Solid-State Nuclear Magnetic Resonance Spectroscopy-Pharmaceutical Applications

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...

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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 spectroscopy), (3) assigning resonances (spectral editing and two-dimensional correlation spectroscopy), 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 The use of nuclear magnetic resonance (NMR) spectroscopy in pharmaceutical research has a long and successful history, primarily in early stages of drug discovery. Most of these NMR studies have been performed with liquid solutions and were conducted primarily to analyze relatively small organic molecules, with significantly fewer applications for macromolecules like proteins, nucleic acids, carbohydrates, and polymers. Studies have included: (a) Elucidation of the structure of compounds,1 (b) investigation of the chirality of drug substances,2 (c) the analysis of cellular metabolism,3,4 and (d) studies of proteins.5–7 In consideration of the later stages of commercial drug development, NMR spectroscopy is traditionally used for conformational analysis, structure elucidation (impurity profiling), and Correspondence to: David E. Bugay (Telephone: 765-4630112; Fax: 765-497-2649; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 92, 441–474 (2003) ß 2003 Wiley-Liss, Inc. and the American Pharmaceutical Association

analytical applications.8 More recently, solidstate NMR spectroscopy has come to the forefront of analytical techniques. Approximately 80–90% of pharmaceutical products on the market exist in the solid form. However, solid-state NMR spectroscopy, in many cases, is just beginning to be applied to pharmaceutical problem solving and methods development. Regulatory documentation is now making specific reference to solid-state NMR spectroscopy. A flow-chart approach to the physical characterization of pharmaceutical solids was first published in 1995.9 In this approach to determining the number of polymorphic forms, spectroscopy, and specifically solid-state NMR spectroscopy, is outlined as a recommended technique. The Food and Drug Administration (FDA) has also recognized the need for solid-state NMR characterization of drug substances or products. In the most recent International Committee of Harmonization (ICH) Q6A guideline for the setting of specifications for drug substance and product, spectroscopy (IR, Raman, and solid-state NMR) is referred to in decision tree four (investigating the need to set

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acceptance criteria for polymorphism in drug substances and drug products).10 Worldwide regulatory authorities now recognize the importance of solid-state NMR spectroscopy and how the technique is intimately tied to the drug development process. During the course of developing pharmaceutical compounds, it is becoming increasingly important to characterize the drug in its dispensed form, which is frequently a solid. It has long been known that drugs may exist in more than one polymorphic form.11 These forms sometimes display very significant differences in solubility, bioavailability, processability, and physical/chemical stability.12 Hence, solid-state analytical techniques are necessary for the characterization of new chemical entities during the drug development process. The study of pharmaceutical compounds in the solid state must take place both at the bulk level and for the dosage form. Sometimes, the extreme conditions of processing the formulation into the dosage form can alter the drug,13 increase its interaction with excipients, or significantly impact the stability properties of the solid.14 These observations reinforce the need for sensitive and specific solid-state analytical techniques. Etter and Vojta published a paper in 1989 on the concurrent use of solid-state NMR spectroscopy and X-ray crystallography to study the structure of pharmaceutical solids.15 Crystallographic effects such as polymorphism, multiple molecules per asymmetric unit cell, disorder, intra- and intermolecular hydrogen bonding, tautomerism, and solvation were all investigated by solid-state NMR spectroscopy. Their work demonstrated that structural and conformational analyses of pharmaceutical solids by spectroscopic and diffraction methods provide complementary data. Therefore, full characterization of drug compounds should include both techniques. It is important to note that solid-state NMR spectroscopy is a nondestructive, multinuclear technique that can probe the chemical environment of specific nuclei within molecules. Additionally, it is a quantitative technique that may be used in conjunction with other solid-state techniques, such as thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), optical microscopy, infrared (IR) and Raman spectroscopy, and X-ray diffraction techniques (single crystal and powder), for the investigation of pharmaceutical solids. Solid-state NMR analysis of different solid forms can provide crucial information that often JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 3, MARCH 2003

resolves conflicting results obtained with other analyses, such as IR, X-ray powder diffraction, and thermal methods.16 In addition, a dictate of formulation technology is that the physical form of the drug substance, after being defined and verified, should not change once the product has been manufactured. Solid-state NMR spectroscopy provides a powerful method for comparing the physical form of the drug substance after pharmaceutical processing or manufacturing. Furthermore, solid-state NMR spectroscopy provides a method for the analysis of mixtures of solid forms in both the pure drug substance and in dosage forms.16 Solid-state NMR spectroscopy has increasingly been used for the analysis of pharmaceutical solids within the last decade. Recent advances in NMR hardware and software have made the acquisition of high-resolution, multinuclear NMR spectra of solids routine. Unfortunately, because of proprietary constraints and possibly underutilization of the technique, relatively few solid-state NMR investigations of pharmaceutical compounds have appeared in the literature. This review is necessarily limited to a selective summary of published solid-state NMR investigations and includes several new examples from our laboratories. Where possible, examples of newer solid-state NMR experiments that include pharmaceutical compounds have been chosen. However, some recent techniques that are not yet commonly used are also included because of the potentially valuable information that may be gained from their wider use in the analysis of pharmaceuticals.

SOLID-STATE NMR SPECTROSCOPY Basic Principles The most obvious difference between NMR spectroscopy of solids and liquids is that solids usually produce much broader peaks. This difference is largely due to strong dipolar coupling interactions and chemical shift anisotropy (CSA) in solids that are eliminated or reduced by the fast random motions of liquid samples. Both dipolar coupling and CSA are orientation-dependent phenomena. Dipolar coupling depends on the orientation of two nuclear dipoles relative to each other. CSA depends on the orientation of a nuclear dipole with respect to the direction of the static magnetic field. The line width problem in solids has been

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substantially reduced by using NMR probes that can handle high decoupling power and by rapidly spinning the sample. High-power proton decoupling effectively removes both homonuclear (1H, 1H) and heteronuclear (1H, 13C) dipolar coupling and results in narrower line widths in the observed carbon spectrum. The homonuclear 13C– 13C dipolar coupling is usually not of concern unless the sample has been 13C-labeled. Magic angle spinning (MAS) was first introduced by Andrew et al. for observing the solid-state 23Na NMR spectrum of a sodium chloride crystal.17 They demonstrated that the minimum line width is obtained when the angle between the sample spinning axis and the direction of the static magnetic field is 548 440 (54.748). A variety of applications for MAS in solid-state NMR spectroscopy is discussed in a dated, but still relevant review by Andrew.18 The dipolar coupling interactions between nuclei in solids can also be an advantage for improving the sensitivity of nuclei with low natural abundance (rare spin). Pines et al. first demonstrated a sensitivity enhancement method by using cross polarization (CP) of magnetization from protons to carbons in solid-state NMR spectroscopy.19 This method has subsequently been used for enhancing the sensitivity of many different nuclei, usually by CP from protons, although fluorine and phosphorus have also been used. In ideal cases, CP can enhance the sensitivity of a rare spin nucleus by a factor that is proportional to the magnetogyric ratios (g) of the nuclei involved (e.g., gH/gC  4 for 1H and 13C). Schaefer and Stejskal combined MAS and CP to obtain the first 13C CP/ MAS NMR spectrum, and this has become the standard experiment for solid-state NMR spectroscopy of organic solids.20,21 The significance of this development is apparent in that a modern solidstate NMR spectrometer can acquire a recognizable 13C spectrum of a crystalline compound with a single scan. The CP from protons provides another advantage because the time between scans is based on the T1 relaxation time of the protons rather than those of 13C nuclei, which are usually much longer. The developments in and applications of solidstate NMR spectroscopy over the last two decades have been discussed in detail in several reviews22,23 and a monograph.24 Jelinski and Melchior have reviewed basic solid-state NMR spectroscopy, including instrumental aspects and a number of useful insights for obtaining high quality spectra.25 Michel and Engelke thoroughly covered the details of CP methods under MAS

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conditions.26 Voelkel reviewed the relaxation processes in solid-state NMR spectroscopy for polymeric systems.27 Solid-state NMR reviews related to pharmaceutical compounds are less extensive because there are relatively few published applications.16,28–30 In addition to the line-narrowing and sensitivity-enhancing methods used for solid-state NMR spectroscopy, accurate temperature control for the sample is required to obtain valuable information in a variety of solids. Variable temperature solidstate NMR spectroscopy is generally a requirement for obtaining relaxation data that is necessary to analyze molecular motions in solids (see Pharmaceutical Applications, Analysis of Molecular Motions—Relaxation). It is generally not a trivial exercise to obtain accurate temperatures, and many reports have been published on the topic of temperature calibration for solid-state NMR spectroscopy.31–46 In particular, it is important to know the design characteristics of the probe and rotor because gas flow into the probe controls the rotor speed and stability as well as regulates the sample temperature.35,44 Some important considerations are the position of the thermocouple, whether or not the bearing gas is used for heating and cooling, the type of gas used, and the type and diameter of the rotor. The spinning speed and radio frequency energy used to obtain experimental data are also important. The importance of these factors indicates that the calibration procedure must be essentially identical to the conditions that will be used for acquiring data. One of the most common temperature calibration standards (PbNO3) has no protons and does not require CP.31–33,35 –38 However, CP can cause temperature changes in a sample, and the calibrations may need to be performed under CP/MAS conditions even if doing so will not enhance the sensitivity. A potential further complication is that the temperature dependence of relaxation times can cause large variations in the signal-to-noise ratio for a particular sample at different temperatures. Optimization of several parameters at each temperature, including the Hartmann–Hahn matching condition, the contact time, and the relaxation delay between scans, will generally alleviate this problem at the expense of a significant amount of time. There are several acceptable standards for calibrating accurate temperatures for solid-state NMR spectroscopy. The first temperature calibration standard (chemical shift thermometer) used for solid-state NMR spectroscopy was samarium JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 3, MARCH 2003

