume.
from its mother liquor, it is typically dried by The lattice energy is expressed as a function of
one of a variety of processes. If the drug is ini- the van der Waals, coulombic, and hydrogen
tially isolated as a hydrated form, it can un- bonding energies, see equation 1. While the van
dergo dehydration and conversion to an anhy- der Waals contribution is not as well described
drous form or perhaps remain in the same crys- as hydrogen bonding interactions for organic
tallographic form, but with altered physical molecules, the relatively large number of van
properties. Figure 6 shows a study conducted der Waals contacts make its contribution to the

Vol. 22 No. 1 2005 7


lattice energy very important in determining the tinuously depending upon the humidity of their
stability of an organic substance. The van der environment [4].
Waals term is often expressed as the Lennard
2.2.3. Monitoring the Influence of Milling of
Jones functional form where r represents the
Active Pharmaceutical Ingredient
interatomic separation and A and B are
After the API is dried, it is typically milled to a
LennardJones coefficients, equation 2. As the
specific particle size by pin milling, ball milling,
crystal lattice of the sample relaxes as its cell
slurry milling or jet milling operations, depen-
volume reduces, heat is given off as the lattice
dent upon material properties and the desired
achieves a more favorable close packing of its
particle size for the API. The milling process can
desolvated structure, see Figure 7.
generate significant amounts of heat and shear
ElatticeEvan der WaalsEcoulombicEhydrogen bonding stress. As a consequence, phase transforma-
(1) tions and amorphous components can be gen-
erated. X-ray diffraction in combination with
Evan der Waals(r )Ar 12Br 6 (2) solid-state NMR is used to understand the im-
pact of milling processes. In Figure 8, the
Similarly, such studies involving humidity-
changes that occurred as the result of milling of
controlled experiments have resulted in better
olanzapine crystals are demonstrated. As the
understanding of cromolyn and cefazolin
particle size is reduced, the intensity of reflec-
sodium non-stoichiometric hydrate drying
tions systematically decreased as preferred ori-
processes, whose unit cell parameters vary con-
entation effects decreased, furthermore broad-
ening of the peaks is observed as the result of
30
Hydrated particle size reduction.
Dried
One can gain better understanding by model-
25
ing such effects by Rietveld refinement. Figure 9
demonstrates the influence of micronization on
20
the X-ray powder diffraction pattern of olanzap-
ine. There is considerable reduction in the peak
15 intensities of the series of reflections relating to
mW/g the crystal morphology. The most developed
10 faces are those whose relative intensity reduce
most dramatically upon milling as preferred ori-
5 entation is reduced, whereas significant degree
of particle size broadening is observed with
0
longer milling times and finer particles.

0 72 144 216 288 360


2.2.4. Monitoring Wet Granulation Processes
time hrs. One often will use X-ray powder diffraction to
assess the processing properties of wet granu-
Fig. 7. Isothermal calorimetry, the change in
power output over time was monitored that corre- lation processes or freeze drying processes. Wet
lated with the reduction in unit cell volume of the ery- granulation involves mixing the active ingredi-
thromycin A dehydrate. ent and possibly some excipients in a mixer. A

Fig. 8. X-ray powder diffraction pattern of olanzapine during milling process.

