Quantitation of crystalline and amorphous forms of anhydrous neotame using 13C CPMAS NMR spectroscopy

Quantitation of Crystalline and Amorphous Forms of Anhydrous Neotame using 13C CPMAS NMR Spectroscopy THOMAS J. OFFERDAHL,1 JONATHON S. SALSBURY,1 ZEDONG DONG,2 DAVID J.W. GRANT,2 STEPHEN A. SCHROEDER,3 INDRA PRAKASH,3 ERIC M. GORMAN,1 DEWEY H. BARICH,1 ERIC J. MUNSON1 1

Department of Pharmaceutical Chemistry, University of Kansas, 2095 Constant Avenue, Lawrence, Kansas 66047

2

Department of Pharmaceutics, University of Minnesota, Weaver-Densford Hall 308 Harvard Street SE, Minneapolis, Minnesota 55455 3

The NutraSweet Company, 1801 Maple Ave., Evanston, Illinois 60201

Received 4 February 2005; accepted 13 July 2005 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20469

ABSTRACT: Although most drugs are formulated in the crystalline state, amorphous or other crystalline forms are often generated during the formulation process. The presence of other forms can dramatically affect the physical and chemical stability of the drug. The identification and quantitation of different forms of a drug is a significant analytical challenge, especially in a formulated product. The ability of solid-state 13C NMR spectroscopy with cross polarization (CP) and magic-angle spinning (MAS) to quantify the amounts of three of the multiple crystalline and amorphous forms of the artificial sweetener neotame is described. It was possible to quantify, in a mixture of two anhydrous polymorphic forms of neotame, the amount of each polymorph within 1–2%. In mixtures of amorphous and crystalline forms of neotame, the amorphous content could be determined within 5%. It was found that the crystalline standards that were used to prepare the mixtures were not pure crystalline forms, but rather a mixture of crystalline and amorphous forms. The effect of amorphous content in the crystalline standards on the overall quantitation of the two crystalline polymorphic forms is discussed. The importance of differences in relaxation parameters and CP efficiencies on quantifying mixtures of different forms using solid-state NMR spectroscopy is also addressed. ß 2005 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 94:2591–2605, 2005

Keywords: solid state NMR; spectroscopy; amorphous; polymorphism; relaxation time; thermal analysis; quantitation

INTRODUCTION

Thomas J. Offerdahl’s present address is Quintiles Inc., 10245 Hickman Mills Dr., P.O. Box 9708, Kansas City, MO 64134. Jonathon S. Salsbury’s present address is Albany Molecular Research, Inc., 21 Corporate Cir., Albany, NY 12212. Zedong Dong’s present address is Pharmaceutical and Analytical Research & Development, Hoffmann-La Roche Inc., 340 Kingsland St., Nutley, NJ 07110. Stephen A. Schroeder’s present address is Baxter Healthcare Corporation, One Baxter Parkway, Deerfield, IL 60015. Correspondence to: Eric J. Munson (Telephone: 785-8643319: Fax: 785-864-5736; E-mail: [email protected],) Journal of Pharmaceutical Sciences, Vol. 94, 2591–2605 (2005) ß 2005 Wiley-Liss, Inc. and the American Pharmacists Association

Approximately 90% of pharmaceuticals are marketed as solids.1 Understanding the solid-state properties of pharmaceuticals is important because changes in these properties may affect solubility, bioavailability, and stability.2 The US Food and Drug Administration (FDA) requires pharmaceutical formulators to monitor and control solid-state properties, since they directly affect drug potency and effectiveness.3 One of the most common changes in the solidstate properties of a drug is conversion to a different polymorphic form.3,4 Polymorphism is defined as the ability of a substance to exist in two

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or more crystalline forms that differ in the conformation and/or arrangement of molecules in the crystal lattice.4,5 An early study by KuhnertBrandsta¨tter and Reidman found that approximately one-third of organic solids exist in more than one polymorphic form.6 Solid-state solvation (solvates or hydrates) also commonly occurs, and is defined as a change in solvation states (e.g., monohydrate vs. dihydrate).4 The physicochemical properties exhibited by two polymorphs or solvates can differ significantly.3,4 Many methods are currently used to identify polymorphs.4 In the pharmaceutical industry, the conversion to a different polymorph is often found during dissolution experiments if the polymorph has a different solubility than the original drug form.7 The overall characterization and quantitation in the solid state of these polymorphs can be accomplished through a variety of analytical techniques. Below is a short review of the quantitative capabilities of differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR), FT-Raman, powder X-ray diffractometry (PXRD), and solid-state nuclear magnetic resonance (SSNMR) spectroscopy to highlight some of the current methods used for quantitation of solid forms and their advantages and disadvantages. A more detailed review of the spectroscopic techniques and quantitation can be found in Bugay8 and Stephenson et al.9 DSC is widely used for characterizing and quantitating mixtures of polymorphs.10–12 Most often, different polymorphs have different melting points and heats of fusion. These differences can easily be observed by DSC. However, sometimes one polymorphic form or amorphous component can recrystallize during the heating process to form a more stable form of the compound, thereby producing an endotherm or exotherm on the DSC scan. McGregor et al.10 report a limit of detection (LOD) of 1%. FTIR can be used to quantitate polymorphs. Bugay et al. found that mixtures of polymorphs of cefepime can be quantified with a limit of quantitation of 1% (w/w) and an LOD of 0.3% (w/w). The working range of this technique is 1 –8% (w/w).13 This work also highlights sample preparation issues because particle size effects can lead to errors in quantitation if not closely monitored. Raman spectroscopy is a valuable technique for quantifying polymorphic mixtures. It has a distinct advantage over FTIR measurements in that sample grinding is unnecessary, so that intact,