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acetate.45 Solid–solid or solid–plastic phase transitions in organic compounds have been used for single-point temperature calibrations.34,39,40 Tautomerism of an organic dye molecule has been used as a chemical shift thermometer for solid-state 15 N NMR spectroscopy.41,42 Lead nitrate has more recently received attention as a chemical shift thermometer because its lineshape and peak position change in a systematic manner with temperature.31–33,35 –38 A significant disadvantage is the high toxicity of lead nitrate, and proper safety precautions should be observed. One of the most commonly used applications of solid-state NMR spectroscopy for pharmaceutical solids involves solid-form characterization (including polymorphs, hydrates, solvates, and amorphous forms) of new chemical entities. The use of solid-state NMR spectroscopy for the investigation of polymorphism can be understood based on the following model. If a compound exists in two, true polymorphic forms labeled as A and B, each form is crystallographically different. Therefore, a carbon nucleus in form A may be situated in a slightly different molecular environment than the same carbon nucleus in form B. Although the chemical bonding of the carbon nucleus is the same in each form, the local environment may be different because of restricted motions in the solid state. These restricted motions cause differences in the local environment for one or more carbons in each polymorph because their spatial position is different with respect to the other nuclei in the molecule. The solid-state NMR spectra show this difference as a change in the isotropic chemical shift of the corresponding carbon in each polymorph. If one is able to obtain pure material for the two forms, analysis and assignment of the solid-state NMR spectra of the two forms can lead to the origin of the crystallographic differences in the two polymorphs. Solid-state NMR spectroscopy is thus an important tool in the multidisciplinary approach to analyzing polymorphism. There are a number of significant advantages to using solid-state NMR spectroscopy for the study of polymorphism. Compared with diffuse reflectance IR, Raman, and X-ray powder diffraction techniques, solid-state NMR spectroscopy is a bulk technique in which particle size effects have little impact on the intensity of the measured signal. In addition, NMR spectroscopy is a quantitative technique under proper data acquisition procedures. Therefore, the intensity of the signal will be directly proportional to the number of nuclei producing it. In some investigations of polyJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 3, MARCH 2003

morphs, a single crystal of sufficient quality may not be available for structure determination by X-ray crystallography. However, by appropriate resonance assignment of the NMR spectrum, the origin of the polymorphism may be inferred from chemical shift differences for identical nuclei in each polymorph. These benefits do not indicate that solid-state NMR spectroscopy can replace X-ray diffraction or any other analytical method. Instead, the advantages of solid-state NMR serve as a reminder to us that a multidisciplinary approach to solid-state characterization should include spectroscopic, diffraction, and thermal methods. The usefulness of solid-state NMR analysis of polymorphs was first demonstrated for hydroquinone by Ripmeester.47 Subsequently, there have been many studies using various solid-state NMR methods to examine polymorphic forms of organic compounds. Analysis of polymorphic systems is currently the predominant use of solid-state NMR spectroscopy in pharmaceutical research (see Pharmaceutical Applications, Polymorphism).

METHODS Quantitative Analysis In most of the publications describing the use of solid-state NMR spectroscopy for the characterization of pharmaceutical compounds, the majority of the work has dealt with qualitative studies, with brief references to the possibility of quantitative analysis. An excellent guide to the utilization of MAS and CP techniques for acquiring quantitative solid-state NMR data has been outlined by Harris.48 To acquire solid-state NMR spectra in which the signal intensities directly reflect the number of nuclei producing them, data acquisition parameters, such as recycle delay time, pulse durations, CP contact time, Hartmann–Hahn matching conditions, and decoupling power, must be explicitly determined for each chemical system. The magic angle and spin rate also must be set accurately to obtain quantitative measurements. Harris also compared the CP experiment with the single-pulse excitation (SPE) experiment for obtaining quantitative solid-state NMR spectra. This comparison was important because the dynamics of CP are different for each carbon within a compound, and the ratios of peak intensities in a CP/MAS spectrum may be significantly different

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from the ratios of the corresponding carbon nuclei.48 The SPE (or Bloch decay) experiment for solids is the application of one excitation pulse followed by acquisition, usually with high-power proton decoupling and MAS without CP. This pulse sequence is useful for systems in which the longitudinal relaxation time (T1) of the observed nucleus is relatively short or there are no nuclei with which to cross polarize the observed nucleus. To get quantitative peak integrals with the SPE experiment, the relaxation delay time must be long enough to allow the magnetization of each nucleus to reach equilibrium prior to acquiring another scan (normally 1–5 times T1). The effects of CP can be determined by acquiring an SPE spectrum with a suitably long relaxation delay and comparing the result to a CP/MAS spectrum of the same compound. The differential NMR relaxation times of nuclei within a molecule present significant problems for obtaining quantitative data using CP. With the CP/MAS experiment, it is necessary to measure the relaxation profiles and the rates of CP for each carbon to obtain quantitative information.49,50 The sample spinning rate is also important because the peak intensity is distributed among the spinning sidebands, and one must account for each sideband to obtain accurate quantitative measurements.50 Quantitative analysis of solid-state NMR spectra has been used to study very difficult cases, such as the components of wood.51 This study demonstrated that quantitative determination of amorphous materials is possible using solid-state NMR spectroscopy. Molecular Motions Molecular motions can be studied by both solidstate NMR spectroscopy and X-ray crystallography. Solid-state NMR spectroscopy offers several unique approaches to studying molecular motions in solids. These approaches include (1) the study of processes that result in coalescence of NMR resonances using variable temperature solid-state NMR spectroscopy, (2) determination of the relaxation processes (T1, T1r, T2) of individual nuclei using variable-temperature solid-state NMR spectroscopy, (3) use of spectral editing methods based on CP rates to detect the different parts of a molecule with unusual mobility, (4) comparison of solid-state MAS spectra measured with and without CP, and (5) various methods to analyze chemical exchange.

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Molecular Motions—Relaxation The relaxation of nuclear magnetism towards its equilibrium value in a static magnetic field is characterized by the spin-lattice relaxation time constant (T1).52–54 In this process, the excess energy from the spin system is transferred to the surroundings or lattice. Interactions with randomly fluctuating magnetic fields at the Larmor frequency of the nucleus stimulate relaxation. These fields arise from motions of other nuclear magnetic moments. Relaxation is most efficient when the maximum number of fluctuating magnetic fields occur at the Larmor frequency of the observed nucleus. Spin-lattice relaxation in the rotating frame (T1r) is similar to T1 relaxation except that it occurs at a much lower field strength during the spinlock time used to obtain the Hartmann–Hahn condition for CP in solidstate NMR spectroscopy. Both of the spin-lattice relaxation times (T1 and T1r) are relatively long for highly mobile liquids and continue to decrease with increasing viscosity until the solid state is reached. A minimum occurs in either relaxation time when the motions in the lattice (the fluctuating magnetic fields at the appropriate frequency) most efficiently relax the observed nucleus. The relaxation times then began to increase again as the lattice motions are no longer efficient at causing relaxation. The transverse or spin–spin relaxation time (T2) is an entropic process in which random spin flips dephase the coherent magnetization produced by input of radio frequency energy. The T2 relaxation time is longer for mobile liquids and decreases asymptotically to a minimum value at the so-called ‘‘rigid lattice limit’’ in solids. NMR relaxation processes in solids can provide information about the molecular motions occurring at the location of the nucleus being observed. In organic solids, 1H and 13C relaxation parameters are generally of most interest, but 15N relaxation measurements are practical in cases where labeling is used. The T1 relaxation time is useful for measuring motions in the megahertz frequency range (Larmor frequency). The T1r relaxation time is sensitive to kilohertz frequency motions because the spinlock field strength is much lower than the static magnetic field strength.20 The T2 relaxation time is similar to T1r in the range of motions that it can measure.55 The methods of inversion recovery and exponential decay-to-zero have been used to measure JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 3, MARCH 2003

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T1 relaxation in solids.56–58 The latter method is more practical for solid-state NMR spectroscopy, where compounds have long T1 relaxation times, because it produces fewer artifacts.56,58 Methods for obtaining and analyzing the T1r relaxation times of 1H and 13C nuclei in polymers have been extensively described.20,59 –63 The 1H T1 and T1r times are generally of less utility for site-specific analyses because the high natural abundance and strong dipolar interactions of protons can lead to spin diffusion and nonspecific relaxation times. MAS may increase or decrease the 1H T1 relaxation times in some solids depending on their proton densities.64 However, 1 H relaxation times are useful if one wants to study mixtures of compounds that have different relaxation properties. A major drawback to relaxation experiments is that they can be very time consuming because multiple measurements at different temperatures are usually required. For 13C and 15N NMR in particular, T1 relaxation times can be minutes to hours in length, especially for highly crystalline material with minimal molecular motions. The application of relaxation time measurements to pharmaceutical solids has not received much attention probably because of the amount of time needed to carry out the experiments. However, a carefully acquired set of relaxation data can provide unique information on molecular motions for a compound that cannot be obtained by other means. This usefulness is apparent in an interesting recent study in which the 13C and 2H T1 relaxation times were used to determine the C--H bond length in ferrocene.65

intramolecular orientational exchange and are not useful for examining intermolecular exchange processes. The method of 2D rotor-synchronized exchange spectroscopy is capable of detecting intramolecular and intermolecular exchange,71 and this method has been used for studying the conformation of a peptide.72 Other methods for 2D exchange spectroscopy (2D EXSY) have been used to study molecular motions in the solid-state. The solid-state 2D EXSY pulse sequence is a modification of the NOESY (nuclear Overhauser effect spectroscopy) experiment used for liquids.73,74 For solids, CP (rather than the usual 908 pulse) is used for the preparation period of the pulse sequence. The resulting spectrum provides a 2D spectrum that has cross peaks corresponding to the chemical shifts of the exchanging nuclei. In all cases, the exchange process must be slow on the NMR time scale to be observed by 1D and 2D exchange spectroscopy. Resonance Assignments A distinct disadvantage of the broad lines observed in most solid-state NMR spectra is that scalar couplings (J-couplings) are generally not resolved. This situation makes it substantially more difficult to unambiguously assign the chemical shifts in the spectra. Tentative resonance assignments can usually be made because many of the chemical shifts of organic solids are not drastically different from those observed for the same compound in solution. However, the direct determination of the solid-state NMR resonance assignments is necessary to obtain accurate structural and conformational information.