8 The Rigaku Journal


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 method is usually satisfactory. It is important to
dissolved in the fluid used for granulating. The understand the phase conversions that occur
granulating solution or suspension is added to during processing. By profiling the process, one
the dry powders in the mixer and mixed until can determine which phases dissolve and then
the desired characteristics are achieved. This recrystallized upon drying. If the API dissolves,
usually produces a granule that is of suitable it is important to understand whether it recrys-
characteristics for producing tablets with ade- tallizes or remains amorphous, or if it forms a
quate hardness, dissolution, content uniformity, hydrate or different polymorph. Certainly if it
and other physical characteristics. After the wet dissolves and recrystallizes, its particle size will
granulation step, the product is most often differ from that of the API introduced into the
dried and then milled after drying to get a major dry blend of the formulation. All of these factors
percentage of the product within a desired size can impact the chemical stability of the drug
range. The dry granulation is then processed to product, and potentially its bioavailability or
get an acceptable size range by first screening tablet disintegration. Figure 10 shows a profil-
with a sieving device, and then milling the over- ing of a formulation that is predominantly com-
sized particles. For normal compressed tablets, posed of lactose monohydrate, a smaller
the broad particle size range produced by this 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- Fig. 11. Evidence of hydrated form of mannitol at
ing Processes initial, low-temperature (1) conditions prior to heating
When one produces a freeze-dried formula- to room temperature (2) versus stick diagrams of
tion such as one for parenteral administration known crystal forms of mannitol.
of a drug substance, a bulking agent is often
added to make a uniform plug that rapidly
dissolves upon reconstitution. One of the most problems was when a protein product was crys-
common bulking agents is mannitol. In one tallizing to a size that was 100 times the product
low-temperature study, X-ray powder diffrac- specification. The only thing unique about this
tion was used to identify a new form of manni- recrystallization was that a new supplier of a
tol, a hemi-hydrate that is stable only at low buffer ingredient, sodium phosphate, had been
temperature, see Figure 11 [5]. This crystalline used. When the sample was examined more
form is thought to be involved in a well known closely by X-ray powder diffraction, it was evi-
vial breakage problem that causes a significant dent that the supplier had formed a significant
percentage of vials to break as they are brought amount, 18 percent, sodium pyrophosphate as
back to room temperature while under vacuum. the result of an errant drying process. Spiking
The mechanical stress afforded by the phase of crystallizations with pyrophosphate resulted
transition combined with defects in the glass in reproduction of the larger crystal size and
cause vials to break as they are heated, under demonstrated the role of pyrophosphate in en-
vacuum, to room temperature. hancing crystal growth.
2.3. X-ray Powder Diffraction of Forensic Sam- 2.4. Reverse Engineering of Drug Products
ples It is often desirable, particularly if one is in
X-ray powder diffraction is not the primary the generic industry, to reverse engineer a com-
means for determination of chemical identifica- petitors formulation. This is particularly useful,
tion of organic molecules, lagging well behind since the innovator company has already estab-
nuclear magnetic resonance and mass spec- lished sufficient compatibility of the API with
trometry. As the proprietor of relatively sophis- the excipient ingredients. As a consequence, it
ticated instrumentation, and perhaps the most can be quite useful to determine the composi-
straightforward method for determining the tion of a competitors product, since it will ad-
structural identity of inorganic materials, X-ray vance ones formulation development consider-
powder diffraction is commonly called upon to ably. Many of the pharmaceutical excipients are
identify inorganic unknowns from manufactur- amorphous, whereas others such as mannitol,
ing processes. Whether the investigation in- lactose monohydrate, and anhydrate are crys-
volves analysis of metals when mechanical fail- talline. A combination of IR, X-ray powder dif-
ure occurs, gasket materials, or common inor- fraction, and SSNMR provide a great under-
ganic salts that are formed as byproducts of re- standing of the composition and solid state
actions, X-ray diffraction is the tool of choice. forms of the components that make up the for-
One of the more interesting identifications mulation.

