Minireview Solid-State Nuclear Magnetic Resonance Spectroscopy-Pharmaceutical Applications

MINIREVIEW
Solid-State Nuclear Magnetic Resonance

Spectroscopy—Pharmaceutical Applications
PATRICK A. TISHMACK,1 DAVID E. BUGAY,1 STEPHEN R. BYRN1,2
1
SSCI, Inc., 3065 Kent Avenue, West Lafayette, Indiana 47906
2
Department of Industrial and Physical Pharmacy, Purdue University, West Lafayette, Indiana 47906
Received 17 April 2002; revised 2 August 2002; accepted 4 September 2002
ABSTRACT: Solid-state nuclear magnetic resonance (NMR) spectroscopy has become an

integral technique in the field of pharmaceutical sciences. This review focuses on the use
of solid-state NMR techniques for the characterization of pharmaceutical solids (drug
substance and dosage form). These techniques include methods for (1) studying structure
and conformation, (2) analyzing molecular motions (relaxation and exchange spectro-
scopy), (3) assigning resonances (spectral editing and two-dimensional correlation spec-
troscopy), and (4) measuring internuclear distances. ß 2003 Wiley-Liss, Inc. and the
American Pharmaceutical Association J Pharm Sci 92:441–474, 2003
Keywords: solid-state NMR; pharmaceutical analysis
INTRODUCTION analytical applications.8 More recently, solid-

state NMR spectroscopy has come to the forefront
The use of nuclear magnetic resonance (NMR) of analytical techniques. Approximately 80–90%
spectroscopy in pharmaceutical research has a of pharmaceutical products on the market exist in
long and successful history, primarily in early the solid form. However, solid-state NMR spectro-
stages of drug discovery. Most of these NMR scopy, in many cases, is just beginning to be
studies have been performed with liquid solutions applied to pharmaceutical problem solving and
and were conducted primarily to analyze rela- methods development.
tively small organic molecules, with significantly Regulatory documentation is now making spe-
fewer applications for macromolecules like pro- cific reference to solid-state NMR spectroscopy. A
teins, nucleic acids, carbohydrates, and polymers. flow-chart approach to the physical characteriza-
Studies have included: (a) Elucidation of the tion of pharmaceutical solids was first published in
structure of compounds,1 (b) investigation of the 1995.9 In this approach to determining the number
chirality of drug substances,2 (c) the analysis of of polymorphic forms, spectroscopy, and specifi-
cellular metabolism,3,4 and (d) studies of pro- cally solid-state NMR spectroscopy, is outlined
teins.5–7 In consideration of the later stages of as a recommended technique. The Food and Drug
commercial drug development, NMR spectroscopy Administration (FDA) has also recognized the
is traditionally used for conformational analysis, need for solid-state NMR characterization of drug
structure elucidation (impurity profiling), and substances or products. In the most recent In-
ternational Committee of Harmonization (ICH)
Q6A guideline for the setting of specifications for
Correspondence to: David E. Bugay (Telephone: 765-463-
0112; Fax: 765-497-2649; E-mail: dbugay@ssci-inc.com)
drug substance and product, spectroscopy (IR,
Journal of Pharmaceutical Sciences, Vol. 92, 441–474 (2003)
Raman, and solid-state NMR) is referred to in
ß 2003 Wiley-Liss, Inc. and the American Pharmaceutical Association decision tree four (investigating the need to set
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 3, MARCH 2003 441

442 TISHMACK, BUGAY, AND BYRN
acceptance criteria for polymorphism in drug resolves conflicting results obtained with other
substances and drug products).10 Worldwide reg- analyses, such as IR, X-ray powder diffraction, and
ulatory authorities now recognize the importance thermal methods.16 In addition, a dictate of form-
of solid-state NMR spectroscopy and how the tech- ulation technology is that the physical form of the
nique is intimately tied to the drug development drug substance, after being defined and verified,
process. should not change once the product has been
During the course of developing pharmaceutical manufactured. Solid-state NMR spectroscopy
compounds, it is becoming increasingly important provides a powerful method for comparing the
to characterize the drug in its dispensed form, physical form of the drug substance after pharma-
which is frequently a solid. It has long been known ceutical processing or manufacturing. Further-
that drugs may exist in more than one polymorphic more, solid-state NMR spectroscopy provides a
form.11 These forms sometimes display very sig- method for the analysis of mixtures of solid forms
nificant differences in solubility, bioavailability, in both the pure drug substance and in dosage
processability, and physical/chemical stability.12 forms.16
Hence, solid-state analytical techniques are neces- Solid-state NMR spectroscopy has increasingly
sary for the characterization of new chemical been used for the analysis of pharmaceutical solids
entities during the drug development process. within the last decade. Recent advances in NMR
The study of pharmaceutical compounds in the hardware and software have made the acquisi-
solid state must take place both at the bulk level tion of high-resolution, multinuclear NMR spectra
and for the dosage form. Sometimes, the extreme of solids routine. Unfortunately, because of pro-
conditions of processing the formulation into the prietary constraints and possibly underutilization
dosage form can alter the drug,13 increase its in- of the technique, relatively few solid-state NMR
teraction with excipients, or significantly impact investigations of pharmaceutical compounds have
the stability properties of the solid.14 These obser- appeared in the literature.
vations reinforce the need for sensitive and specific This review is necessarily limited to a selective
solid-state analytical techniques. summary of published solid-state NMR investiga-
Etter and Vojta published a paper in 1989 on the tions and includes several new examples from our
concurrent use of solid-state NMR spectroscopy laboratories. Where possible, examples of newer
and X-ray crystallography to study the structure solid-state NMR experiments that include phar-
of pharmaceutical solids.15 Crystallographic ef- maceutical compounds have been chosen. How-
fects such as polymorphism, multiple molecules ever, some recent techniques that are not yet
per asymmetric unit cell, disorder, intra- and commonly used are also included because of the
intermolecular hydrogen bonding, tautomerism, potentially valuable information that may be
and solvation were all investigated by solid-state gained from their wider use in the analysis of
NMR spectroscopy. Their work demonstrated that pharmaceuticals.
structural and conformational analyses of phar-
maceutical solids by spectroscopic and diffraction
methods provide complementary data. Therefore,
SOLID-STATE NMR SPECTROSCOPY
full characterization of drug compounds should
include both techniques.
Basic Principles
It is important to note that solid-state NMR
spectroscopy is a nondestructive, multinuclear The most obvious difference between NMR spec-
technique that can probe the chemical environ- troscopy of solids and liquids is that solids usually
ment of specific nuclei within molecules. Addition- produce much broader peaks. This difference is
ally, it is a quantitative technique that may be used largely due to strong dipolar coupling interactions
in conjunction with other solid-state techniques, and chemical shift anisotropy (CSA) in solids that
such as thermogravimetric analysis (TGA), differ- are eliminated or reduced by the fast random
ential scanning calorimetry (DSC), optical micro- motions of liquid samples. Both dipolar coupling
scopy, infrared (IR) and Raman spectroscopy, and and CSA are orientation-dependent phenomena.
X-ray diffraction techniques (single crystal and Dipolar coupling depends on the orientation of
powder), for the investigation of pharmaceutical two nuclear dipoles relative to each other. CSA
solids. depends on the orientation of a nuclear dipole
Solid-state NMR analysis of different solid with respect to the direction of the static magnetic
forms can provide crucial information that often field. The line width problem in solids has been
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 3, MARCH 2003

PHARMACEUTICAL USES FOR NMR 443
substantially reduced by using NMR probes that conditions.26 Voelkel reviewed the relaxation
can handle high decoupling power and by rapidly processes in solid-state NMR spectroscopy for
spinning the sample. High-power proton decoupl- polymeric systems.27 Solid-state NMR reviews
ing effectively removes both homonuclear (1H, 1H) related to pharmaceutical compounds are less
and heteronuclear (1H, 13C) dipolar coupling and extensive because there are relatively few pub-
results in narrower line widths in the observed lished applications.16,28–30
carbon spectrum. The homonuclear 13C– 13C di- In addition to the line-narrowing and sensitiv-
polar coupling is usually not of concern unless the ity-enhancing methods used for solid-state NMR
sample has been 13C-labeled. Magic angle spin- spectroscopy, accurate temperature control for the
ning (MAS) was first introduced by Andrew et al. sample is required to obtain valuable information
for observing the solid-state 23Na NMR spectrum in a variety of solids. Variable temperature solid-
of a sodium chloride crystal.17 They demonstrated state NMR spectroscopy is generally a require-
that the minimum line width is obtained when the ment for obtaining relaxation data that is neces-
angle between the sample spinning axis and the sary to analyze molecular motions in solids (see
direction of the static magnetic field is 548 440 Pharmaceutical Applications, Analysis of Molec-
(54.748). A variety of applications for MAS in ular Motions—Relaxation). It is generally not a
solid-state NMR spectroscopy is discussed in a trivial exercise to obtain accurate temperatures,
dated, but still relevant review by Andrew.18 The and many reports have been published on the topic
dipolar coupling interactions between nuclei in of temperature calibration for solid-state NMR
solids can also be an advantage for improving the spectroscopy.31–46 In particular, it is important to
sensitivity of nuclei with low natural abundance know the design characteristics of the probe and
(rare spin). Pines et al. first demonstrated a sensi- rotor because gas flow into the probe controls the
tivity enhancement method by using cross polar- rotor speed and stability as well as regulates the
ization (CP) of magnetization from protons to sample temperature.35,44 Some important consid-
carbons in solid-state NMR spectroscopy.19 This erations are the position of the thermocouple,
method has subsequently been used for enhanc- whether or not the bearing gas is used for heating
ing the sensitivity of many different nuclei, us- and cooling, the type of gas used, and the type and
ually by CP from protons, although fluorine and diameter of the rotor. The spinning speed and
phosphorus have also been used. In ideal cases, radio frequency energy used to obtain experimen-
CP can enhance the sensitivity of a rare spin tal data are also important. The importance of
nucleus by a factor that is proportional to the these factors indicates that the calibration proce-
magnetogyric ratios (g) of the nuclei involved (e.g., dure must be essentially identical to the conditions
gH/gC 4 for 1H and 13C). Schaefer and Stejskal that will be used for acquiring data. One of the
combined MAS and CP to obtain the first 13C CP/ most common temperature calibration standards
MAS NMR spectrum, and this has become the (PbNO3) has no protons and does not require
standard experiment for solid-state NMR spectro- CP.31–33,35 –38 However, CP can cause tempera-
scopy of organic solids.20,21 The significance of this ture changes in a sample, and the calibrations may
development is apparent in that a modern solid- need to be performed under CP/MAS conditions
state NMR spectrometer can acquire a recogniz- even if doing so will not enhance the sensitivity. A
able 13C spectrum of a crystalline compound with potential further complication is that the tempera-
a single scan. The CP from protons provides ano- ture dependence of relaxation times can cause
ther advantage because the time between scans is large variations in the signal-to-noise ratio for a
based on the T1 relaxation time of the protons particular sample at different temperatures. Opti-
rather than those of 13C nuclei, which are usually mization of several parameters at each tempera-
much longer. ture, including the Hartmann–Hahn matching
The developments in and applications of solid- condition, the contact time, and the relaxation
state NMR spectroscopy over the last two de- delay between scans, will generally alleviate this
cades have been discussed in detail in several problem at the expense of a significant amount of
reviews22,23 and a monograph.24 Jelinski and time.
Melchior have reviewed basic solid-state NMR There are several acceptable standards for
spectroscopy, including instrumental aspects and calibrating accurate temperatures for solid-state
a number of useful insights for obtaining high NMR spectroscopy. The first temperature calibra-
quality spectra.25 Michel and Engelke thoroughly tion standard (chemical shift thermometer) used
covered the details of CP methods under MAS for solid-state NMR spectroscopy was samarium