Molecular Motions—Exchange Spectroscopy One of the most useful applications for NMR spectroscopy is in the analysis of dynamic processes in molecules for both liquids and solids. A large time-scale range (109 –102 s) for molecular motions can be studied by NMR spectroscopy by using pulse sequences to analyze chemical exchange or relaxation processes (see Methods, Molecular Motions—Relaxation). Very slow motions in solids have been studied with rotorsynchronized one-dimensional exchange spectroscopy by sideband alternation (ODESSA),66 the time-reversed ODESSA,67,68 or exchange-induced sidebands (EIS).69,70 Motional correlation times and activation energies for certain slow exchange processes can be extracted from the spectra.69 However, these methods are generally limited to JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 3, MARCH 2003

Resonance Assignments—Spectral Editing A number of ‘‘spectral editing’’ pulse sequences have been developed to aid in the assignment of solid-state NMR spectra.75 One of the first editing sequences, based on a modification of the standard 13C CP/MAS sequence, was dipolar dephasing or interrupted decoupling.76,77 In this pulse sequence, the 1H and 13C radio frequency fields are removed for a brief period (40–100 ms) before acquisition to allow the carbon magnetization to decay because of 1H/13C dipolar coupling. The difference in the strength of the dipolar coupling for each type of carbon (CH2 > CH > C, CH3) results in a spectrum in which the CH and CH2 carbon resonances are substantially reduced or eliminated. The methyl carbons are not greatly

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affected because their rapid rotational motion reduces the dipolar coupling interaction. The quaternary carbons are only slightly affected because dipolar coupling to them is much weaker without a directly bonded proton. Dipolar dephasing is useful for both crystalline and amorphous materials and has been successfully used to study the complex mixture of components in coal that have very broad peaks.78 In an analogous method, Peng and Frydman have used differences in the chemical shift anisotropies (CSA-dephasing) of carbons for spectral editing of 3-methylsalicylic acid and p-methoxybenzoate. However, the results were generally poorer compared with dipolar dephasing spectra.79 Relaxation differences between individual nuclei or regions of a sample can be used to perform spectral editing. The delayed contact pulse sequence is one method based on relaxation differences that has been used to determine amorphous and crystalline regions in polymers.59 In this modification of the standard 13C CP/MAS sequence, the 1H spinlock field is turned on for a short time prior to turning on the 13C spinlock field to allow the magnetization to decay for protons with short T1r relaxation times. An alternative method is to begin with standard 1H– 13C CP, and then remove the 1H spinlock field while maintaining 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. Indeed, 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 synchronization 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 polarization inversion is accomplished by changing the phase of either the 1H or 13C spinlock pulse by 1808 to reverse the direction of magnetization transfer between spins, which results in either the disappearance or inversion of the observed signal depending on the particular characteristics of the sample. A series of pulse sequences based on this method have been developed to differentiate between C, CH, CH2, and CH3 groups in organic solids.87–89

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The spectral editing methods just discussed rely on dipolar coupling of nuclei through space to differentiate between types of carbons in a compound. One disadvantage of these techniques is their sensitivity to molecular motion that is apparent in the minimal response of rotating methyl groups to the editing schemes described. This effect can also be observed by the lack of selectivity for some resonances in a CPPI spectrum (see Figure 1 and Pharmaceutical Applications, Conformation and Stereochemistry). Several editing pulse sequences have been developed to use scalar coupling that acts through chemical bonds rather than through space as for the dipolar coupling interactions.90–92 The method of Terao was demonstrated for solid camphor, a nearly spherical molecule that has unusually small dipolar couplings and very narrow line widths due to rapid molecular motion.92 Many organic solids do not have such narrow resonances, which limits the usefulness of Terao’s technique. Emsley’s group has recently exploited J-coupling for editing of solid-state 13C NMR spectra of organic compounds with broader line widths.91 The SS-APT pulse sequence, analogous to the attached proton test for solution 13C NMR, can be used to differentiate between CH and CH2 groups in solids. The SS-APT spectra (Figures 2

Figure 1. 13C CPPI edited spectra of lisinopril dihydrate at 100.6 MHz (the CP/MAS and TOSS spectra are shown for comparison): (A) C and aliphatic CH  0, aromatic CH positive, CH2 negative; (B) C positive, CH and CH2 negative; (C) C  0, CH and CH2 positive; (D) TOSS spectrum; and (E) CP/MAS spectrum. The resonance assignments are given for the TOSS spectrum and correspond to the structure in Figure 8C. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 3, MARCH 2003

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Figure 2. (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. Some of the higher frequency resonance assignments of the C and CH groups are shown. (Reprinted with permission from ref. 91; copyright 1998; American Chemical Society.)

and 3) of cholesteryl acetate demonstrate the potential of the technique for assigning chemical shifts. The SS-APT experiment worked best with several special decoupling methods and at spinning speeds of 10–16 kHz. It is useful only for samples with 13C at natural abundance because 13 C– 13C dipole couplings interfere with the method. The frequency-switched Lee–Goldburg (FSLG) sequence was used for 1H homonuclear decoupling to obtain narrow carbon line widths and to resolve the 1H– 13C J-couplings.93–95 The two-pulse phase modulation (TPPM) sequence was used during acquisition to remove heteronuclear coupling during the acquisition time.96 Through-bond 1H– 13C coupling has also been used for spectral editing by creating multiple JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 3, MARCH 2003

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 Chemical Society.)

quantum coherence (1Q, 2Q, and 3Q) to discriminate between the types of carbons in a protected tripeptide.90 This ‘‘J-MQ filter’’ removes quaternary carbon resonances in the 1Q experiment and quaternary and methine carbons in the 2Q experiment, thereby leaving only methyl carbons in the 3Q experiment. However, sensitivity gets progressively worse for the higher quantum filtering experiments. The authors state that the method should work well for amorphous solids, but no examples have been demonstrated so far. Resonance Assignments—2D Correlation Spectroscopy Correlation spectroscopy is routinely used for analyzing the structure of organic molecules in solution. Both homonuclear and heteronuclear NMR correlation experiments in solutions are valuable methods for determining the chemical bonding pattern of a compound and thus its

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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 interaction to correlate the 1H and 13C spins. These correlation pulse sequences use a series of multiple 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 ˚ (usually 5–6 A ˚ ). Spiess’ group distance of <10 A 1 has used H spin diffusion to extend this range ˚ .101 This technique may be useful for up to 200 A 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 experiments.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. One such technique is the solid-state TOBSY (total through-bond-correlation spectroscopy) that was demonstrated for 13C-labeled calcium acetate and 13 C– 15N-labeled arginine.103,104 Other methods include the solid-state INADEQUATE105 and refocused INADEQUATE106 NMR experiments that were used to study 13C-labeled samples of isoleucine, cellulose, and wood chips. Of the two sequences, the refocused INADEQUATE proved to be more sensitive for compounds with greater line widths. However, both experiments are very time consuming and impractical without labeling of the sample. A very promising 2D correlation technique (MAS-J-HMQC) has been developed in Emsley’s group and does not require labeling of the sample to obtain data in a reasonable amount of time.107 The MAS-J-HMQC and dipolar HETCOR spectra of L-tyrosine hydrochloride are compared in Figure 4. The MAS-J-HMQC spectrum has approximately half the sensitivity of the dipolar HETCOR spectrum.107 However, the selectivity for one-bond J-coupled correlations is apparent

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Figure 4. The 2D (a) MAS-J-HMQC spectrum and (b) dipolar HETCOR spectrum of natural abundance L-tyrosine hydrochloride. (Reprinted with permission from ref. 107; copyright 1998; American Chemical Society.)

compared with the dipolar HETCOR spectrum. The MAS-J-HMQC spectrum for a more complicated tripeptide molecule is shown in Figure 5. The complete 1H, 13C, and 15N assignments for this tripeptide were subsequently obtained using MAS-J-HMQC NMR spectroscopy.108 The authors also demonstrated a modification of the technique to obtain multiple-bond correlations similar to the data from the HMBC sequence for liquids. Experiments based on scalar and dipolar coupling interactions offer complementary information that can be used to obtain unambiguous chemical shift assignments for solid materials without resorting to tentative assignments based on solution spectra. These data are necessary for determining the conformations of compounds in the solid state. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 3, MARCH 2003

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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.)