10 The Rigaku Journal


2.5. Quantitative Phase Analysis Using X-ray is particularly troublesome during the earliest
Powder Diffraction stages of development when only limited
Because the physical form of a drug can im- amounts of unmilled material are available
pact pharmaceutical drug product performance, and lot sizes are smaller. As the process is
there are occasions when it is not sufficient to scaled up and controls are made on the particle
merely qualitatively identify the forms present size of the API, the precision and accuracy of
in the API or final product. In such cases, one the method tends to improve. There are numer-
may need to develop a quantitative method to ous considerations that must be made when
monitor the production process and ensure that considering exactly what method is best to de-
the active pharmaceutical ingredient remains velop [9]. Most whole pattern methods require
within manufacturing control limits and the a greater level of knowledge about the phases
drug product performance is not compromised. to be analyzed than the single peak methods.
To meet regulatory requirements for drug prod- Two exceptions would be the whole pattern ap-
uct registration, flow charts were constructed proaches such as factor-based Partial Least
for investigators to use as guidelines for charac- Squares (PLS) or the whole pattern method de-
terizing compounds under development [6]. Ac- scribed by Smith et al. Factor-based PLS is a
cording to Byrn et al., quantitative methods are multivariate method that has found widespread
called for only in cases where mixtures of poly- analytical application [10]. Such methods in-
morphs or hydrates, that are known to have dif- volve establishing a calibration set that is used
ferent physical properties that are relevant to to derive a predictive model for analysis of fu-
dosage form performance (bioavailability or ture data sets. The calibration set should con-
chemical stability) or manufacturing repro- tain as many sources of sample variation as
ducibility, cannot be avoided. In such cases, val- possible. In doing so, one might expect to be
idated methods would be needed to ensure that able to empirically correct for (or at least par-
the ratio of forms is reproducible and the pro- tially compensate for) the influence of preferred
duction process is controlled. A variety of physi- orientation [11]. Such methods require no more
cal techniques (crystallography, spectroscopy, information than single peak methods because
thermal analysis, and microscopy) are useful for they rely on empirically derived correlation of
characterizing the solid forms of pharmaceuti- intensity/composition through training sets.
cals and have recently been reviewed [7]. The program GMQUANT, developed by
X-ray powder diffraction has been used ex- Smith et al., uses a whole powder pattern ap-
tensively for quantitative analysis of mixtures of proach that does not require indexation of the
crystal forms and to a lesser extent the determi- individual components of the mixture [12]. In-
nation of the degree of crystallinity. There are dexation of pharmaceuticals can be highly
two primary methods for quantification; using problematic due to their low symmetry and
either individual peaks or the whole patterns to commonly large unit cell axes. This require-
establish the relationship between phase com- ment is perhaps the greatest limitation to the
position and the intensity of individual peaks or utility of the whole powder pattern approaches
of patterns of the phases being quantified. Klug described later. GMQUANT uses least squares
and Alexander first outlined the basic mathe- minimization of the difference between the digi-
matical relationships for quantitative analysis of tized experimental pattern of a mixture and that
powder mixtures in 1948 [8]. The primary as- of a convolution of the digitized pattern of the
sumption of the diffraction method relies on the individual phases related by weighting factors.
particle size to be sufficiently small that extinc- This approach represents perhaps the most eas-
tion and micro-absorption effects are negligible. ily applied whole pattern method and is suitable
Furthermore, accurate quantification relies for quality control applications, since it requires
heavily on our ability to minimize the effects of minimal interaction of the analyst.
preferred orientation. With inorganic samples,
Whole Powder-pattern Decomposition Meth-
this is typically accomplished by grinding of the
ods (WPPD)
sample. With organic compounds, this may not
In WPPD the integrated intensity parameters,
be so readily accomplished. The potential of
unit-cell parameters, and the peak profile para-
phase inter-conversion while reducing particle
meters are refined by least squares fitting pro-
size is of major concern. Oftentimes crystalline
cedures along with an overall scale-factor relat-
samples can be made amorphous, solvates des-
ing the individual phases (or even amorphous
olvate, and metastable phases convert to more
background). intensity of the diffraction pattern
thermodynamically stable forms. This problem