acetate.45 Solid–solid or solid–plastic phase tran- morphs, a single crystal of sufficient quality may
sitions in organic compounds have been used for not be available for structure determination by
single-point temperature calibrations.34,39,40 Tau- X-ray crystallography. However, by appropriate
tomerism of an organic dye molecule has been used resonance assignment of the NMR spectrum, the
as a chemical shift thermometer for solid-state origin of the polymorphism may be inferred from
15
N NMR spectroscopy.41,42 Lead nitrate has more chemical shift differences for identical nuclei
recently received attention as a chemical shift in each polymorph. These benefits do not indicate
thermometer because its lineshape and peak posi- that solid-state NMR spectroscopy can replace
tion change in a systematic manner with tempera- X-ray diffraction or any other analytical method.
ture.31–33,35 –38 A significant disadvantage is the Instead, the advantages of solid-state NMR serve
high toxicity of lead nitrate, and proper safety as a reminder to us that a multidisciplinary ap-
precautions should be observed. proach to solid-state characterization should
One of the most commonly used applications of include spectroscopic, diffraction, and thermal
solid-state NMR spectroscopy for pharmaceutical methods.
solids involves solid-form characterization (includ- The usefulness of solid-state NMR analysis of
ing polymorphs, hydrates, solvates, and amor- polymorphs was first demonstrated for hydroqui-
phous forms) of new chemical entities. The use of none by Ripmeester.47 Subsequently, there have
solid-state NMR spectroscopy for the investigation been many studies using various solid-state NMR
of polymorphism can be understood based on the methods to examine polymorphic forms of organic
following model. If a compound exists in two, true compounds. Analysis of polymorphic systems is
polymorphic forms labeled as A and B, each form is currently the predominant use of solid-state NMR
crystallographically different. Therefore, a carbon spectroscopy in pharmaceutical research (see
nucleus in form A may be situated in a slightly Pharmaceutical Applications, Polymorphism).
different molecular environment than the same
carbon nucleus in form B. Although the chemical
bonding of the carbon nucleus is the same in each
METHODS
form, the local environment may be different
because of restricted motions in the solid state.
Quantitative Analysis
These restricted motions cause differences in
the local environment for one or more carbons in In most of the publications describing the use
each polymorph because their spatial position is of solid-state NMR spectroscopy for the char-
different with respect to the other nuclei in the acterization of pharmaceutical compounds, the
molecule. The solid-state NMR spectra show this majority of the work has dealt with qualitative
difference as a change in the isotropic chemical studies, with brief references to the possibility
shift of the corresponding carbon in each poly- of quantitative analysis. An excellent guide to
morph. If one is able to obtain pure material for the utilization of MAS and CP techniques for ac-
the two forms, analysis and assignment of the quiring quantitative solid-state NMR data has
solid-state NMR spectra of the two forms can lead been outlined by Harris.48 To acquire solid-state
to the origin of the crystallographic differences in NMR spectra in which the signal intensities
the two polymorphs. Solid-state NMR spectro- directly reflect the number of nuclei producing
scopy is thus an important tool in the multidis- them, data acquisition parameters, such as re-
ciplinary approach to analyzing polymorphism. cycle delay time, pulse durations, CP contact
There are a number of significant advantages to time, Hartmann–Hahn matching conditions, and
using solid-state NMR spectroscopy for the study decoupling power, must be explicitly determin-
of polymorphism. Compared with diffuse reflec- ed for each chemical system. The magic angle and
tance IR, Raman, and X-ray powder diffraction spin rate also must be set accurately to obtain
techniques, solid-state NMR spectroscopy is a bulk quantitative measurements.
technique in which particle size effects have little Harris also compared the CP experiment with
impact on the intensity of the measured signal. the single-pulse excitation (SPE) experiment for
In addition, NMR spectroscopy is a quantitative obtaining quantitative solid-state NMR spectra.
technique under proper data acquisition proce- This comparison was important because the dy-
dures. Therefore, the intensity of the signal will namics of CP are different for each carbon within a
be directly proportional to the number of nuclei compound, and the ratios of peak intensities in a
producing it. In some investigations of poly- CP/MAS spectrum may be significantly different

from the ratios of the corresponding carbon Molecular Motions—Relaxation

nuclei.48 The SPE (or Bloch decay) experiment
for solids is the application of one excitation pulse The relaxation of nuclear magnetism towards its
followed by acquisition, usually with high-power equilibrium value in a static magnetic field is
proton decoupling and MAS without CP. This characterized by the spin-lattice relaxation time
pulse sequence is useful for systems in which the constant (T1).52–54 In this process, the excess
longitudinal relaxation time (T1) of the observed energy from the spin system is transferred to the
nucleus is relatively short or there are no nuclei surroundings or lattice. Interactions with ran-
with which to cross polarize the observed nucleus. domly fluctuating magnetic fields at the Larmor
To get quantitative peak integrals with the SPE frequency of the nucleus stimulate relaxation.
experiment, the relaxation delay time must be long These fields arise from motions of other nuclear
enough to allow the magnetization of each nucleus magnetic moments. Relaxation is most efficient
to reach equilibrium prior to acquiring another when the maximum number of fluctuating mag-
scan (normally 1–5 times T1). The effects of CP can netic fields occur at the Larmor frequency of the
be determined by acquiring an SPE spectrum with observed nucleus. Spin-lattice relaxation in the
a suitably long relaxation delay and comparing rotating frame (T1r) is similar to T1 relaxation
the result to a CP/MAS spectrum of the same except that it occurs at a much lower field
compound. strength during the spinlock time used to obtain
The differential NMR relaxation times of nuclei the Hartmann–Hahn condition for CP in solid-
within a molecule present significant problems for state NMR spectroscopy. Both of the spin-lattice
obtaining quantitative data using CP. With the relaxation times (T1 and T1r) are relatively long
CP/MAS experiment, it is necessary to measure for highly mobile liquids and continue to decrease
the relaxation profiles and the rates of CP for each with increasing viscosity until the solid state is
carbon to obtain quantitative information.49,50 reached. A minimum occurs in either relaxation
The sample spinning rate is also important time when the motions in the lattice (the fluctuat-
because the peak intensity is distributed among ing magnetic fields at the appropriate frequency)
the spinning sidebands, and one must account for most efficiently relax the observed nucleus. The
each sideband to obtain accurate quantitative relaxation times then began to increase again as
measurements.50 the lattice motions are no longer efficient at caus-
Quantitative analysis of solid-state NMR spec- ing relaxation. The transverse or spin–spin re-
tra has been used to study very difficult cases, such laxation time (T2) is an entropic process in which
as the components of wood.51 This study demon- random spin flips dephase the coherent magne-
strated that quantitative determination of amor- tization produced by input of radio frequency
phous materials is possible using solid-state NMR energy. The T2 relaxation time is longer for mobile
spectroscopy. liquids and decreases asymptotically to a mini-
mum value at the so-called ‘‘rigid lattice limit’’ in
solids.
Molecular Motions
NMR relaxation processes in solids can provide
Molecular motions can be studied by both solid- information about the molecular motions occur-
state NMR spectroscopy and X-ray crystallo- ring at the location of the nucleus being observed.
graphy. Solid-state NMR spectroscopy offers In organic solids, 1H and 13C relaxation para-
several unique approaches to studying molec- meters are generally of most interest, but 15N
ular motions in solids. These approaches include relaxation measurements are practical in cases
(1) the study of processes that result in coales- where labeling is used. The T1 relaxation time
cence of NMR resonances using variable tem- is useful for measuring motions in the mega-
perature solid-state NMR spectroscopy, (2) deter- hertz frequency range (Larmor frequency). The
mination of the relaxation processes (T1, T1r, T2) T1r relaxation time is sensitive to kilohertz
of individual nuclei using variable-temperature frequency motions because the spinlock field
solid-state NMR spectroscopy, (3) use of spectral strength is much lower than the static magne-
editing methods based on CP rates to detect tic field strength.20 The T2 relaxation time is
the different parts of a molecule with unusual similar to T1r in the range of motions that it can
mobility, (4) comparison of solid-state MAS measure.55
spectra measured with and without CP, and (5) The methods of inversion recovery and expo-
various methods to analyze chemical exchange. nential decay-to-zero have been used to measure