Internuclear Distance Measurements The dipolar coupling interactions that are partly responsible for the broad resonances in solidstate NMR spectra also can be used to determine internuclear distances in addition to obtaining homonuclear and heteronuclear correlations. For most cases, rapid-sample spinning removes or drastically reduces the dipolar couplings along with the chemical shift anisotropy to give the isotropic chemical shifts of each nuclei. The distance information provided by dipolar coupling is lost unless it can be reintroduced prior to acquiring a spectrum. A number of pulse sequences have been designed to recover the dipolar coupling information, to determine internuclear distances, and to obtain correlation spectra.109–111 The REDOR112 (rotational-echo double-resonance) NMR sequence is one of the many examples of dipolar recoupling experiments and has been used ˚ with to measure 13C– 15N distances up to 6.3 A 113,114 ˚ an accuracy of 0.1 A. Some limitations of these dipolar recoupling techniques include (1) only a single spin pair can be easily studied, (2) selective labeling is needed to provide an isolated spin pair, (3) total acquisition times are usually long, and (4) molecular motions and exchange can average the dipolar couplings and prevent accurate distance measurements.111 Uniformly labeled samples can be used if they are diluted to 10–20% in the unlabeled sample. This method was demonstrated with the 2D JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 3, MARCH 2003

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C– 13C correlation spectrum of 13C-labeled erythromycin109 and the 13C, 15N-labeled achatin-II tetrapeptide.115 Double-quantum dipolar recoupling pulse sequences have also been used to obtain accurate 13 C– 13C bond distance measurements. The doublequantum methods are better for samples with relatively large chemical shift anisotropies and can minimize interference from natural abundance 13C nuclei.116 A REDOR variant, termed REPT-HMQC (recoupled polarization transfer–heteronuclear multiple-quantum correlation), has been used to obtain 2D correlated 13C– 1H chemical shifts, to measure internuclear distances, and to perform 1D spectral editing.117 The results of the REPTHMQC experiment for L-tyrosine hydrochloride at two different field strengths are shown in Figure 6. These spectra should be compared with the MASJ-HMQC of the same molecule in Figure 4. The advantage of higher field strength is also apparent in the spectra shown in Figure 6. According to Saalwa¨chter et al., crystal packing effects are apparent in the 1H chemical shifts of the aromatic ring of tyrosine.117 The authors state that REPTHMQC does not require some of the special spectrometer setup procedures that other HETCOR experiments require. However, spinning at 30 kHz is necessary to obtain reasonable 1H resolution, and certain correlations are dependent on the multiple quantum recoupling time used (see Figure 6).

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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 The chemical shift anisotropy of a nucleus can be a useful means of extracting conformational data from solids because the CSA of each nucleus depends on its orientation with respect to the static magnetic field. Therefore, orientations of the nuclei relative to other nuclei in the molecule can also be determined. Several methods of analyzing CSA tensors have been used to examine both polymorphs and stereochemistry. The 2DTOSS118 experiment uses MAS at 1.5 kHz. The similar 2D FIREMAT119 experiment also relies on the spinning sideband pattern

produced at low MAS speeds (500 Hz). In both experiments, the anisotropic tensor patterns for each nucleus are separated into a second dimension by the differences in their isotropic chemical shifts. A requirement for these techniques is prior assignment of each isotropic chemical shift by spectral editing or correlation spectroscopy. Solid-State 1H NMR Spectroscopy The 1H-detected 1D and 2D NMR spectroscopy methods are standard for studying liquid solutions because of the high natural abundance and JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 3, MARCH 2003

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sensitivity of protons. However, these methods are not standard for studying solids because the strong 1H homonuclear dipolar couplings (40 kHz) and large chemical shift anisotropies (20 ppm) result in very broad line widths in the spectra. Similar difficulties arise for solid-state 19 F NMR spectroscopy, although most fluorinated organic compounds have relatively few fluorine atoms. Useful information can be obtained by using very fast MAS, special decoupling methods, similar to those used for the correlation spectroscopy experiments, or a combination of both. Gerstein et al. first applied the combined-rotation and multiple-pulse NMR spectroscopy (CRAMPS) method to obtain resolved chemical shifts in a fluorocarbon.120 Subsequently, CRAMPS has been developed for solid-state 1H NMR spectroscopy. However, solid-state 1H NMR still remains a relatively difficult experiment because of the long spectrometer ‘‘tune-up’’ time necessary to ensure that pulse widths and power levels are appropriately adjusted to obtain good data. A constant-time CRAMPS pulse sequence using FSLG decoupling has recently been developed where much narrower 1H line widths were obtained.121 However, the sensitivity is substantially lower than for the standard CRAMPS experiment, and some proton resonances may not be observed. Solid-state 1H NMR spectroscopy of pharmaceutical compounds is likely to become more widely used only when very high spinning speeds and the special multiple-pulse sequences are more easily implemented.

PHARMACEUTICAL APPLICATIONS There are a variety of different applications of solid-state NMR spectroscopy that are relevant to pharmaceutical research. Some of these include analysis of (1) solid forms (polymorphs, solvates), (2) hydrogen bonding and crystal packing, (3) amorphous solids, (4) stereochemistry, and (5) solid–solid interactions (phase transformations, activation energies of molecular motions, and solid-state reactions). Aboul-Enein has outlined some of the general uses of solidstate NMR spectroscopy for pharmaceutical research.28 General Applications An example of solid-state 31P CP/MAS NMR spectroscopy was the study of the dehydration of JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 3, MARCH 2003

disodium clodronate.122 A fast rise in temperature reveals that disodium clodronate loses lattice water and only one 31P resonance is measured, whereas a slow increase in temperature converts the crystalline form to an anhydrous form that displays two nonequivalent phosphorus atoms (two separate resonances). Some studies using solid-state NMR of pharmaceutical compounds involved characterizing the structures of N-desmethylnefopam hydrochloride,123 patellin,124 and erythromycin A dihydrate,125 and the amorphous nature of ursodeoxycholic acid.126 The conformations of 30 amino-30 -deoxythymidine,127 gramicidin A,128 and amiodarone hydrochloride129 have also been confirmed by solid-state NMR spectroscopy. Because of the specificity of solid-state NMR spectroscopy, it is an ideal technique to study inclusion complexes, drug–excipient interactions, or the effect of moisture on the drug substance or formulation. Studies on inclusion complexes include gliclazide-b-cyclodextrin130 and hydrocortisone butyrate.131 Makriyannis utilized NMR spectroscopy for investigating drug–membrane interactions.132 Other interactions studied by solid-state NMR spectroscopy include polyethylene glycol with griseofulvin133 and the moistureinduced interaction with trospectomycin sulfate that affects the equilibrium between the 30 -gemdiol and 30 -keto forms in the drug substance and the freeze-dried formulation.134 Polymorphism The study of polymorphism appears to be one of the most common applications of solid-state NMR spectroscopy for pharmaceutical compounds.29,135 Pharmaceutical solids can exist in a number of solid forms, each having different properties of pharmaceutical importance, including stability and bioavailability. The number of these forms and their properties are largely unpredictable and vary considerably from case to case. Pharmaceutical solids can be divided into crystalline and amorphous solids based on X-ray powder diffraction and/or microscopic examination. Crystalline solids can then be further classified into (1) polymorphs, forms having the same chemical composition but different crystal structures and therefore different densities, melting points, solubilities, and other properties; and (2) solvates, forms containing solvent molecules within the crystal structure, giving rise to unique differences in solubility, response to atmospheric moisture,

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loss of solvent, and other properties. Sometimes a drug substance may be a desolvated solvate that is formed when solvent is removed from a specific crystalline solvate while retaining a crystalline structure. Many important properties are unique to such a form. Different physical forms of a drug substance can display radically different solubilities, which directly affects the dissolution and bioavailability characteristics of the compound. In addition, the chemical stability of one form, as compared with another, may vary. The physical stability of polymorphs is also crucial. During various processing steps (grinding, mixing, tablet pressing, etc.), the physical form of the drug substance may be compromised, subsequently leading to dissolution problems. For these reasons, the full characterization of polymorphic systems is critical to numerous groups within commercial drug development; namely, preformulation/physical pharmacy, chemical process development, regulatory affairs, intellectual property, and analytical development. The solid-state 13C CP/MAS spectra of two flufenamic acid polymorphs are shown in Figure 7. The structure of the analgesic, flufenamic acid, is given in Figure 8F. The resonance assignments in the solid state have not been unambiguously assigned for the polymorphs because they are quite different from the solution 13C NMR positions.136 In general, only tentative resonance assignments are possible when comparing

Figure 7. 13C CP/MAS NMR spectra of flufenamic acid polymorphs: (A) Form III and (B) Form I.