Vol. 22 No. 1 2005 11


profile intensity, Y, at individual steps, i, of 2q is sideration of many factors and oftentimes de-
decomposed pends on trade-off between ease and sophisti-
N cation of approach.
Y (2i )  B (2i )  S I
k 1
k
j
jk P (2 i ) jk (3)
2.5.1. Applications of Quantitative Analysis to
Pharmaceuticals in the Solid-state
where B(2q i ) is the background function, P(2q i )
is the profile function and Sk is the scale factor. Quantification of mixtures of crystalline forms
There are a number of different background Christ et al. reported the first application of
functions and profile functions that describe the quantitative X-ray powder diffraction to a phar-
diffraction profile [13]. maceutical system in 1948 [15]. In this work, X-
In the WPPD as implemented by Toraya et al. ray powder diffraction was used to quantify the
[14], the peak profiles and background functions amount of crystalline sodium penicillin G in
are decomposed to obtain the best fit to the ex- samples containing a mixture of five other re-
perimental powder pattern of the individual lated substances from the fermentation
pure phase data by least-squares refinement. process. Measuring the intensity of a single re-
The integrated intensities of the pure phases flection against an external standard and plot-
are then stored with the other refined parame- ting its ratio versus concentration determined
ters, such as the profile parameters and the sodium penicillin G content. A straight-line
unit-cell parameters of the phases to be quanti- working curve was obtained. They found that
fied. During quantification step, the integrated addition of carbon black to the sample reduced
intensity of the phase being quantified is scaled, the effect of preferred orientation. We have
as defined in equation 3, such that the total of found this approach effective for minimizing
the scale factors for the component phases sum preferred orientation of a number of other phar-
to unity. The scale factors of the individual com- maceuticals. The advantage of carbon black is
ponents are then refined by least-squares meth- that its color provides a visible indication of the
ods until a best fit is observed with respect to homogeneity of mixing. Being relatively inert
the pattern of unknown composition. In the Ri- and amorphous, it is non-reactive and disfavors
etveld method, essentially the same approach is successive layering of platy crystals, as was the
used as in Torayas method except a structural case with penicillin G samples. In 1963, Shell
model (typically from a crystallographic deter- studied the application of quantitative XRPD to
mination) is used to calculate the intensities of pamoic acid, sulfonamide, tetracycline, and
the individual phases. novobiocin. He further surveyed the use of di-
There seems to be an endless number of ap- rect simple calibration curves and the use of in-
proaches to quantification by X-ray powder dif- ternal standards. In this work, Shell demon-
fraction, some of which have been briefly dis- strated the use of quantitative XRPD for the
cussed herein. When deciding what approach to completion of a salt forming reaction. Duddu
use there are many considerations one must and coworkers studied the reaction between
take into account; are you developing a method two enantiomers to form a racemic crystal [16].
to guide the development of a process, are you In the method, physical mixtures were made of
developing a method that will ultimately serve the racemic crystalline form and one of the
as a quality control application. Many factors in- enantiomers. A linear relationship was ob-
fluence the lab-to-lab or instrument-to-instru- served in a plot of the peak area versus the
ment transferability of a diffraction method. For weight fraction. The standard curve was used to
instance, it might not be advisable for one to determine the amount of crystalline racemate
transfer a Rietveld method to a quality control formed during the solid-state reaction.
laboratory since the success of quantification More recently, the development of quantita-
relies heavily on obtaining a global minimum tive methods by the group of Suryarananan to
from a non-linear least squares refinement investigate carbamazepine has occurred. A sin-
process. The robustness of such a methodology gle-peak powder diffraction method was devel-
is highly dependent upon the skill level of the oped to quantify the relative amounts of anhy-
analyst and may not be readily automated. drous carbamazepine and carbamezapine dihy-
Since organic compounds may decompose with drate to study the kinetics of transformation
time, consideration must also be made about upon suspending the anhydrous form in water
the long-term availability of standards if stan- [17]. Although the study used a single-peak
dard curves are to be used. Development of a method, which may be most affected by pre-
good quantitative method requires careful con- ferred orientation, its influence was minimized

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

14 The Rigaku Journal


in the pharmaceutical industry. While it does [12] Smith, D. K.; Johnson, G. G., Jr.; Scheible, A.; Wims,
serve as a means for identification of primarily A. M.; Johnson, J. L.; Ullmann, G. Quantitative X-ray
Powder Diffraction Method Using the Full Diffraction
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screening for crystal form in the discovery Using the Whole-Powder-Pattern Decomposition
Method. I. Solution from Knowledge of Chemical
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increased availability of single crystal data on Method for Studying the Reaction between Pseu-
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ability to quantify phase purity and to solve Amounts of Anhydrous Carbamazepine and Carba-
mazepine Dihydrate in a Mixture by Powder X-ray Dif-
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Analysis of the Active Tablet Ingredient by Powder X-
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Vol. 22 No. 1 2005 15