T1 relaxation in solids.56–58 The latter method is intramolecular orientational exchange and are
more practical for solid-state NMR spectroscopy, not useful for examining intermolecular exchange
where compounds have long T1 relaxation times, processes. The method of 2D rotor-synchroniz-
because it produces fewer artifacts.56,58 Methods ed exchange spectroscopy is capable of detecting
for obtaining and analyzing the T1r relaxation intramolecular and intermolecular exchange,71
times of 1H and 13C nuclei in polymers have been and this method has been used for studying the
extensively described.20,59 –63 conformation of a peptide.72 Other methods for 2D
The 1H T1 and T1r times are generally of less exchange spectroscopy (2D EXSY) have been used
utility for site-specific analyses because the high to study molecular motions in the solid-state. The
natural abundance and strong dipolar interactions solid-state 2D EXSY pulse sequence is a modifica-
of protons can lead to spin diffusion and nonspe- tion of the NOESY (nuclear Overhauser effect
cific relaxation times. MAS may increase or de- spectroscopy) experiment used for liquids.73,74 For
crease the 1H T1 relaxation times in some solids solids, CP (rather than the usual 908 pulse) is
depending on their proton densities.64 However, used for the preparation period of the pulse
1
H relaxation times are useful if one wants to study sequence. The resulting spectrum provides a 2D
mixtures of compounds that have different relaxa- spectrum that has cross peaks corresponding to
tion properties. the chemical shifts of the exchanging nuclei. In all
A major drawback to relaxation experiments is cases, the exchange process must be slow on the
that they can be very time consuming because NMR time scale to be observed by 1D and 2D
multiple measurements at different temperatures exchange spectroscopy.
are usually required. For 13C and 15N NMR in
particular, T1 relaxation times can be minutes to
hours in length, especially for highly crystalline Resonance Assignments
material with minimal molecular motions. The A distinct disadvantage of the broad lines ob-
application of relaxation time measurements to served in most solid-state NMR spectra is that
pharmaceutical solids has not received much scalar couplings (J-couplings) are generally not
attention probably because of the amount of time resolved. This situation makes it substantially
needed to carry out the experiments. However, a more difficult to unambiguously assign the che-
carefully acquired set of relaxation data can pro- mical shifts in the spectra. Tentative resonance
vide unique information on molecular motions for assignments can usually be made because many
a compound that cannot be obtained by other of the chemical shifts of organic solids are not
means. This usefulness is apparent in an interest- drastically different from those observed for the
ing recent study in which the 13C and 2H T1 re- same compound in solution. However, the direct
laxation times were used to determine the C--H determination of the solid-state NMR resonance
bond length in ferrocene.65 assignments is necessary to obtain accurate struc-
tural and conformational information.
Molecular Motions—Exchange Spectroscopy
Resonance Assignments—Spectral Editing
One of the most useful applications for NMR
spectroscopy is in the analysis of dynamic pro- A number of ‘‘spectral editing’’ pulse sequences
cesses in molecules for both liquids and solids. A have been developed to aid in the assignment of
large time-scale range (109 –102 s) for molecu- solid-state NMR spectra.75 One of the first editing
lar motions can be studied by NMR spectroscopy sequences, based on a modification of the stan-
by using pulse sequences to analyze chemical dard 13C CP/MAS sequence, was dipolar dephas-
exchange or relaxation processes (see Methods, ing or interrupted decoupling.76,77 In this pulse
Molecular Motions—Relaxation). Very slow mo- sequence, the 1H and 13C radio frequency fields
tions in solids have been studied with rotor- are removed for a brief period (40–100 ms) before
synchronized one-dimensional exchange spectro- acquisition to allow the carbon magnetization to
scopy by sideband alternation (ODESSA),66 the decay because of 1H/13C dipolar coupling. The
time-reversed ODESSA,67,68 or exchange-induced difference in the strength of the dipolar coupling
sidebands (EIS).69,70 Motional correlation times for each type of carbon (CH2 > CH > C, CH3) re-
and activation energies for certain slow exchange sults in a spectrum in which the CH and CH2
processes can be extracted from the spectra.69 carbon resonances are substantially reduced or
However, these methods are generally limited to eliminated. The methyl carbons are not greatly

affected because their rapid rotational motion re- The spectral editing methods just discussed rely
duces the dipolar coupling interaction. The qua- on dipolar coupling of nuclei through space to dif-
ternary carbons are only slightly affected because ferentiate between types of carbons in a compound.
dipolar coupling to them is much weaker without One disadvantage of these techniques is their
a directly bonded proton. Dipolar dephasing is sensitivity to molecular motion that is apparent in
useful for both crystalline and amorphous materi- the minimal response of rotating methyl groups to
als and has been successfully used to study the the editing schemes described. This effect can also
complex mixture of components in coal that be observed by the lack of selectivity for some
have very broad peaks.78 In an analogous method, resonances in a CPPI spectrum (see Figure 1 and
Peng and Frydman have used differences in the Pharmaceutical Applications, Conformation and
chemical shift anisotropies (CSA-dephasing) of Stereochemistry). Several editing pulse sequences
carbons for spectral editing of 3-methylsalicylic have been developed to use scalar coupling that
acid and p-methoxybenzoate. However, the results acts through chemical bonds rather than through
were generally poorer compared with dipolar space as for the dipolar coupling interactions.90–92
dephasing spectra.79 The method of Terao was demonstrated for solid
Relaxation differences between individual nu- camphor, a nearly spherical molecule that has
clei or regions of a sample can be used to perform unusually small dipolar couplings and very nar-
spectral editing. The delayed contact pulse se- row line widths due to rapid molecular motion.92
quence is one method based on relaxation differ- Many organic solids do not have such narrow
ences that has been used to determine amorphous resonances, which limits the usefulness of Terao’s
and crystalline regions in polymers.59 In this technique. Emsley’s group has recently exploited
modification of the standard 13C CP/MAS se- J-coupling for editing of solid-state 13C NMR
quence, the 1H spinlock field is turned on for a spectra of organic compounds with broader line
short time prior to turning on the 13C spinlock field widths.91 The SS-APT pulse sequence, analogous
to allow the magnetization to decay for protons to the attached proton test for solution 13C NMR,
with short T1r relaxation times. An alternative can be used to differentiate between CH and CH2
method is to begin with standard 1H– 13C CP, and groups in solids. The SS-APT spectra (Figures 2
then remove the 1H spinlock field while maintain-
ing the 13C spinlock field for a short time prior to
acquisition with high-power proton decoupling.80
These approaches select for specific carbons based
on differences in their T1r relaxation times. Inde-
ed, similar pulse sequences have been used to
determine 13C T1r values.60,61,81
Another editing sequence, referred to as
WIMSE (windowless isotropic mixing for spectral
editing), was designed to identify methylene
carbons. However, the necessity for rotor synchro-
nization and multiple spectra for subtraction
make this method less convenient to use.82
One of the most successful editing methods for
solid-state NMR spectroscopy incorporates CP and
polarization inversion or cross depolarization and
is commonly referred to as CPPI.83–86 The polar-
ization inversion is accomplished by changing the
phase of either the 1H or 13C spinlock pulse by 1808
to reverse the direction of magnetization transfer
Figure 1. 13C CPPI edited spectra of lisinopril dihy-
between spins, which results in either the disap-
drate at 100.6 MHz (the CP/MAS and TOSS spectra are
pearance or inversion of the observed signal de- shown for comparison): (A) C and aliphatic CH 0,
pending on the particular characteristics of the aromatic CH positive, CH2 negative; (B) C positive, CH
sample. A series of pulse sequences based on this and CH2 negative; (C) C 0, CH and CH2 positive;
method have been developed to differentiate be- (D) TOSS spectrum; and (E) CP/MAS spectrum. The
tween C, CH, CH2, and CH3 groups in organic resonance assignments are given for the TOSS spectrum
solids.87–89 and correspond to the structure in Figure 8C.

Figure 3. Expansion of the spectra shown in Figure 2

(12–45 ppm): (a) 1D 13C CP/MAS spectrum of cholesteryl
acetate; (b) SS-APT spectrum of cholesteryl acetate
recorded in 35 min. Spinning sidebands are indicated
with an asterisk. The resonance assignments of C,
CH, CH2, and CH3 groups are shown. (Reprinted with
permission from ref. 91; copyright 1998; American
Figure 2. (a) 1D 13C CP/MAS spectrum of cholesteryl Chemical Society.)
acetate. (b) SS-APT spectrum of cholesteryl acetate
recorded in 35 min. Spinning sidebands are indicated
with an asterisk. Some of the higher frequency reso-
nance assignments of the C and CH groups are shown. quantum coherence (1Q, 2Q, and 3Q) to discrimi-
(Reprinted with permission from ref. 91; copyright 1998; nate between the types of carbons in a protected
American Chemical Society.) tripeptide.90 This ‘‘J-MQ filter’’ removes quatern-
ary carbon resonances in the 1Q experiment and
quaternary and methine carbons in the 2Q ex-
and 3) of cholesteryl acetate demonstrate the periment, thereby leaving only methyl carbons
potential of the technique for assigning chemical in the 3Q experiment. However, sensitivity gets
shifts. The SS-APT experiment worked best with progressively worse for the higher quantum filter-
several special decoupling methods and at spin- ing experiments. The authors state that the
ning speeds of 10–16 kHz. It is useful only for method should work well for amorphous solids,
samples with 13C at natural abundance because but no examples have been demonstrated so far.
13
C– 13C dipole couplings interfere with the
method. The frequency-switched Lee–Goldburg
Resonance Assignments—2D Correlation
(FSLG) sequence was used for 1H homonuclear
Spectroscopy
decoupling to obtain narrow carbon line widths
and to resolve the 1H– 13C J-couplings.93–95 The Correlation spectroscopy is routinely used for
two-pulse phase modulation (TPPM) sequence was analyzing the structure of organic molecules in
used during acquisition to remove heteronuclear solution. Both homonuclear and heteronuclear
coupling during the acquisition time.96 NMR correlation experiments in solutions are
Through-bond 1H– 13C coupling has also been valuable methods for determining the chemical
used for spectral editing by creating multiple bonding pattern of a compound and thus its

conformation. Similar correlation spectroscopy

has been implemented in the solid state. Ernst’s
group demonstrated a 2D 1H– 13C HETCOR
experiment initially using single crystals without
MAS,97 and subsequently for powdered threonine
spinning at 2.6 kHz.98 This experiment is widely
known as dipolar HETCOR spectroscopy because
it uses the through-space dipolar coupling inter-
action to correlate the 1H and 13C spins. These
correlation pulse sequences use a series of multi-
ple pulses that are applied simultaneously to the
1
H and 13C radio frequency channels. Therefore,
each channel must be adjusted carefully to obtain
the best quality data.99,100
Most solid-state NMR experiments that rely
on dipolar coupling interactions are effective at
distance of <10 Å (usually 5–6 Å). Spiess’ group
has used 1H spin diffusion to extend this range
up to 200 Å.101 This technique may be useful for
polymers and biological macromolecules, but it
requires labeling of a portion of the sample.
A recently developed method for indirect 1H
detection of 15N and 13C solid-state CP/MAS NMR
spectra has been used for 2D HETCOR experi-
ments.102 Sensitivity enhancement factors of up to
3 have been demonstrated for an organic polymer
and a polypeptide. One requirement that limits the
general application of this technique is that MAS
speeds of 30 kHz or greater are required.
Heteronuclear correlation spectroscopy of solids
has also been performed using scalar couplings.
Figure 4. The 2D (a) MAS-J-HMQC spectrum and
One such technique is the solid-state TOBSY (total
(b) dipolar HETCOR spectrum of natural abundance
through-bond-correlation spectroscopy) that was L-tyrosine hydrochloride. (Reprinted with permission
demonstrated for 13C-labeled calcium acetate and from ref. 107; copyright 1998; American Chemical
13
C– 15N-labeled arginine.103,104 Other methods Society.)
include the solid-state INADEQUATE105 and
refocused INADEQUATE106 NMR experiments
that were used to study 13C-labeled samples of compared with the dipolar HETCOR spectrum.
isoleucine, cellulose, and wood chips. Of the two The MAS-J-HMQC spectrum for a more com-
sequences, the refocused INADEQUATE proved to plicated tripeptide molecule is shown in Figure 5.
be more sensitive for compounds with greater The complete 1H, 13C, and 15N assignments for
line widths. However, both experiments are very this tripeptide were subsequently obtained using
time consuming and impractical without labeling MAS-J-HMQC NMR spectroscopy.108 The authors
of the sample. also demonstrated a modification of the technique
A very promising 2D correlation technique to obtain multiple-bond correlations similar to the
(MAS-J-HMQC) has been developed in Emsley’s data from the HMBC sequence for liquids.
group and does not require labeling of the sample Experiments based on scalar and dipolar coupl-
to obtain data in a reasonable amount of time.107 ing interactions offer complementary informa-
The MAS-J-HMQC and dipolar HETCOR spec- tion that can be used to obtain unambiguous
tra of L-tyrosine hydrochloride are compared in chemical shift assignments for solid materials
Figure 4. The MAS-J-HMQC spectrum has ap- without resorting to tentative assignments based
proximately half the sensitivity of the dipolar on solution spectra. These data are necessary for
HETCOR spectrum.107 However, the selectivity determining the conformations of compounds in
for one-bond J-coupled correlations is apparent the solid state.