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solution and solid-state NMR spectra. Assignments may be confirmed with solid-state spectral editing or correlation spectroscopy (see Methods, Resonance Assignments). The solution 13C NMR spectrum and solid-state 13 C CP/MAS NMR spectra of two crystalline and an amorphous form of lisinopril, an angiotensin converting enzyme inhibitor, are compared in Figure 9.136 The structure of lisinopril is shown in Figure 8C. In this case, the chemical shift dispersion is quite similar for the solution and solid forms. Removing the water from the dihydrate form causes subtle changes in the peaks at 55 and 175 ppm that are probably due to changes in hydrogen bonding. The small peaks in the solution NMR spectrum are due to a minor conformation of lisinopril in exchange with the major conformation due to restricted rotation about the amide bond. Only one conformation is observed in the solidstate NMR spectra because the rotation is frozen out at room temperature. The solid-state 15N CP/ MAS spectra of crystalline and amorphous lisinopril, shown in Figure 10; demonstrates that useful natural abundance 15N NMR data can be obtained for both crystalline and amorphous pharmaceutical compounds with relatively low nitrogen densities (10% of the sample mass).136 Polymorphs may be studied using solid-state NMR spectroscopy of less common nuclei, such as 15 N, to provide additional information or to determine specific details within the molecule. Solid–solid phase transitions have been observed by 13C and 15N solid-state variable temperature NMR spectroscopy of 15N-labeled 2-(2,4-dintrobenzyl)-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 determined to be in the keto tautomeric form using 13 C solid-state NMR data and X-ray crystallography.138 In a second study, the three polymorphs of tedisamil dihydrochloride were studied by Burger’s group using variable-temperature solidstate 13C NMR spectroscopy and X-ray powder diffraction.139 The study was mainly of academic interest because the two high-temperature forms were not stable at room temperature and could not be isolated. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 3, MARCH 2003

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Figure 8. Chemical structures of (A) prednisolone tert-butylacetate, (B) 21cyclopentyl ester of cortisol, (C) lisinopril, (D) peri-substituted naphthalenes, (E) ibuprofen, (F) flufenamic acid, and (G) [5-methyl-2-(2-nitrophenyl)amino]-3-thiophenecarbonitrile (ROY).

The potential for polymorphism in anhydrous theophylline was examined by theoretical structure calculations using X-ray powder diffraction data and ab initio calculations of NMR shielding tensors.140 In this study, the hydrogen bonding in theophylline was determined by 13C and 15N NMR spectroscopy. The results eliminated one of the two possible hydrogen bonding configurations, and the remaining structure was similar to the crystal structure of anhydrous theophylline. Munson and co-workers have studied uniformly 13 C-labeled aspartame polymorphs using 13C labeling, fast MAS (up to 28 kHz), very high decoupling power (up to 263 kHz), and a dipolar recoupling JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 3, MARCH 2003

pulse sequence to observe short-range and longerrange couplings.141 Aspartame exists in three hydrates (two hemihydrates and a dihemihydrate). Two of these hydrates apparently contain three molecules per asymmetric unit and show three resolvable resonances for each carbon atom. This complex crystal packing prevented assignments of the resonances using techniques based on the number of attached protons or J-couplings. Typical MAS experiments at spinning rates of 7 kHz and proton decoupling powers of 63 kHz gave broad peaks due to dipolar coupling. However, increasing the spinning rate to 28 kHz and the decoupling power to 263 kHz gave narrow

PHARMACEUTICAL USES FOR NMR

Figure 9. Comparison of the solution and solid-state 13 C NMR spectra of lisinopril at 100.6 MHz (solids) and 150.9 MHz (solution). The total time for data acquisition is given for each spectrum: (A) amorphous form CP/MAS spectrum (17 min); (B) anhydrous crystalline form CP/ MAS spectrum (54 min); (C) dihydrate crystalline form, CP/MAS spectrum (54 min); (D) D2O solution spectrum with 100 Hz line broadening (2 h); and (E) D2O solution spectrum with 5 Hz line broadening.

resonances that allowed the assignment of the crystallographically inequivalent sites. For one of the forms, peak assignments could be made for all three molecules in the asymmetric unit of the crystal. The five known polymorphs of sulfathiazole were distinguished by solid-state 13C NMR spectroscopy.142 One observation to note in this study was that polymorph I was partially converted to polymorph IV during solid-state NMR spectroscopy, and this transformation was attributed to sample spinning. However, the forces transferred to a solid sample in a rapidly spinning rotor are generally much less than the forces applied during tablet formation. The most obvious possible cause for a transformation would be sample heating due to either spinning or radio frequency energy input, which is certainly possible given the changes shown for the variable temperature spectra in Figure 3 of that article.142 Medek and Frydman analyzed vitamin B12 polymorphs with solid-state 13C, 15N, 31P, and 59 Co NMR spectroscopy.143 The 13C NMR data were the most informative because the highly crystalline material produced sharp resonances

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Figure 10. 15N CP/MAS NMR spectra of lisinopril at 40.54 MHz for (A) amorphous form and (B) dihydrate crystalline form. Each spectrum was acquired in 11 h. The chemical shift assignments correspond to the labeled lisinopril structure in Figure 8C.

for most of the carbons in this rather complex organic molecule. The solid-state 13C NMR analysis of several amphetamines and their mixtures with lactose monohydrate have been reported.144 The lactose monohydrate mixture of one amphetamine (3,4methylenedioxyamphetamine hydrochloride) produced distinct chemical shift differences for the amphetamine resonances, and the authors attribute the changes to relaxed crystal packing forces rather than hydrogen bonding. Two racemic polymorphs and an enantiomorph of tazefelone, an antioxidant and 5-lipoxygenase inhibitor, were studied by solid-state 13C and 15N NMR spectroscopy and X-ray crystallography.145 The different forms produced very distinct NMR spectra, and the unique hydrogen-bonding pattern of each form resulted in chemical shift differences, especially for the 15N NMR spectra. Intramolecular hydrogen bonding, tautomerization, and polymorphism in several Schiff’s bases have been studied with natural abundance 15N and 13C solid-state NMR spectroscopy.146 The 15N NMR spectra were more sensitive for detecting tautomers because of the large chemical shifts of 15 N that depend on specific interactions with other nuclei. The majority of applications of solid-state NMR spectroscopy that have been used in the investigation of pharmaceutical polymorphs are performed JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 3, MARCH 2003

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in conjunction with other analytical techniques. Byrn et al. have reported differences in the solidstate NMR spectra for different polymorphic forms of benoxaprofen, nabilone, and pseudopolymorphic forms of cefazolin.147 Although singlecrystal X-ray diffraction was initially used to study the polymorphs, the solid-state 13C CP/MAS NMR spectrum of each form was distinctly different. These studies primarily focused on the bulk drug material, although a granulation of benoxaprofen was also studied. The authors concluded that solidstate NMR spectroscopy could be used to differentiate the form present in the granulation, even with excipients. In other studies at Purdue University, the crystalline forms of prednisolone tert-butylacetate,148 cefaclor dihydrate,149 and glyburide150 were studied. The five crystal forms of prednisolone tert-butylacetate were again determined by single-crystal X-ray diffraction, and solid-state NMR spectroscopy was used to determine the effect of crystal packing on the 13C chemical shifts of the different steroid forms. Although conformational changes were observed in the ester side chain by X-ray crystallography, no major differences were noted in the NMR spectra, indicating that the environment remains relatively unchanged. However, significant chemical shift differences were noted for carbonyl atoms involved in hydrogen bonding. This observation is consistent with the NMR study of cefaclor dihydrate. Again, the effects of hydrogen bonding were discernible by solid-state NMR spectroscopy. The study of glyburide was principally concerned with the molecular conformation of the compound in solution and in the solid state. The conformation in solution was determined by 1H and 13C NMR spectroscopy and in the solid by single-crystal X-ray diffraction, IR, and solid-state 13C NMR spectroscopy. The solid-state NMR results suggested that this method would be useful for comparing conformations of molecules both in solution and in the solid state. In a series of publications by Harris and Fletton, solid-state NMR spectroscopy was used to investigate the structures of several polymorphs.151 –155 The majority of these studies included X-ray diffraction and IR techniques and also addressed the possibility of quantitative measurements of polymorphic mixtures. The three pseudopolymorphic forms of testosterone were examined by IR and 13C CP/MAS NMR spectroscopy.151 The two forms of molecular spectroscopy were able to differentiate the forms, but NMR spectroscopy could be used to investigate nonequivalent JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 3, MARCH 2003

molecules in a given unit cell. A series of doublet resonances were noted for several different carbon atoms. This result implies that a specific carbon atom within the molecule may resonate at two different frequencies corresponding to each crystallographic site of the molecule (crystallographic splitting). In addition to the hydrogen bonding explanation of the crystallographic splittings, the use of NMR spectroscopy to quantitatively determine the amount of each pseudopolymorph present in a mixture was addressed. In the study of androstanolone,152 high-quality 13C NMR spectra were obtained by the CP/MAS technique, which permitted the characterization of the anhydrous and monohydrate forms. In this case, the crystallographic splittings were noted for the two forms and were correlated with hydrogen bonding. An identical approach was used to study a pharmaceutical polymorphic structure in the investigation of the two polymorphs of 40 -methyl20 -nitroacetanilide.153 An additional study of cortisone acetate154,155 by solid-state NMR spectroscopy revealed differences in the NMR spectra for the six crystalline forms. All of these studies indicate that conformational differences, hydrogen bonding, solvated forms, and polymorphs in solids are readily analyzed by solid-state NMR spectroscopy. Further multidisciplinary approaches to the physical characterization of pharmaceutical compounds are detailed in separate studies on cyclopenthiazide,156 the excipient lactose,157 frusemide,158 losartan,159 fosinopril sodium,160 leukotriene antagonists MK-679 and MK-571,161 L-660,711,162 captopril,163 diphenhydramine hydrochloride,164 mofebutazone,165 phenylbutazone,165 oxyphenbutazone,165 cimetidine,166 and the iron chelator 1,2 dimethyl-3-hydroxy-4-pyridone.167 In each case, solid-state NMR spectroscopy was used in conjunction with other techniques such as DSC, IR, X-ray diffraction, microscopy, and solubility/dissolution studies to fully characterize the polymorphic systems. In these studies, solid-state NMR spectroscopy is one of several complementary techniques for fully characterizing pharmaceutical solids. NMR spectroscopy fits into the niche between the techniques of X-ray diffraction for analysis of longrange order and vibrational (IR and Raman) spectroscopy for analysis of organic functional groups. In other studies of polymorphism by solid-state NMR spectroscopy, conversion from one solid form to another by ultraviolet irradiation168 and variable-temperature techniques169 are outlined.