Figure 5. The 2D MAS-J-HMQC spectrum of a natural abundance sample of the

tripeptide Boc-Ala-Ala-Pro-O-Bzl. (Reprinted with permission from ref. 107; copyright
1998; American Chemical Society.)
13
Internuclear Distance Measurements C– 13C correlation spectrum of 13C-labeled ery-
thromycin109 and the 13C, 15N-labeled achatin-II
The dipolar coupling interactions that are partly tetrapeptide.115
responsible for the broad resonances in solid- Double-quantum dipolar recoupling pulse se-
state NMR spectra also can be used to determine quences have also been used to obtain accurate
13
internuclear distances in addition to obtaining C– 13C bond distance measurements. The double-
homonuclear and heteronuclear correlations. For quantum methods are better for samples with
most cases, rapid-sample spinning removes or relatively large chemical shift anisotropies and
drastically reduces the dipolar couplings along can minimize interference from natural abun-
with the chemical shift anisotropy to give the dance 13C nuclei.116
isotropic chemical shifts of each nuclei. The dis- A REDOR variant, termed REPT-HMQC
tance information provided by dipolar coupling is (recoupled polarization transfer–heteronuclear
lost unless it can be reintroduced prior to acquir- multiple-quantum correlation), has been used to
ing a spectrum. A number of pulse sequences have obtain 2D correlated 13C– 1H chemical shifts, to
been designed to recover the dipolar coupling in- measure internuclear distances, and to perform
formation, to determine internuclear distances, 1D spectral editing.117 The results of the REPT-
and to obtain correlation spectra.109–111 The HMQC experiment for L-tyrosine hydrochloride at
REDOR112 (rotational-echo double-resonance) two different field strengths are shown in Figure 6.
NMR sequence is one of the many examples of These spectra should be compared with the MAS-
dipolar recoupling experiments and has been used J-HMQC of the same molecule in Figure 4. The
to measure 13C– 15N distances up to 6.3 Å with advantage of higher field strength is also apparent
an accuracy of 0.1 Å.113,114 Some limitations in the spectra shown in Figure 6. According to
of these dipolar recoupling techniques include Saalwächter et al., crystal packing effects are
(1) only a single spin pair can be easily studied, apparent in the 1H chemical shifts of the aromatic
(2) selective labeling is needed to provide an ring of tyrosine.117 The authors state that REPT-
isolated spin pair, (3) total acquisition times are HMQC does not require some of the special
usually long, and (4) molecular motions and spectrometer setup procedures that other HET-
exchange can average the dipolar couplings and COR experiments require. However, spinning
prevent accurate distance measurements.111 Uni- at 30 kHz is necessary to obtain reasonable 1H
formly labeled samples can be used if they are resolution, and certain correlations are dependent
diluted to 10–20% in the unlabeled sample. on the multiple quantum recoupling time used (see
This method was demonstrated with the 2D Figure 6).

Figure 6. REPT-HMQC spectra of L-tyrosine hydrochloride at 30 kHz MAS and

magnetic field strengths corresponding to proton Larmor frequencies of 300 (I, III) and
700 (II, IV) MHz. Skyline projections along the 1H and 13C dimensions are shown. The
spectral assignments in IV are according to ref. 201. The multiple quantum recoupling
times in I and II are half as long as for III and IV. (Reprinted with permission from ref. 117;
copyright 1999; Elsevier Science.)
Spinning Sideband Analysis produced at low MAS speeds (500 Hz). In both
experiments, the anisotropic tensor patterns for
The chemical shift anisotropy of a nucleus can
each nucleus are separated into a second dimen-
be a useful means of extracting conformational
sion by the differences in their isotropic chemical
data from solids because the CSA of each nucleus
shifts. A requirement for these techniques is prior
depends on its orientation with respect to the
assignment of each isotropic chemical shift by
static magnetic field. Therefore, orientations of
spectral editing or correlation spectroscopy.
the nuclei relative to other nuclei in the mole-
cule can also be determined. Several methods
of analyzing CSA tensors have been used to Solid-State 1H NMR Spectroscopy
examine both polymorphs and stereochemistry.
The 2DTOSS118 experiment uses MAS at The 1H-detected 1D and 2D NMR spectroscopy
1.5 kHz. The similar 2D FIREMAT119 experi- methods are standard for studying liquid solu-
ment also relies on the spinning sideband pattern tions because of the high natural abundance and

sensitivity of protons. However, these methods disodium clodronate.122 A fast rise in temperature
are not standard for studying solids because reveals that disodium clodronate loses lattice
the strong 1H homonuclear dipolar couplings water and only one 31P resonance is measured,
(40 kHz) and large chemical shift anisotropies whereas a slow increase in temperature converts
(20 ppm) result in very broad line widths in the the crystalline form to an anhydrous form that
spectra. Similar difficulties arise for solid-state displays two nonequivalent phosphorus atoms
19
F NMR spectroscopy, although most fluorinated (two separate resonances).
organic compounds have relatively few fluorine Some studies using solid-state NMR of
atoms. Useful information can be obtained by pharmaceutical compounds involved character-
using very fast MAS, special decoupling methods, izing the structures of N-desmethylnefopam
similar to those used for the correlation spec- hydrochloride,123 patellin,124 and erythromycin
troscopy experiments, or a combination of both. A dihydrate,125 and the amorphous nature of
Gerstein et al. first applied the combined-rotation ursodeoxycholic acid.126 The conformations of 30 -
and multiple-pulse NMR spectroscopy (CRAMPS) amino-30 -deoxythymidine,127 gramicidin A,128 and
method to obtain resolved chemical shifts in a amiodarone hydrochloride129 have also been con-
fluorocarbon.120 Subsequently, CRAMPS has firmed by solid-state NMR spectroscopy.
been developed for solid-state 1H NMR spectro- Because of the specificity of solid-state NMR
scopy. However, solid-state 1H NMR still remains spectroscopy, it is an ideal technique to study
a relatively difficult experiment because of the inclusion complexes, drug–excipient interactions,
long spectrometer ‘‘tune-up’’ time necessary to or the effect of moisture on the drug substance
ensure that pulse widths and power levels are or formulation. Studies on inclusion complexes
appropriately adjusted to obtain good data. include gliclazide-b-cyclodextrin130 and hydrocor-
A constant-time CRAMPS pulse sequence using tisone butyrate.131 Makriyannis utilized NMR
FSLG decoupling has recently been developed spectroscopy for investigating drug–membrane
where much narrower 1H line widths were interactions.132 Other interactions studied by
obtained.121 However, the sensitivity is substan- solid-state NMR spectroscopy include polyethyl-
tially lower than for the standard CRAMPS ex- ene glycol with griseofulvin133 and the moisture-
periment, and some proton resonances may not induced interaction with trospectomycin sulfate
be observed. that affects the equilibrium between the 30 -gem-
Solid-state 1H NMR spectroscopy of pharma- diol and 30 -keto forms in the drug substance and
ceutical compounds is likely to become more the freeze-dried formulation.134
widely used only when very high spinning speeds
and the special multiple-pulse sequences are more
Polymorphism
easily implemented.
The study of polymorphism appears to be one of
the most common applications of solid-state NMR
PHARMACEUTICAL APPLICATIONS
spectroscopy for pharmaceutical compounds.29,135
Pharmaceutical solids can exist in a number of
There are a variety of different applications of
solid forms, each having different properties of
solid-state NMR spectroscopy that are relevant
pharmaceutical importance, including stability
to pharmaceutical research. Some of these in-
and bioavailability. The number of these forms
clude analysis of (1) solid forms (polymorphs,
and their properties are largely unpredictable and
solvates), (2) hydrogen bonding and crystal pack-
vary considerably from case to case. Pharmaceu-
ing, (3) amorphous solids, (4) stereochemistry,
tical solids can be divided into crystalline and
and (5) solid–solid interactions (phase trans-
amorphous solids based on X-ray powder dif-
formations, activation energies of molecular
fraction and/or microscopic examination. Crystal-
motions, and solid-state reactions). Aboul-Enein
line solids can then be further classified into
has outlined some of the general uses of solid-
(1) polymorphs, forms having the same chemical
state NMR spectroscopy for pharmaceutical
composition but different crystal structures and
research.28
therefore different densities, melting points, solu-
bilities, and other properties; and (2) solvates,
General Applications
forms containing solvent molecules within the
An example of solid-state 31P CP/MAS NMR crystal structure, giving rise to unique differences
spectroscopy was the study of the dehydration of in solubility, response to atmospheric moisture,