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In the first study,168 NMR spectroscopy was employed to follow the chemical transformation within the organic crystals of p-formyl-transcinnamic acid ( p-FCA). A photoreactive b-phase can be crystallized from ethanol, whereas a photostable g-phase is produced from acetone. After irradiation of the b-phase with UV radiation and subsequent acquisition of the solid-state 13C NMR spectrum, the photoproduct was easily identified. The second conversion study169 investigated the four forms of p-amino-benzenesulphonamide sulphanilamide (a, b, g, and d). The first three forms were investigated by solid-state 13C NMR spectroscopy and X-ray crystallography techniques. Subsequent variable-temperature studies monitored the interconversion of the a and b forms to the g form. Coalescence of some NMR signals in the g form also suggested that phenyl ring motion occurred within the crystal. Conclusions from the study indicated that solid-state NMR spectroscopy could differentiate pharmaceutical polymorphs, determine asymmetry in the unit cell, and analyze molecular motion within the solid state. Drug Substance Quantitative Analysis A few quantitative solid-state NMR analyses of pharmaceutical compounds have been published.170,171 In one study, a glycine internal standard was used to analyze mixtures of carbamazepine anhydrate and carbamazepine dihydrate.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 spinlattice 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 be measured and related through a calibration curve to the amount of anhydrous and dihydrate content in mixtures of carbamazepine. In another study, the detection limit for quantifying de-

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lavirdine mesylate polymorph and pseudopolymorph mixtures by 13C CP/MAS was 2–3% by weight.171 Both of these studies took into account the differential CP dynamics of each form, but peak intensities were used in the latter study whereas peak integrals were used in the former study. In solid-state 13C CP/MAS NMR spectroscopy, it is generally necessary to do peak deconvolution to obtain accurate integrals for quantitative analysis because peak overlap is quite common and small errors in the magic angle setting can lead to additional peak broadening that will cause peak intensities to be inaccurate.48 The detection limit is an important factor to consider when developing a quantitative method. For solid-state NMR spectroscopy, crystalline compounds usually permit a much lower detection limit than amorphous compounds because of better resolution in the spectrum. The overall signal-tonoise ratio in a spectrum is also important, but differentiating one component from another generally requires high resolution. A set of spectra for 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 at 165 ppm and 4% for compound I based on the peak at 170 ppm. However the relaxation delay

Figure 11. 13C CP/MAS NMR spectra at 100.6 MHz for analgesic I and II. Spinning sideband suppression (TOSS) was used for each spectrum. The mole fractions (I:II) of each compound are (A) 98:2, (B) 97:3, (C) 60:40, (D) 7:93, and (E) 4:96. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 3, MARCH 2003

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time is optimized for compound II, and the CP contact time is a compromise between the optimum values for both compounds. The peak integral for compound I at the relaxation delay time used in the spectra is 20% of its value at the optimum delay time. Therefore, the actual detection limit for compound I is expected to be the same as that for compound II under optimized conditions. Dosage Form Analysis NMR spectroscopy is generally quite useful for examining mixtures of compounds in solutions or in solids. Therefore, it can be used to analyze formulated drug products for transformations and interactions or reactions with excipients. A series of papers has been published on the solid-state NMR spectra of a number of analgesic drugs. Jagannathan recorded the solid-state 13C NMR spectrum of acetaminophen in bulk and dosage forms.173 From the solution NMR spectrum, assignments of the solid-state NMR resonances could be inferred in addition to explanations for the doublet structure of some resonances. Spectra of the dosage product from two sources showed identical drug substance but different levels of excipients.

The topic of drug–excipient interactions was addressed by solid-state 13C NMR spectroscopy in the investigation of different commercially available aspirin samples.174,175 In each commercial aspirin product, the only difference in the measured NMR spectrum was due to variations in the excipients, indicating that there were no interactions between the drug and the excipients under dry blending conditions. After lyophilization of two of the products, one aspirin sample had a different NMR spectrum, indicating a possible interaction during lyophilization or conversion to a different solid form during processing. Solid-state NMR spectroscopy has been used to analyze a number of different drugs in tablet form.176 The ten different drugs that were analyzed and the dosages in the tablets are summarized in Table 1. The NMR analysis could discern the active component for the listed drugs, except for the low dose enalapril maleate tablets. Excipients obscured some drug resonances that appear at the same chemical shift value but have little effect on resonances at other chemical shifts. This result indicates that the excipients are probably not interacting with the drug. Similar results were obtained for tablets of another closely related HMG-CoA reductase inhibitor, simvastatin (Table 1).

Table 1. Tablets and Capsules of Drugs Investigated in Our Laboratory by Solid-State

Drug Enalapril maleate

Lovastatin Simvastatin Ibuprofen tablets

Sulindac tablets Flurbiprofen tablets Diflunisal tablets Indomethacin capsules

Mefenamic acid capsules Piroxicam capsules

Dose (mg)

Total Weight of Dosage Form (mg)

1.25 2.5 5 20 20 40 200

230 230 230 200 400 400 320

200 400 200 100 500 50 75 50 50

330 620 330 420 840 350 280 380 480

250 20

350 300

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Manufacturer and Trade Name Merck (Vasotec) Merck (Vasotec) Merck (Vasotec) Merck (Vasotec) Merck (Mevacor) Merck (Zocor) Bristol-Myers Squibb (Nuprin) Upjohn (Haltran) Geneva Generics Merck (Clinoril) Upjohn (Ansaid) Merck, (Dolobid) Merck (Indocin) Merck (Indocin-SR) Geneva Generics United Research Laboratories Parke-Davis (Ponstel) Pfizer (Feldene)

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C-NMR Spectroscopy

Number of NMR Scans (# of Scans for Interrupted Decoupling Experiment) 16,000 16,000 16,000 16,000 4000 4000 2000

(33,576) (33,576) (33,576) (33,576) (4000) (4000) (4048)

7264 6000 3000 4000 10,000 12,000 12,000 10,000 10,000

(2692) (9084) (8104) (10,000) (20,492) (20,000) (28,248) (27,772) (30,000)

7000 (6000) 14,000 (19,556)

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The solid-state 13C NMR spectra of a number of tablets and capsules of various nonsteroidal antiinflammatory agents were also obtained (Table 1). The spectra of the ibuprofen tablets showed narrow lines with excellent signal-to-noise ratios. This result indicates that the drug itself is crystalline and is present at a high dose in the tablets. In general, most of the resonances due to the other nonsteroidal drugs were also clearly distinguishable in the spectra of the dosage forms. The interrupted decoupling pulse sequence was used to simplify the appearance of the spectra by suppressing excipient peaks and to aid in signal assignment. These studies showed that solid-state 13C NMR spectroscopy can be used to determine the

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crystal form present in low-dose tablets and to identify whether a tablet is a placebo or contains the drug. Solid-state NMR spectroscopy offers a useful alternative to X-ray diffraction for direct determination of the crystal form present in the final dosage form. As previously mentioned, prednisolone tertbutylacetate (Figure 8A) exists in at least five crystalline forms. Solid-state NMR spectroscopy has been used to show that different brands of prednisolone tablets contained different polymorphs.176 The 13C CP/MAS spectra of prednisolone tert-butylacetate that was removed from various commercial suspensions by filtration are shown in Figure 12. Solid-state NMR spectroscopy

Figure 12. Solid-state 13C CP/MAS NMR spectra of filtered suspensions of prednisolone tert-butyl acetate obtained from different vendors. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 3, MARCH 2003

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was used to determine which crystal form was present. Note that the bulk drug obtained from Vendor Y is an unequal mixture of compounds, as demonstrated by 10 additional smaller peaks in its spectrum. Thus, solid-state NMR spectroscopy is also useful for analyzing the solids obtained from filtered suspensions.

Analysis of Molecular Motions—Relaxation Examining the various motions of solid pharmaceutical compounds may lead to a better understanding of solid-state stability and reactivity by examining processes such as solid–solid transformations and intramolecular and intermolecular interactions. For solid-state NMR spectroscopy, variable-temperature methods are generally used along with relaxation methods to study molecular motions. Variable-temperature solid-state NMR spectroscopy has been shown to be a powerful technique for the study of molecular motion in a variety of compounds, including cortisone,177 cholesterol,178 proteins,179 polymers,61,180 –182 and amino acids.57 The effects of water on the molecular mobility of galacturonic acid,183 b-cyclodextrin,184 poly(vinyl alcohol) hydrogels,185 polyaspartic acid,186 and scleroglucan,187 have been studied by 1H and 13 C relaxation times. In general, the hydrated materials had increased molecular mobility, which is consistent with the effect of water in many solids. The effects of hydration on hydrogen bonding in collagen and several collagen-like polypeptides have been analyzed by 15N CP/MAS and 1 H T1r relaxation time measurements.188 The use of nuclei-specific relaxation measurements makes it possible to examine different types of motions or motions restricted to one area of the molecule. Solid-state NMR spectroscopy has also been used to study protein hydration and stability189 and the activation energies of spinning methyl groups in amino acids.190 Relaxation analyses of peri-substituted naphthalenes (Figure 8D) were used to determine the barrier to methyl rotation when substituents at the peri position were --H, --CH3, --Cl and --Br.191 The observed barriers were H, 9.7 kJ/mol; CH3, 13.5 kJ/mol; Cl, 15.2 kJ/mol; and Br 18.4 kJ/mol. The barriers sequentially follow the size of the van der Waals radii and indicate that a large group adjacent to the methyl group can restrict its rotation. This type of information is useful for examining potential reaction mechanisms in the solid-state. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 3, MARCH 2003

One of the earliest solid-state NMR studies of the molecular mobility of pharmaceuticals involved the steroid ester containing the cyclopentyl group shown in Figure 8B.148 Crystallographic studies showed that the cyclopentyl group of the side chain had an extremely large thermal motion. Studies comparing the CP and SPE NMR spectra clearly showed that the cyclopentyl group was the most intense peak in the SPE spectrum. However, in the CP spectrum, there were several signals with greater or equal intensity. The enhancement of this signal in the SPE spectrum is consistent with the large amount of thermal motion observed in the crystal structure. Potentially related observations on simvastatin were made by Cauchon.192 A signal from one of the side chain methylene carbon atoms was present in the interrupted decoupling spectrum that normally shows the presence of only quaternary carbon atoms and methyl groups having significant thermal motion. This result was interpreted to mean that the mobility of the methylene carbon atom was similar to that of a methyl group, presumably because of side chain motion. This explanation is consistent with the crystallographic studies of simvastatin in which the side chain could not be refined because of a large amount of thermal motion. Additional studies are needed to verify the usefulness of interrupted decoupling and other editing sequences as well as SPE NMR spectroscopy for the study of motion in pharmaceutical solids. However, one should note that molecular motions affect the dipolar coupling interaction on which many editing sequences are based, which would indicate that these methods could be readily applied to analysis of many types of motions in solids. The activation energies for the molecular motions of methyl groups in ibuprofen (Figure 8E) were measured by solid-state 13C CP/MAS NMR spectroscopy. The spectra of ibuprofen at different temperatures are shown in Figure 13. The spin-lattice relaxation times were measured at various temperatures for the three methyl groups in ibuprofen using the pulse sequence of Torchia,58 and their activation energies were derived from Arrhenius plots of ln T1 versus T1. The semilog plots of signal intensity versus the relaxation delay times that were used to calculate the T1 values were all linear. This result indicates that the relaxation function for each individual carbon atom is governed by a single exponential correlation time. There was no evidence of nonexponential behavior in this case,

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Table 2. Activation Energies from Solid-State NMR Spectroscopy for the Methyl Carbon Atoms in Ibuprofen

Figure 13. Solid-state 13C NMR spectra of ibuprofen at several temperatures. The isotropic resonances at 120–150 ppm produce first-order spinning sideband peaks at 70–95 ppm and 180–200 ppm in the 198 K spectrum. Second-order spinning sidebands are even smaller peaks at 25–40 ppm and are nearly unnoticeable under the larger peaks. Differences in the spinning sideband positions in the other three spectra are due to differences in the spinning speeds. The sharp peak at 183.3 ppm is due to the carbonyl resonance.

even at long relaxation delay times. The low observed standard deviations are a consequence of the good signal-to-noise ratio that reflects the highly crystalline nature of the sample. A summary of the solid-state NMR relaxation measurements for the three methyl groups in ibuprofen is given in Table 2. In this case, the methyl

Carbon

Chemical Shift (ppm)

Ea (kJ/mol)

C3 C12/C13 C12/C13

15.4 22.1 25.1

10.0 8.8 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 increased 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 polymorphs. 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 relaxation times can be exploited is for the analysis of binary eutectic mixtures.172 If there are distinct differences in the relaxation times of the two components, a correlation can be made beween the integrated ratios of the resonances for each solid in the eutectic compared to a physical mixture of the same compounds. The 1H T1 relaxation time approaches the shorter value between the two components for eutectic mixtures, but is different than either compound alone. In the physical mixture, no significant differences in the relaxation times are apparent. Few solidstate NMR studies have been published on eutectics, particularly involving pharmaceutical compounds. However, NMR relaxation was used for analyzing eutectics at least 30 years ago. Clifford used 1H T1 relaxation measurements to analyze phase changes in mixtures of glycerides.196 McGavock and Harlowe recognized the usefulness of T1 relaxation differences for analyzing the phases of LiCl/CrCl3 mixtures.197 In JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 3, MARCH 2003

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this case, 7Li NMR relaxation measurements were performed. Eutectic formation during formulation or processing of pharmaceutical solids can be either beneficial or undesirable. Solid-state NMR spectroscopy may be a useful means of analyzing for eutectics in tablets and powders as part of the preformulation process. Analysis of Molecular Motions—Exchange Spectroscopy Only a few examples of exchange spectroscopy techniques have been reported in the literature for solids, and no relevant pharmaceutical applications were found at the time of this review. Several interesting studies by Riddell et al.198,199 have determined the rates of phenyl and tertbutyl group rotations and the activation energies for the processes by 2D EXSY, in addition to relaxation and line shape analyses. These types of analyses could be implemented for a variety of pharmaceutical compounds in the pure state as well as in formulations.

Conformation and Stereochemistry The structural analysis of chiral compounds is an important issue in drug development. Solidstate NMR spectroscopy can be used to directly measure the enantiomeric purity of a sample in certain cases. It is important to remember that enantiomers are indistinguishable by NMR spectroscopy. However, diastereomers have different NMR spectra, and NMR spectra of racemic mixtures in the solid state are generally different from either enantiomer. A number of reports in the literature demonstrate the ability of 13C or 31P solid-state NMR spectroscopy to differentiate between optically pure material and racemic species.200 In one report, tartaric acid was studied by 13C CP/MAS NMR spectroscopy.201 For each of the optically pure (2R, 3R), racemic, and meso-tartaric acid materials studied, two molecules exist per asymmetric unit cell. Because the carbonyl and a-carbon atoms in a single molecule are not symmetry related, one would expect two resonances for each carbon (crystallographic splitting). This crystallographic splitting was observed for the carbonyl and a-carbon atoms in the 13C NMR spectra in all three cases, and distinct isotropic chemical shift values were observed as well. These data demonstrate the potential for solid-state NMR spectroscopy to JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 3, MARCH 2003

differentiate between racemic and enantiomeric crystallites. In another example, a series of racemic and optically pure organophosphorus samples were studied by solid-state 31P NMR spectroscopy.202 Analogous to the tartaric acid work, the two optical isomers (þ and ) had identical 31P NMR spectra that were distinct from the spectrum of the racemic material. The issue of quantitative analysis of optical purity has been addressed by solid-state 13C NMR spectroscopy in the conformational analysis of DL-, 203 L-, and D-methionine. The data in this study indicate that each crystalline form of DL-methionine consists of a single conformer, which is in agreement with X-ray diffraction data. Both the D and L conformers of methionine have two forms according to their solid-state NMR spectra, which is consistent with the X-ray crystal structures that show two molecules per asymmetric unit. The conformation of a molecule in the solid state can only be determined by NMR spectroscopy if 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 experiment and its variants have been used to analyze a wide range of compounds, including amorphous humic materials,204 crystalline calcipotriol monohydrate and vitamin D,205 crystalline and amorphous forms of delavirdine mesylate,206 and crystalline L-leucinamide.207 A series of editing spectra, obtained for lisinopril dihydrate, are compared with the conventional 13C CP/MAS and TOSS spectra in Figure 1.136 In this example, most of the tentative resonance assignments that were made based on the 13C NMR spectrum of lisinopril in solution (Figure 9) can be confirmed in the solidstate NMR spectrum. Two-dimensional solid-state correlation NMR spectroscopy has also been applied to pharmaceutical systems.99,208 The overall goal of these methods is to obtain information on the conformation of solid compounds, which is particularly useful when a crystal structure cannot be determined. The 2D dipolar HETCOR experiment has been used for studying ibuprofen,100 aspirin tablet and sucrose,95,99 amino acids,209 and a variety of small organic compounds and polymers.100,210 A number of 15N– 13C solid-state NMR correlation experiments have been developed for analyzing amino acids and peptides.211 –215 As many as four dimensions have been used to obtain greater