loss of solvent, and other properties. Sometimes a solution and solid-state NMR spectra. Assign-
drug substance may be a desolvated solvate that ments may be confirmed with solid-state spectral
is formed when solvent is removed from a specific editing or correlation spectroscopy (see Methods,
crystalline solvate while retaining a crystalline Resonance Assignments).
structure. Many important properties are unique The solution 13C NMR spectrum and solid-state
13
to such a form. C CP/MAS NMR spectra of two crystalline and
Different physical forms of a drug substance an amorphous form of lisinopril, an angiotensin
can display radically different solubilities, which converting enzyme inhibitor, are compared in
directly affects the dissolution and bioavailability Figure 9.136 The structure of lisinopril is shown
characteristics of the compound. In addition, the in Figure 8C. In this case, the chemical shift dis-
chemical stability of one form, as compared with persion is quite similar for the solution and solid
another, may vary. The physical stability of poly- forms. Removing the water from the dihydrate
morphs is also crucial. During various processing form causes subtle changes in the peaks at 55 and
steps (grinding, mixing, tablet pressing, etc.), the 175 ppm that are probably due to changes in
physical form of the drug substance may be hydrogen bonding. The small peaks in the solution
compromised, subsequently leading to dissolu- NMR spectrum are due to a minor conformation of
tion problems. For these reasons, the full char- lisinopril in exchange with the major conformation
acterization of polymorphic systems is critical due to restricted rotation about the amide bond.
to numerous groups within commercial drug Only one conformation is observed in the solid-
development; namely, preformulation/physical state NMR spectra because the rotation is frozen
pharmacy, chemical process development, regula- out at room temperature. The solid-state 15N CP/
tory affairs, intellectual property, and analytical MAS spectra of crystalline and amorphous lisino-
development. pril, shown in Figure 10; demonstrates that useful
The solid-state 13C CP/MAS spectra of two flu- natural abundance 15N NMR data can be obtained
fenamic acid polymorphs are shown in Figure 7. for both crystalline and amorphous pharmaceuti-
The structure of the analgesic, flufenamic acid, cal compounds with relatively low nitrogen den-
is given in Figure 8F. The resonance assign- sities (10% of the sample mass).136
ments in the solid state have not been unambi- Polymorphs may be studied using solid-state
guously assigned for the polymorphs because NMR spectroscopy of less common nuclei, such as
they are quite different from the solution 13C 15
N, to provide additional information or to
NMR positions.136 In general, only tentative reso- determine specific details within the molecule.
nance assignments are possible when comparing Solid–solid phase transitions have been observed
by 13C and 15N solid-state variable temperature
NMR spectroscopy of 15N-labeled 2-(2,4-dintro-
benzyl)-3-methylpyridine.137 In this study, two
of the polymorphs coexisted over a temperature
range of 8 K. The mole fraction of each solid
phase at each temperature was also determined by
using the integrated peak areas in the 15N NMR
spectra.
Solid-state NMR and X-ray crystallography
are complimentary techniques for solid-state
characterization studies. The two polymorphs of
acetohexamide, an antidiabetic agent, were deter-
mined to be in the keto tautomeric form using
13
C solid-state NMR data and X-ray crystallo-
graphy.138 In a second study, the three polymorphs
of tedisamil dihydrochloride were studied by
Burger’s group using variable-temperature solid-
state 13C NMR spectroscopy and X-ray powder
diffraction.139 The study was mainly of academic
interest because the two high-temperature forms
Figure 7. 13C CP/MAS NMR spectra of flufenamic were not stable at room temperature and could not
acid polymorphs: (A) Form III and (B) Form I. be isolated.

Figure 8. Chemical structures of (A) prednisolone tert-butylacetate, (B) 21-

cyclopentyl ester of cortisol, (C) lisinopril, (D) peri-substituted naphthalenes,
(E) ibuprofen, (F) flufenamic acid, and (G) [5-methyl-2-(2-nitrophenyl)amino]-3-thiophe-
necarbonitrile (ROY).
The potential for polymorphism in anhydrous pulse sequence to observe short-range and longer-
theophylline was examined by theoretical struc- range couplings.141 Aspartame exists in three
ture calculations using X-ray powder diffraction hydrates (two hemihydrates and a dihemihy-
data and ab initio calculations of NMR shielding drate). Two of these hydrates apparently contain
tensors.140 In this study, the hydrogen bonding in three molecules per asymmetric unit and show
theophylline was determined by 13C and 15N NMR three resolvable resonances for each carbon atom.
spectroscopy. The results eliminated one of the two This complex crystal packing prevented assign-
possible hydrogen bonding configurations, and the ments of the resonances using techniques based on
remaining structure was similar to the crystal the number of attached protons or J-couplings.
structure of anhydrous theophylline. Typical MAS experiments at spinning rates of
Munson and co-workers have studied uniformly 7 kHz and proton decoupling powers of 63 kHz
13
C-labeled aspartame polymorphs using 13C label- gave broad peaks due to dipolar coupling. How-
ing, fast MAS (up to 28 kHz), very high decoupling ever, increasing the spinning rate to 28 kHz and
power (up to 263 kHz), and a dipolar recoupling the decoupling power to 263 kHz gave narrow

Figure 10. 15N CP/MAS NMR spectra of lisinopril at

Figure 9. Comparison of the solution and solid-state 40.54 MHz for (A) amorphous form and (B) dihydrate
13
C NMR spectra of lisinopril at 100.6 MHz (solids) and crystalline form. Each spectrum was acquired in 11 h.
150.9 MHz (solution). The total time for data acquisition The chemical shift assignments correspond to the
is given for each spectrum: (A) amorphous form CP/MAS labeled lisinopril structure in Figure 8C.
spectrum (17 min); (B) anhydrous crystalline form CP/
MAS spectrum (54 min); (C) dihydrate crystalline form, for most of the carbons in this rather complex
CP/MAS spectrum (54 min); (D) D2O solution spectrum organic molecule.
with 100 Hz line broadening (2 h); and (E) D2O solution The solid-state 13C NMR analysis of several
spectrum with 5 Hz line broadening.
amphetamines and their mixtures with lactose
monohydrate have been reported.144 The lactose
resonances that allowed the assignment of the monohydrate mixture of one amphetamine (3,4-
crystallographically inequivalent sites. For one of methylenedioxyamphetamine hydrochloride) pro-
the forms, peak assignments could be made for all duced distinct chemical shift differences for the
three molecules in the asymmetric unit of the amphetamine resonances, and the authors attri-
crystal. bute the changes to relaxed crystal packing forces
The five known polymorphs of sulfathiazole rather than hydrogen bonding.
were distinguished by solid-state 13C NMR spec- Two racemic polymorphs and an enantiomorph
troscopy.142 One observation to note in this study of tazefelone, an antioxidant and 5-lipoxygenase
was that polymorph I was partially converted to inhibitor, were studied by solid-state 13C and 15N
polymorph IV during solid-state NMR spectro- NMR spectroscopy and X-ray crystallography.145
scopy, and this transformation was attributed to The different forms produced very distinct NMR
sample spinning. However, the forces transferred spectra, and the unique hydrogen-bonding pattern
to a solid sample in a rapidly spinning rotor are of each form resulted in chemical shift differences,
generally much less than the forces applied during especially for the 15N NMR spectra.
tablet formation. The most obvious possible cause Intramolecular hydrogen bonding, tautomeri-
for a transformation would be sample heating zation, and polymorphism in several Schiff’s bases
due to either spinning or radio frequency energy have been studied with natural abundance 15N
input, which is certainly possible given the changes and 13C solid-state NMR spectroscopy.146 The 15N
shown for the variable temperature spectra in NMR spectra were more sensitive for detecting
Figure 3 of that article.142 tautomers because of the large chemical shifts of
15
Medek and Frydman analyzed vitamin B12 N that depend on specific interactions with other
polymorphs with solid-state 13C, 15N, 31P, and nuclei.
59
Co NMR spectroscopy.143 The 13C NMR data The majority of applications of solid-state NMR
were the most informative because the highly spectroscopy that have been used in the investiga-
crystalline material produced sharp resonances tion of pharmaceutical polymorphs are performed

in conjunction with other analytical techniques. molecules in a given unit cell. A series of doublet
Byrn et al. have reported differences in the solid- resonances were noted for several different carbon
state NMR spectra for different polymorphic atoms. This result implies that a specific carbon
forms of benoxaprofen, nabilone, and pseudopoly- atom within the molecule may resonate at two
morphic forms of cefazolin.147 Although single- different frequencies corresponding to each crys-
crystal X-ray diffraction was initially used to study tallographic site of the molecule (crystallographic
the polymorphs, the solid-state 13C CP/MAS NMR splitting). In addition to the hydrogen bonding
spectrum of each form was distinctly different. explanation of the crystallographic splittings, the
These studies primarily focused on the bulk drug use of NMR spectroscopy to quantitatively deter-
material, although a granulation of benoxaprofen mine the amount of each pseudopolymorph pre-
was also studied. The authors concluded that solid- sent in a mixture was addressed. In the study of
state NMR spectroscopy could be used to differ- androstanolone,152 high-quality 13C NMR spectra
entiate the form present in the granulation, even were obtained by the CP/MAS technique, which
with excipients. In other studies at Purdue Uni- permitted the characterization of the anhydrous
versity, the crystalline forms of prednisolone and monohydrate forms. In this case, the crystal-
tert-butylacetate,148 cefaclor dihydrate,149 and lographic splittings were noted for the two forms
glyburide150 were studied. The five crystal forms and were correlated with hydrogen bonding. An
of prednisolone tert-butylacetate were again de- identical approach was used to study a pharma-
termined by single-crystal X-ray diffraction, and ceutical polymorphic structure in the investi-
solid-state NMR spectroscopy was used to de- gation of the two polymorphs of 40 -methyl-
termine the effect of crystal packing on the 13C 20 -nitroacetanilide.153 An additional study of
chemical shifts of the different steroid forms. cortisone acetate154,155 by solid-state NMR spec-
Although conformational changes were observed troscopy revealed differences in the NMR spectra
in the ester side chain by X-ray crystallography, no for the six crystalline forms. All of these studies
major differences were noted in the NMR spectra, indicate that conformational differences, hydro-
indicating that the environment remains rela- gen bonding, solvated forms, and polymorphs in
tively unchanged. However, significant chemical solids are readily analyzed by solid-state NMR
shift differences were noted for carbonyl atoms spectroscopy.
involved in hydrogen bonding. This observation is Further multidisciplinary approaches to the
consistent with the NMR study of cefaclor dihy- physical characterization of pharmaceutical com-
drate. Again, the effects of hydrogen bonding were pounds are detailed in separate studies on cyclope-
discernible by solid-state NMR spectroscopy. The nthiazide,156 the excipient lactose,157 frusemide,158
study of glyburide was principally concerned with losartan,159 fosinopril sodium,160 leukotriene anta-
the molecular conformation of the compound in gonists MK-679 and MK-571,161 L-660,711,162
solution and in the solid state. The conformation captopril,163 diphenhydramine hydrochloride,164
in solution was determined by 1H and 13C NMR mofebutazone,165 phenylbutazone,165 oxyphenbu-
spectroscopy and in the solid by single-crystal tazone,165 cimetidine,166 and the iron chelator 1,2
X-ray diffraction, IR, and solid-state 13C NMR dimethyl-3-hydroxy-4-pyridone.167 In each case,
spectroscopy. The solid-state NMR results sug- solid-state NMR spectroscopy was used in conjunc-
gested that this method would be useful for com- tion with other techniques such as DSC, IR, X-ray
paring conformations of molecules both in solution diffraction, microscopy, and solubility/dissolution
and in the solid state. studies to fully characterize the polymorphic
In a series of publications by Harris and Fletton, systems. In these studies, solid-state NMR spectro-
solid-state NMR spectroscopy was used to inves- scopy is one of several complementary techniques
tigate the structures of several polymorphs.151 –155 for fully characterizing pharmaceutical solids.
The majority of these studies included X-ray NMR spectroscopy fits into the niche between the
diffraction and IR techniques and also addres- techniques of X-ray diffraction for analysis of long-
sed the possibility of quantitative measurements range order and vibrational (IR and Raman)
of polymorphic mixtures. The three pseudopoly- spectroscopy for analysis of organic functional
morphic forms of testosterone were examined by groups.
IR and 13C CP/MAS NMR spectroscopy.151 The In other studies of polymorphism by solid-state
two forms of molecular spectroscopy were able to NMR spectroscopy, conversion from one solid
differentiate the forms, but NMR spectroscopy form to another by ultraviolet irradiation168 and
could be used to investigate nonequivalent variable-temperature techniques169 are outlined.

In the first study,168 NMR spectroscopy was em- lavirdine mesylate polymorph and pseudopoly-
ployed to follow the chemical transformation morph mixtures by 13C CP/MAS was 2–3% by
within the organic crystals of p-formyl-trans- weight.171 Both of these studies took into account
cinnamic acid ( p-FCA). A photoreactive b-phase the differential CP dynamics of each form, but
can be crystallized from ethanol, whereas a peak intensities were used in the latter study
photostable g-phase is produced from acetone. whereas peak integrals were used in the former
After irradiation of the b-phase with UV radiation study. In solid-state 13C CP/MAS NMR spec-
and subsequent acquisition of the solid-state 13C troscopy, it is generally necessary to do peak
NMR spectrum, the photoproduct was easily deconvolution to obtain accurate integrals for
identified. quantitative analysis because peak overlap is
The second conversion study169 investigated quite common and small errors in the magic
the four forms of p-amino-benzenesulphonamide angle setting can lead to additional peak broad-
sulphanilamide (a, b, g, and d). The first three ening that will cause peak intensities to be
forms were investigated by solid-state 13C NMR inaccurate.48
spectroscopy and X-ray crystallography techni- The detection limit is an important factor to
ques. Subsequent variable-temperature studies consider when developing a quantitative method.
monitored the interconversion of the a and b forms For solid-state NMR spectroscopy, crystalline
to the g form. Coalescence of some NMR signals in compounds usually permit a much lower detection
the g form also suggested that phenyl ring motion limit than amorphous compounds because of better
occurred within the crystal. Conclusions from the resolution in the spectrum. The overall signal-to-
study indicated that solid-state NMR spectroscopy noise ratio in a spectrum is also important, but
could differentiate pharmaceutical polymorphs, differentiating one component from another gen-
determine asymmetry in the unit cell, and analyze erally requires high resolution. A set of spectra for
molecular motion within the solid state. different mixtures of two analgesic compounds is
shown in Figure 11.172 The detection limit is <2%
(mole fraction) for compound II based on the peak
Drug Substance Quantitative Analysis
at 165 ppm and 4% for compound I based on the
A few quantitative solid-state NMR analyses peak at 170 ppm. However the relaxation delay
of pharmaceutical compounds have been pub-
lished.170,171 In one study, a glycine internal
standard was used to analyze mixtures of carba-
mazepine anhydrate and carbamazepine dihy-
drate.170 The 13C CP/MAS NMR spectra for
carbamazepine anhydrate and carbamazepine
dihydrate were essentially the same, although a
sufficient signal-to-noise ratio for the spectrum of
the anhydrous form required long accumulation
times.170 This requirement was determined to be
due to the long proton T1 relaxation time for this
form. Utilizing the fact that different proton spin-
lattice relaxation times exist for the two different
pseudopolymorphic forms, a quantitative method
was developed. The dihydrate form displayed a
relatively short relaxation time, and a relaxation
delay time of only 10 s was sufficient to obtain full
intensity spectra of the dihydrate form with no
signal due to the anhydrous form. By utilizing
glycine as an internal standard and accounting
for the differences in the relaxation rates of the
two forms, the peak area of the dihydrate could Figure 11. 13C CP/MAS NMR spectra at 100.6 MHz
be measured and related through a calibration for analgesic I and II. Spinning sideband suppression
curve to the amount of anhydrous and dihydrate (TOSS) was used for each spectrum. The mole fractions
content in mixtures of carbamazepine. In another (I:II) of each compound are (A) 98:2, (B) 97:3, (C) 60:40,
study, the detection limit for quantifying de- (D) 7:93, and (E) 4:96.

time is optimized for compound II, and the CP The topic of drug–excipient interactions was
contact time is a compromise between the opti- addressed by solid-state 13C NMR spectroscopy in
mum values for both compounds. The peak the investigation of different commercially avail-
integral for compound I at the relaxation delay able aspirin samples.174,175 In each commercial
time used in the spectra is 20% of its value at the aspirin product, the only difference in the mea-
optimum delay time. Therefore, the actual detec- sured NMR spectrum was due to variations in the
tion limit for compound I is expected to be the excipients, indicating that there were no interac-
same as that for compound II under optimized tions between the drug and the excipients under
conditions. dry blending conditions. After lyophilization of two
of the products, one aspirin sample had a different
NMR spectrum, indicating a possible interaction
Dosage Form Analysis
during lyophilization or conversion to a different
NMR spectroscopy is generally quite useful for solid form during processing.
examining mixtures of compounds in solutions Solid-state NMR spectroscopy has been used
or in solids. Therefore, it can be used to analyze to analyze a number of different drugs in tablet
formulated drug products for transformations and form.176 The ten different drugs that were ana-
interactions or reactions with excipients. A series lyzed and the dosages in the tablets are summar-
of papers has been published on the solid-state ized in Table 1. The NMR analysis could discern
NMR spectra of a number of analgesic drugs. the active component for the listed drugs, except
Jagannathan recorded the solid-state 13C NMR for the low dose enalapril maleate tablets. Ex-
spectrum of acetaminophen in bulk and dosage cipients obscured some drug resonances that
forms.173 From the solution NMR spectrum, appear at the same chemical shift value but have
assignments of the solid-state NMR resonances little effect on resonances at other chemical shifts.
could be inferred in addition to explanations for This result indicates that the excipients are
the doublet structure of some resonances. Spectra probably not interacting with the drug. Similar
of the dosage product from two sources showed results were obtained for tablets of another closely
identical drug substance but different levels of related HMG-CoA reductase inhibitor, simvas-
excipients. tatin (Table 1).
13
Table 1. Tablets and Capsules of Drugs Investigated in Our Laboratory by Solid-State C-NMR Spectroscopy
Number of NMR Scans

Total Weight of Manufacturer (# of Scans for Interrupted
Drug Dose (mg) Dosage Form (mg) and Trade Name Decoupling Experiment)
Enalapril maleate 1.25 230 Merck (Vasotec) 16,000 (33,576)
2.5 230 Merck (Vasotec) 16,000 (33,576)
5 230 Merck (Vasotec) 16,000 (33,576)
20 200 Merck (Vasotec) 16,000 (33,576)
Lovastatin 20 400 Merck (Mevacor) 4000 (4000)
Simvastatin 40 400 Merck (Zocor) 4000 (4000)
Ibuprofen tablets 200 320 Bristol-Myers Squibb 2000 (4048)
(Nuprin)
200 330 Upjohn (Haltran) 7264 (2692)
400 620 Geneva Generics 6000 (9084)
Sulindac tablets 200 330 Merck (Clinoril) 3000 (8104)
Flurbiprofen tablets 100 420 Upjohn (Ansaid) 4000 (10,000)
Diflunisal tablets 500 840 Merck, (Dolobid) 10,000 (20,492)
Indomethacin capsules 50 350 Merck (Indocin) 12,000 (20,000)
75 280 Merck (Indocin-SR) 12,000 (28,248)
50 380 Geneva Generics 10,000 (27,772)
50 480 United Research 10,000 (30,000)
Laboratories
Mefenamic acid capsules 250 350 Parke-Davis (Ponstel) 7000 (6000)
Piroxicam capsules 20 300 Pfizer (Feldene) 14,000 (19,556)

The solid-state 13C NMR spectra of a number of crystal form present in low-dose tablets and to
tablets and capsules of various nonsteroidal anti- identify whether a tablet is a placebo or contains
inflammatory agents were also obtained (Table 1). the drug. Solid-state NMR spectroscopy offers a
The spectra of the ibuprofen tablets showed useful alternative to X-ray diffraction for direct
narrow lines with excellent signal-to-noise ratios. determination of the crystal form present in the
This result indicates that the drug itself is crystal- final dosage form.
line and is present at a high dose in the tablets. As previously mentioned, prednisolone tert-
In general, most of the resonances due to the butylacetate (Figure 8A) exists in at least five
other nonsteroidal drugs were also clearly distin- crystalline forms. Solid-state NMR spectroscopy
guishable in the spectra of the dosage forms. The has been used to show that different brands of
interrupted decoupling pulse sequence was used to prednisolone tablets contained different poly-
simplify the appearance of the spectra by suppres- morphs.176 The 13C CP/MAS spectra of predniso-
sing excipient peaks and to aid in signal assign- lone tert-butylacetate that was removed from
ment. These studies showed that solid-state 13C various commercial suspensions by filtration are
NMR spectroscopy can be used to determine the shown in Figure 12. Solid-state NMR spectroscopy
Figure 12. Solid-state 13C CP/MAS NMR spectra of filtered suspensions of predniso-
lone tert-butyl acetate obtained from different vendors.