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resolution.215 These experiments may eventually lead to the solid-state structure determination of polypeptides, proteins, and nucleotides. These compounds are becoming more important as active pharmaceutical components. Limitations of these NMR experiments include the necessity for 15N and 13C labeling and the fact that the compounds used in the cited studies have been single crystals. No current publications have been found in which the more advanced solid-state NMR spectroscopy experiments, like SS-APT and MASJ-HMQC, have been used for pharmaceutical compounds. Presumably, these methods will find wider use in pharmaceutical research in the future. Internuclear Distance Measurements Measurement of internuclear distances has only recently been applied to pharmaceutical compounds. Many of the difficulties involved in applying the necessary recoupling techniques were already outlined. The few examples cited are either biologically or pharmaceutically active molecules, but they provide an indication of the potential value of these techniques. Internuclear distances, obtained by 13C– 15N REDOR NMR spectroscopy for melanostatin, were used to create a set of structures with root-mean˚ compared with square-deviation of 0.4370.224 A 216 the X-ray structure. However, three separate 13 C, 15N-labeled melanostatin tripeptides were needed to obtain the constraints used to determine the structures. In another study, solvates of singly and doubly labeled (13C, 15N) Leu-enkephalin and Met-enkephalin were analyzed by REDOR NMR spectroscopy, relaxation methods, and variabletemperature 13C CP/MAS to determine the structural effects of solvent molecules.217 The conformational changes due to dehydration were readily observed in the solid-state NMR spectra of each compound, and site-specific differences in molecular motions were detected in the relaxation measurements. The rotational resonance and rotor-driven magnetization exchange recoupling methods were used to measure internuclear 13C– 13C distances in 13 C2-labeled retinal, and the results compared well with X-ray diffraction data (within 0.04– ˚ ).218 A rotor synchronized recoupling pulse 0.05 A sequence was used to determine the 15N– 1H bond lengths in crystalline [U-13C, 15N]-L-histidine, which enabled the authors to detect an intermolecular hydrogen bond.219 The polymorphic

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forms of 13C2-labeled cimetidine were studied using distance measurements via rotational resonance magnetization exchange, and relative 13 C– 1H bond orientations were determined by double quantum heteronuclear local field NMR spectroscopy.220 In an application of a double-quantum recoupling method, the relative orientations of purine and pyrimidine rings in a nucleic acid dodecamer were determined.221 This result indicates that it may be practical to determine secondary and tertiary structures of relatively large molecules with solid-state NMR spectroscopy. Spinning Sideband Analysis Raftery et al. have used 2D solid-state NMR spectroscopy to study the red, orange, and yellow conformational polymorphs of ROY (5-methyl2-(2-nitrophenyl)amino]-3-thiophenecarbonitrile (Figure 8G).222 The chemical shift anisotropy of the C3 carbon atom in ROY could be readily distinguished with the 2DTOSS pulse sequence, and the differences in the chemical environments among the three polymorphs were examined. The chemical shift anisotropy for C3 in the three polymorphic forms is shown in Table 3. There is a large difference between the isotropic chemical shifts (diso) among the three forms. The diso increases by almost 10 ppm between the red and yellow polymorphs. Similarly, the chemical shift anisotropies differ by 30 ppm between the red and yellow forms. This study demonstrated that CSA tensor analysis can distinguish between conformational polymorphs and is therefore a powerful method for analyzing such systems. Raftery’s group is now combining information from the 2DTOSS experiments with ab initio or density functional calculations to attempt to obtain quantitative structural information. Similar experiments involving spinning side band analysis have been performed by Harper et al. to examine the stereochemistry of the natural product, terrein.119 The tensor principle values were determined with the 2D FIREMAT Table 3. Chemical Shift Tensor Values for the Three Forms of ROY Solid Form

d11

d22

d33

diso

Red (R) Orange (O) Yellow (Y)

49.2 50.2 43.3

90.7 102.7 105.8

155.0 165.7 179.2

98.3 106.2 109.4

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technique, and the authors note that diastereomers of terrein were differentiated, but enantiomers cannot be resolved as expected. The analysis of spinning sidebands was developed relatively recently, and it is anticipated that many pharmaceutical compounds could be analyzed in this manner. The experimental method is not difficult, but the data must be correctly analyzed to extract the necessary information. These methods, along with solid-state NMR correlation experiments, promise to provide conformational and stereochemical information for solids whose crystal structures cannot be determined by X-ray diffraction. Solid-State 1H NMR Spectroscopy As noted earlier in this review, very few 1H NMR spectra of solid pharmaceutical compounds have been obtained because of the difficulties inherent in the experiment as well as the general lack of useful data available in the spectrum. However, some recent applications have been demonstrated, in which reasonably good results were obtained. 1 H CRAMPS has been applied to the study of hydrogen bonding in a number of organic solids.223,224 Hydrogen bonding and polymorphs of solid amino acids225 and the secondary structural conformation of polypeptides up to six amino acids long226 have been examined by 1H CRAMPS. Hydrogen bonding interactions in the polypeptides could be determined by the characteristic chemical shifts of the observed resonances.

useful for analyzing peak doubling (crystallographic splitting) to determine if multiple molecules per asymmetric unit are independent or if there is exchange between the different conformations. Solid-state NMR spectroscopy can be used to study interactions between the active drug and excipients in tablets or capsules. It is also possible to study solid-state chemical reactions and their mechanisms as well as solid–solid phase transformations with solid-state NMR spectroscopy. Use of internuclear distance measurements may enable the conformational and structural analysis of amorphous pharmaceutical solids. NMR relaxation parameters could also be correlated with thermal data in the analysis of amorphous compounds. Many drugs are biological macromolecules or modifications of them (e.g., proteins, nucleic acids, carbohydrates) that are likely to be amorphous, and solid-state NMR spectroscopy would be a much more useful analytical tool for them than X-ray diffraction. Magnetic resonance imaging of solid dosage forms may also provide useful information that cannot be obtained by other techniques. Note Added in Proof In a continuing effort to provide the most up-todate references on solid-state NMR, the following manuscripts appeared in the literature during the revision and proofing stages of this article preparation. Additional references include manuscripts on quantitative analysis,227–229 conformation analysis,230–232 molecular motion,232 and amorphous/crystalline characterization.233

Future Developments Although solid-state NMR spectroscopy can be considered a routine analytical method at this time, it is not trivial to obtain high quality spectra. A significant barrier to using solid-state NMR spectroscopy may be the perception that it does not provide any information that cannot be obtained more easily by another technique. Many of the examples given in this review demonstrate that solid-state NMR spectroscopy does provide unique data for a variety of pharmaceutical compounds. There are a number of other potential applications of solid-state NMR spectroscopy that may have implications for future pharmaceutical research. However, it should be noted that these applications are not new to NMR spectroscopists who have used them in other fields of study. For instance, 2D correlation spectroscopy may be JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 3, MARCH 2003

SUMMARY This review has covered some of the recent methods and applications of solid-state NMR spectroscopy that will hopefully become more widely used for studying pharmaceutical solids. Within various pharmaceutical laboratories (industrial and academic), the multinuclear technique of solid-state NMR spectroscopy has primarily been applied to the study of polymorphism at the qualitative and quantitative levels. Although the technique ideally lends itself to the determination of conformations of drug compounds in the solid state, it is anticipated that in the future, solid-state NMR spectroscopy will become routinely used for method development and problem solving activities in the analytical/

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materials science/physical pharmacy areas of pharmaceutical science. During the past few years, an increasing number of publications have emerged in which solid-state NMR spectroscopy is an invaluable method for solving difficult problems in many different areas. With the continuing development of solid-state NMR pulse sequences, along with hardware improvements that increase sensitivity and resolution, solidstate NMR spectroscopy will provide more detailed molecular information for the characterization of pharmaceutical solids. Some of the areas that will continue to progress rapidly include (1) higher field strengths, (2) faster sample spinning, (3) improved decoupling methods, (4) advanced pulse sequences, and (5) improved probes and other electronic components of spectrometers. However, increased use of solid-state NMR spectroscopy in analyzing pharmaceutical solids will

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come about only if scientists view it as a useful part of the array of analytical methods available to them. The future advancement of solid-state NMR spectroscopy in pharmaceutical applications depends more on the creative use of it for solving specific problems rather than on technological advances in the spectrometer hardware and software.

ACKNOWLEDGMENTS We thank Patricia Saindon Miyake, Gregory Stephenson (Eli Lilly), Pascal Toma (Abbott Laboratories), and Joseph G. Stowell (Purdue University) for generously providing some of the data used in this review. We also thank Alan D. Ronemus for his valuable advice and assistance with the solid-state NMR applications.

APPENDIX Table A1. Summary of NMR-Related Acronyms Used in This Review Acronym 1Q, 2Q, 3Q, MQ 2DTOSS CP CPPI CRAMPS CSA DECRA EIS EXSY FSLG HETCOR HMBC HMQC INADEQUATE J-coupling MAS MAS-J-HMQC NMR NOESY ODESSA REDOR REPT-HMQC SPE SS-APT TOBSY TOSS TPPM WIMSE

Description Single, double, triple, and multiple quantum 2-Dimensional total sideband suppression Cross polarization Cross polarization/polarization inversion Combined rotation and multiple pulse spectroscopy Chemical shift anisotropy Direct exponential curve resolution algorithm Exchange-induced sidebands Exchange spectroscopy Frequency-switched Lee–Goldberg Heteronuclear correlation Heteronuclear multiple bond correlation Heteronuclear multiple quantum coherence Incredible natural abundance double quantum transfer Through-bond coupling Magic-angle spinning Magic-angle spinning heteronuclear multiple quantum coherence using scalar coupling Nuclear magnetic resonance Nuclear overhauser effect spectroscopy One-dimensional exchange spectroscopy by sideband alternation Retational echo double resonance Recoupled polarization transfer-heteronuclear multiple quantum correlation (coherence) Single-pulse excitation Solid-state attached proton test Total through-bond correlation spectroscopy Total sideband suppression Two-pulse phase modulation Windowless isotropic mixing for spectral editing JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 3, MARCH 2003

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