was used to determine which crystal form was One of the earliest solid-state NMR studies
present. Note that the bulk drug obtained from of the molecular mobility of pharmaceuticals
Vendor Y is an unequal mixture of compounds, as involved the steroid ester containing the cyclopen-
demonstrated by 10 additional smaller peaks in tyl group shown in Figure 8B.148 Crystallographic
its spectrum. Thus, solid-state NMR spectroscopy studies showed that the cyclopentyl group of the
is also useful for analyzing the solids obtained from side chain had an extremely large thermal motion.
filtered suspensions. Studies comparing the CP and SPE NMR spectra
clearly showed that the cyclopentyl group was the
most intense peak in the SPE spectrum. However,
Analysis of Molecular Motions—Relaxation in the CP spectrum, there were several signals
with greater or equal intensity. The enhancement
Examining the various motions of solid pharma- of this signal in the SPE spectrum is consistent
ceutical compounds may lead to a better under- with the large amount of thermal motion observed
standing of solid-state stability and reactivity by in the crystal structure. Potentially related obser-
examining processes such as solid–solid transfor- vations on simvastatin were made by Cauchon.192
mations and intramolecular and intermolecular A signal from one of the side chain methylene
interactions. For solid-state NMR spectroscopy, carbon atoms was present in the interrupted
variable-temperature methods are generally used decoupling spectrum that normally shows the
along with relaxation methods to study molecular presence of only quaternary carbon atoms and
motions. Variable-temperature solid-state NMR methyl groups having significant thermal motion.
spectroscopy has been shown to be a powerful This result was interpreted to mean that the
technique for the study of molecular motion in mobility of the methylene carbon atom was similar
a variety of compounds, including cortisone,177 to that of a methyl group, presumably because
cholesterol,178 proteins,179 polymers,61,180 –182 and of side chain motion. This explanation is consis-
amino acids.57 tent with the crystallographic studies of simvas-
The effects of water on the molecular mobility of tatin in which the side chain could not be refined
galacturonic acid,183 b-cyclodextrin,184 poly(vinyl because of a large amount of thermal motion.
alcohol) hydrogels,185 polyaspartic acid,186 and Additional studies are needed to verify the useful-
scleroglucan,187 have been studied by 1H and ness of interrupted decoupling and other editing
13
C relaxation times. In general, the hydrated sequences as well as SPE NMR spectroscopy for
materials had increased molecular mobility, which the study of motion in pharmaceutical solids.
is consistent with the effect of water in many However, one should note that molecular motions
solids. The effects of hydration on hydrogen bond- affect the dipolar coupling interaction on which
ing in collagen and several collagen-like poly- many editing sequences are based, which would
peptides have been analyzed by 15N CP/MAS and indicate that these methods could be readily
1
H T1r relaxation time measurements.188 The use applied to analysis of many types of motions in
of nuclei-specific relaxation measurements makes solids.
it possible to examine different types of motions or The activation energies for the molecular mo-
motions restricted to one area of the molecule. tions of methyl groups in ibuprofen (Figure 8E)
Solid-state NMR spectroscopy has also been were measured by solid-state 13C CP/MAS NMR
used to study protein hydration and stability189 spectroscopy. The spectra of ibuprofen at differ-
and the activation energies of spinning methyl ent temperatures are shown in Figure 13. The
groups in amino acids.190 Relaxation analyses of spin-lattice relaxation times were measured
peri-substituted naphthalenes (Figure 8D) were at various temperatures for the three methyl
used to determine the barrier to methyl rotation groups in ibuprofen using the pulse sequence of
when substituents at the peri position were --H, Torchia,58 and their activation energies were
--CH3, --Cl and --Br.191 The observed barriers were derived from Arrhenius plots of ln T1 versus
H, 9.7 kJ/mol; CH3, 13.5 kJ/mol; Cl, 15.2 kJ/mol; T1. The semilog plots of signal intensity versus
and Br 18.4 kJ/mol. The barriers sequentially the relaxation delay times that were used to
follow the size of the van der Waals radii and calculate the T1 values were all linear. This result
indicate that a large group adjacent to the methyl indicates that the relaxation function for each
group can restrict its rotation. This type of in- individual carbon atom is governed by a single
formation is useful for examining potential reac- exponential correlation time. There was no evi-
tion mechanisms in the solid-state. dence of nonexponential behavior in this case,

Table 2. Activation Energies from Solid-State

NMR Spectroscopy for the Methyl Carbon Atoms in
Ibuprofen
Carbon Chemical Shift (ppm) Ea (kJ/mol)

C3 15.4 10.0
C12/C13 22.1 8.8
C12/C13 25.1 9.1
carbon atom (C3) had the highest activation energy

based on the solid-state NMR study. The C12/C13
methyls had lower activation energies.
Because solid-state NMR spectroscopy can be
used to study pharmaceutical dosage forms and
other mixtures, this method should find increas-
ed application in the analysis of the molecular
motions for these systems.
In addition to the differences in chemical shifts
displayed by polymorphs, their NMR relaxation
properties may be quite different. For example,
the 13C T1 relaxation time for the carbonyl carbon
of g-glycine is about five times longer than for
a-glycine, and the 1H T1 relaxation time is about
10 times longer.193 The differences in relaxation
times can be exploited to enable the deconvolution
of solid-state NMR spectra of mixtures of poly-
morphs. One useful method, the direct exponential
curve resolution algorithm (DECRA), can be used
to separate spectra of mixed components when
their relaxation times differ by as little as
20%.194,195
Another case where differences in 1H T1 re-
laxation times can be exploited is for the analysis
of binary eutectic mixtures.172 If there are dis-
tinct differences in the relaxation times of the
Figure 13. Solid-state 13C NMR spectra of ibuprofen two components, a correlation can be made be-
at several temperatures. The isotropic resonances at ween the integrated ratios of the resonances for
120–150 ppm produce first-order spinning sideband each solid in the eutectic compared to a physical
peaks at 70–95 ppm and 180–200 ppm in the 198 K mixture of the same compounds. The 1H T1 rela-
spectrum. Second-order spinning sidebands are even xation time approaches the shorter value between
smaller peaks at 25–40 ppm and are nearly unnotice- the two components for eutectic mixtures, but
able under the larger peaks. Differences in the spinning
is different than either compound alone. In the
sideband positions in the other three spectra are due to
differences in the spinning speeds. The sharp peak at
physical mixture, no significant differences in
183.3 ppm is due to the carbonyl resonance. the relaxation times are apparent. Few solid-
state NMR studies have been published on
eutectics, particularly involving pharmaceutical
even at long relaxation delay times. The low ob- compounds. However, NMR relaxation was used
served standard deviations are a consequence of for analyzing eutectics at least 30 years ago.
the good signal-to-noise ratio that reflects the Clifford used 1H T1 relaxation measurements to
highly crystalline nature of the sample. analyze phase changes in mixtures of glycer-
A summary of the solid-state NMR relaxation ides.196 McGavock and Harlowe recognized the
measurements for the three methyl groups in ibu- usefulness of T1 relaxation differences for ana-
profen is given in Table 2. In this case, the methyl lyzing the phases of LiCl/CrCl3 mixtures.197 In

this case, 7Li NMR relaxation measurements differentiate between racemic and enantiomeric
were performed. Eutectic formation during crystallites.
formulation or processing of pharmaceutical In another example, a series of racemic and
solids can be either beneficial or undesirable. optically pure organophosphorus samples were
Solid-state NMR spectroscopy may be a useful studied by solid-state 31P NMR spectroscopy.202
means of analyzing for eutectics in tablets and Analogous to the tartaric acid work, the two optical
powders as part of the preformulation process. isomers (þ and ) had identical 31P NMR spectra
that were distinct from the spectrum of the race-
mic material.
Analysis of Molecular Motions—Exchange The issue of quantitative analysis of optical
Spectroscopy purity has been addressed by solid-state 13C NMR
Only a few examples of exchange spectroscopy spectroscopy in the conformational analysis of DL-,
203
techniques have been reported in the literature L-, and D-methionine. The data in this study
for solids, and no relevant pharmaceutical appli- indicate that each crystalline form of DL-methio-
cations were found at the time of this review. nine consists of a single conformer, which is in
Several interesting studies by Riddell et al.198,199 agreement with X-ray diffraction data. Both the D
have determined the rates of phenyl and tert- and L conformers of methionine have two forms
butyl group rotations and the activation energies according to their solid-state NMR spectra, which
for the processes by 2D EXSY, in addition to is consistent with the X-ray crystal structures that
relaxation and line shape analyses. These types show two molecules per asymmetric unit.
of analyses could be implemented for a variety of The conformation of a molecule in the solid state
pharmaceutical compounds in the pure state as can only be determined by NMR spectroscopy if
well as in formulations. each resonance is assigned to the corresponding
nucleus. As discussed earlier, a number of useful
solid-state NMR methods are now available to
perform resonance assignments. The CPPI experi-
Conformation and Stereochemistry
ment and its variants have been used to analyze
The structural analysis of chiral compounds is a wide range of compounds, including amor-
an important issue in drug development. Solid- phous humic materials,204 crystalline calcipotriol
state NMR spectroscopy can be used to directly monohydrate and vitamin D,205 crystalline and
measure the enantiomeric purity of a sample in amorphous forms of delavirdine mesylate,206 and
certain cases. It is important to remember that crystalline L-leucinamide.207 A series of editing
enantiomers are indistinguishable by NMR spec- spectra, obtained for lisinopril dihydrate, are com-
troscopy. However, diastereomers have different pared with the conventional 13C CP/MAS and
NMR spectra, and NMR spectra of racemic mix- TOSS spectra in Figure 1.136 In this example, most
tures in the solid state are generally different of the tentative resonance assignments that were
from either enantiomer. A number of reports in made based on the 13C NMR spectrum of lisinopril
the literature demonstrate the ability of 13C in solution (Figure 9) can be confirmed in the solid-
or 31P solid-state NMR spectroscopy to differen- state NMR spectrum.
tiate between optically pure material and race- Two-dimensional solid-state correlation NMR
mic species.200 In one report, tartaric acid was spectroscopy has also been applied to pharma-
studied by 13C CP/MAS NMR spectroscopy.201 ceutical systems.99,208 The overall goal of these
For each of the optically pure (2R, 3R), racemic, methods is to obtain information on the confor-
and meso-tartaric acid materials studied, two mation of solid compounds, which is particular-
molecules exist per asymmetric unit cell. Be- ly useful when a crystal structure cannot be
cause the carbonyl and a-carbon atoms in a single determined.
molecule are not symmetry related, one would The 2D dipolar HETCOR experiment has been
expect two resonances for each carbon (crystal- used for studying ibuprofen,100 aspirin tablet and
lographic splitting). This crystallographic split- sucrose,95,99 amino acids,209 and a variety of small
ting was observed for the carbonyl and a-carbon organic compounds and polymers.100,210
atoms in the 13C NMR spectra in all three cases, A number of 15N– 13C solid-state NMR correla-
and distinct isotropic chemical shift values were tion experiments have been developed for analyz-
observed as well. These data demonstrate the ing amino acids and peptides.211 –215 As many as
potential for solid-state NMR spectroscopy to four dimensions have been used to obtain greater

resolution.215 These experiments may eventually forms of 13C2-labeled cimetidine were studied
lead to the solid-state structure determination using distance measurements via rotational re-
of polypeptides, proteins, and nucleotides. These sonance magnetization exchange, and relative
13
compounds are becoming more important as active C– 1H bond orientations were determined by
pharmaceutical components. Limitations of these double quantum heteronuclear local field NMR
NMR experiments include the necessity for 15N spectroscopy.220
and 13C labeling and the fact that the compounds In an application of a double-quantum recou-
used in the cited studies have been single crystals. pling method, the relative orientations of purine
No current publications have been found in and pyrimidine rings in a nucleic acid dodecamer
which the more advanced solid-state NMR spec- were determined.221 This result indicates that
troscopy experiments, like SS-APT and MAS- it may be practical to determine secondary and
J-HMQC, have been used for pharmaceutical tertiary structures of relatively large molecules
compounds. Presumably, these methods will with solid-state NMR spectroscopy.
find wider use in pharmaceutical research in
the future.
Spinning Sideband Analysis
Raftery et al. have used 2D solid-state NMR
Internuclear Distance Measurements
spectroscopy to study the red, orange, and yellow
Measurement of internuclear distances has only conformational polymorphs of ROY (5-methyl-
recently been applied to pharmaceutical com- 2-(2-nitrophenyl)amino]-3-thiophenecarbonitrile
pounds. Many of the difficulties involved in (Figure 8G).222 The chemical shift anisotropy of
applying the necessary recoupling techniques the C3 carbon atom in ROY could be readily dis-
were already outlined. The few examples cited tinguished with the 2DTOSS pulse sequence,
are either biologically or pharmaceutically active and the differences in the chemical environments
molecules, but they provide an indication of the among the three polymorphs were examined. The
potential value of these techniques. chemical shift anisotropy for C3 in the three poly-
Internuclear distances, obtained by 13C– 15N morphic forms is shown in Table 3. There is a
REDOR NMR spectroscopy for melanostatin, were large difference between the isotropic chemical
used to create a set of structures with root-mean- shifts (diso) among the three forms. The diso in-
square-deviation of 0.4370.224 Å compared with creases by almost 10 ppm between the red and
the X-ray structure.216 However, three separate yellow polymorphs. Similarly, the chemical shift
13
C, 15N-labeled melanostatin tripeptides were anisotropies differ by 30 ppm between the red
needed to obtain the constraints used to determine and yellow forms. This study demonstrated that
the structures. In another study, solvates of singly CSA tensor analysis can distinguish between con-
and doubly labeled (13C, 15N) Leu-enkephalin and formational polymorphs and is therefore a power-
Met-enkephalin were analyzed by REDOR NMR ful method for analyzing such systems. Raftery’s
spectroscopy, relaxation methods, and variable- group is now combining information from the
temperature 13C CP/MAS to determine the struc- 2DTOSS experiments with ab initio or density
tural effects of solvent molecules.217 The confor- functional calculations to attempt to obtain quan-
mational changes due to dehydration were readily titative structural information.
observed in the solid-state NMR spectra of each Similar experiments involving spinning side
compound, and site-specific differences in molec- band analysis have been performed by Harper
ular motions were detected in the relaxation et al. to examine the stereochemistry of the
measurements. natural product, terrein.119 The tensor principle
The rotational resonance and rotor-driven values were determined with the 2D FIREMAT
magnetization exchange recoupling methods were
used to measure internuclear 13C– 13C distances in
13 Table 3. Chemical Shift Tensor Values for the Three
C2-labeled retinal, and the results compared
Forms of ROY
well with X-ray diffraction data (within 0.04–
0.05 Å).218 A rotor synchronized recoupling pulse Solid Form d11 d22 d33 diso
sequence was used to determine the 15N– 1H bond
lengths in crystalline [U-13C, 15N]-L-histidine, Red (R) 49.2 90.7 155.0 98.3
Orange (O) 50.2 102.7 165.7 106.2
which enabled the authors to detect an inter-
Yellow (Y) 43.3 105.8 179.2 109.4
molecular hydrogen bond.219 The polymorphic

technique, and the authors note that diastereo- useful for analyzing peak doubling (crystallo-
mers of terrein were differentiated, but enantio- graphic splitting) to determine if multiple mole-
mers cannot be resolved as expected. cules per asymmetric unit are independent or if
The analysis of spinning sidebands was devel- there is exchange between the different conforma-
oped relatively recently, and it is anticipated that tions. Solid-state NMR spectroscopy can be used to
many pharmaceutical compounds could be ana- study interactions between the active drug and
lyzed in this manner. The experimental method excipients in tablets or capsules. It is also possible
is not difficult, but the data must be correctly to study solid-state chemical reactions and their
analyzed to extract the necessary information. mechanisms as well as solid–solid phase trans-
These methods, along with solid-state NMR cor- formations with solid-state NMR spectroscopy.
relation experiments, promise to provide confor- Use of internuclear distance measurements may
mational and stereochemical information for solids enable the conformational and structural analysis
whose crystal structures cannot be determined by of amorphous pharmaceutical solids. NMR rela-
X-ray diffraction. xation parameters could also be correlated with
thermal data in the analysis of amorphous com-
pounds. Many drugs are biological macromolec-
Solid-State 1H NMR Spectroscopy
ules or modifications of them (e.g., proteins,
As noted earlier in this review, very few 1H NMR nucleic acids, carbohydrates) that are likely to be
spectra of solid pharmaceutical compounds have amorphous, and solid-state NMR spectroscopy
been obtained because of the difficulties inherent would be a much more useful analytical tool for
in the experiment as well as the general lack of them than X-ray diffraction. Magnetic resonance
useful data available in the spectrum. How- imaging of solid dosage forms may also provide
ever, some recent applications have been demons- useful information that cannot be obtained by
trated, in which reasonably good results were other techniques.
obtained.
1
H CRAMPS has been applied to the study
Note Added in Proof
of hydrogen bonding in a number of organic
solids.223,224 Hydrogen bonding and polymorphs In a continuing effort to provide the most up-to-
of solid amino acids225 and the secondary struc- date references on solid-state NMR, the following
tural conformation of polypeptides up to six amino manuscripts appeared in the literature during
acids long226 have been examined by 1H CRAMPS. the revision and proofing stages of this article
Hydrogen bonding interactions in the polypep- preparation. Additional references include manu-
tides could be determined by the characteristic scripts on quantitative analysis,227–229 conforma-
chemical shifts of the observed resonances. tion analysis,230–232 molecular motion,232 and
amorphous/crystalline characterization.233
Future Developments
Although solid-state NMR spectroscopy can be SUMMARY
considered a routine analytical method at this
time, it is not trivial to obtain high quality spectra. This review has covered some of the recent
A significant barrier to using solid-state NMR methods and applications of solid-state NMR
spectroscopy may be the perception that it does spectroscopy that will hopefully become more
not provide any information that cannot be obtain- widely used for studying pharmaceutical solids.
ed more easily by another technique. Many of the Within various pharmaceutical laboratories
examples given in this review demonstrate that (industrial and academic), the multinuclear tech-
solid-state NMR spectroscopy does provide unique nique of solid-state NMR spectroscopy has pri-
data for a variety of pharmaceutical compounds. marily been applied to the study of polymorphism
There are a number of other potential applica- at the qualitative and quantitative levels. Al-
tions of solid-state NMR spectroscopy that may though the technique ideally lends itself to the
have implications for future pharmaceutical re- determination of conformations of drug com-
search. However, it should be noted that these ap- pounds in the solid state, it is anticipated that
plications are not new to NMR spectroscopists who in the future, solid-state NMR spectroscopy will
have used them in other fields of study. For become routinely used for method development
instance, 2D correlation spectroscopy may be and problem solving activities in the analytical/

materials science/physical pharmacy areas of come about only if scientists view it as a useful
pharmaceutical science. During the past few part of the array of analytical methods available
years, an increasing number of publications have to them. The future advancement of solid-state
emerged in which solid-state NMR spectroscopy NMR spectroscopy in pharmaceutical applica-
is an invaluable method for solving difficult pro- tions depends more on the creative use of it for
blems in many different areas. With the conti- solving specific problems rather than on techno-
nuing development of solid-state NMR pulse logical advances in the spectrometer hardware
sequences, along with hardware improvements and software.
that increase sensitivity and resolution, solid-
state NMR spectroscopy will provide more detail-
ed molecular information for the characterization ACKNOWLEDGMENTS
of pharmaceutical solids. Some of the areas
that will continue to progress rapidly include We thank Patricia Saindon Miyake, Gregory
(1) higher field strengths, (2) faster sample spin- Stephenson (Eli Lilly), Pascal Toma (Abbott Labo-
ning, (3) improved decoupling methods, (4) ad- ratories), and Joseph G. Stowell (Purdue Uni-
vanced pulse sequences, and (5) improved probes versity) for generously providing some of the
and other electronic components of spectrometers. data used in this review. We also thank Alan D.
However, increased use of solid-state NMR spec- Ronemus for his valuable advice and assistance
troscopy in analyzing pharmaceutical solids will with the solid-state NMR applications.
APPENDIX
Table A1. Summary of NMR-Related Acronyms Used in This Review
Acronym Description
1Q, 2Q, 3Q, MQ Single, double, triple, and multiple quantum
2DTOSS 2-Dimensional total sideband suppression
CP Cross polarization
CPPI Cross polarization/polarization inversion
CRAMPS Combined rotation and multiple pulse spectroscopy
CSA Chemical shift anisotropy
DECRA Direct exponential curve resolution algorithm
EIS Exchange-induced sidebands
EXSY Exchange spectroscopy
FSLG Frequency-switched Lee–Goldberg
HETCOR Heteronuclear correlation
HMBC Heteronuclear multiple bond correlation
HMQC Heteronuclear multiple quantum coherence
INADEQUATE Incredible natural abundance double quantum transfer
J-coupling Through-bond coupling
MAS Magic-angle spinning
MAS-J-HMQC Magic-angle spinning heteronuclear multiple quantum coherence
using scalar coupling
NMR Nuclear magnetic resonance
NOESY Nuclear overhauser effect spectroscopy
ODESSA One-dimensional exchange spectroscopy by sideband alternation
REDOR Retational echo double resonance
REPT-HMQC Recoupled polarization transfer-heteronuclear multiple quantum
correlation (coherence)
SPE Single-pulse excitation
SS-APT Solid-state attached proton test
TOBSY Total through-bond correlation spectroscopy
TOSS Total sideband suppression
TPPM Two-pulse phase modulation
WIMSE Windowless isotropic mixing for spectral editing

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MINIREVIEW

Solid-State Nuclear Magnetic Resonance

Received 17 April 2002; revised 2 August 2002; accepted 4 September 2002

ABSTRACT: Solid-state nuclear magnetic resonance (NMR) spectroscopy has become an

INTRODUCTION analytical applications.8 More recently, solid-

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from the ratios of the corresponding carbon Molecular Motions—Relaxation

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Figure 3. Expansion of the spectra shown in Figure 2

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conformation. Similar correlation spectroscopy

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Figure 5. The 2D MAS-J-HMQC spectrum of a natural abundance sample of the

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Figure 6. REPT-HMQC spectra of L-tyrosine hydrochloride at 30 kHz MAS and

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 3, MARCH 2003

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JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 3, MARCH 2003

Figure 8. Chemical structures of (A) prednisolone tert-butylacetate, (B) 21-

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Figure 10. 15N CP/MAS NMR spectra of lisinopril at

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Number of NMR Scans

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Table 2. Activation Energies from Solid-State

Carbon Chemical Shift (ppm) Ea (kJ/mol)

carbon atom (C3) had the highest activation energy

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Table A1. Summary of NMR-Related Acronyms Used in This Review

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REFERENCES 13. Otsuka M, Matsumoto T, Kaneniwa N. 1989. Ef-

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JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 3, MARCH 2003

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JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 3, MARCH 2003

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 3, MARCH 2003

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 3, MARCH 2003

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