formulated tablets can be directly analyzed. An inherent problem with this technique is that the high-intensity laser beam is focused on a small area of the sample (
100 mm diameter) and sample heating can cause changes in crystal form. This type of unwanted effect can be eliminated through either rotation of the sample or by reducing the laser power. Taylor and Zografi14 used FT-Raman spectroscopy to determine the degree of crystallinity in a sample of indomethacin and were able to quantitate amorphous and crystalline content as low as 1%. Roberts et al.15 quantitated two polymorphs of mannitol at the 2% level. The most widely used methods for the characterization and structure determination of different polymorphs are based on X-ray diffraction. In single-crystal X-ray diffractometry, an exact structure of the polymorph can be determined. In PXRD a powder sample is analyzed and the resulting X-ray diffraction profile can be used as a qualitative and sometimes quantitative test for a specific polymorph. PXRD is sensitive to the longrange order in a crystal lattice. Because polymorphs differ in the conformation/arrangement of molecules in the crystal lattice, polymorphs usually have distinct PXRD patterns. Alexander and Klug16 first introduced the use of PXRD as a method for quantitation. Suryanarayanan and coworkers recently investigated the quantification of carbamazepine using PXRD and were able to quantify crystalline drug form content at the 1% level, and to establish a LOD around 0.1%.17 Cao et al.18 utilized parallel-beam PXRD for the quantitation of compacted samples. The use of a parallel-beam source, rather than the standard Bragg-Brentano para-focusing geometry, reduces the effects of surface irregularities so that intact tablets, with either flat or curved edges, can be examined. The resulting quantitative data on mixtures of polymorphic forms, when plotted as integrated intensity versus the ratio of polymorphic components, yielded linear plots with R2 values approaching unity. Some deviation in the quantitation results was ascribed to a preferred orientation effect that occurred during tablet compression. The preferred orientation effect cannot be ignored, because it affects the intensities of certain peaks in the diffraction pattern. This effect is greater in samples with plate or needle morphology. Because PXRD typically does not include three-dimensional rotation, the peaks observed in the diffraction profile from two crystal axes may be much larger than those due to the third axis. The sample is often ground to reduce or

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to eliminate this effect, which may lead to changes in crystal form. All of these techniques have been widely utilized for quantitation of pharmaceutical solids but have certain drawbacks. In all these techniques, standard curves must be prepared to obtain quantitative results. However, to construct a standard curve a pure form is required, which sometimes cannot be isolated or synthesized. Another drawback is the presence of excipients in the formulation, which can greatly reduce the overall resolution of the data and hinder the quantitation of the drug form in the formulated tablet or capsules. Another problem can be introduced when extensive sample preparation is required prior to analysis. Grinding is often used to prepare the sample for analysis and can generate metastable drug forms or can lead to desolvation or dehydration of the product. SSNMR spectroscopy has emerged as a powerful technique for the characterization of pharmaceutical solids.1,19–27 It is mainly used to characterize compounds by their isotropic and anisotropic chemical shifts. The chemical shift reflects the local environment of the nuclei in the sample, such that minute changes in the molecular conformation are apparent in the SSNMR spectrum. A recent review by Bugay et al. describes some of the basic SSNMR techniques and their application to pharmaceutical systems.27 NMR spectroscopy is inherently quantitative because the amount of signal observed in an NMR spectrum is proportional to the number of distinct nuclei that resonate at a given frequency. To increase the sensitivity of a 13C NMR signal in most SSNMR spectra of pharmaceuticals, cross polarization (CP) is used to transfer the magnetization from an abundant spin (1H) to a dilute spin (13C).28,29 Magnetization transfer during a CP period depends upon two rate constants. One is the CP rate constant (TCH), which determines the rate of increase in 13C magnetization. The other is the proton spin-lattice relaxation time in the rotating laboratory frame (T1r), which determines how quickly the magnetization decays. These two rate constants are experimentally determined for each sample. Unless these rate constants are identical for each form, the relative peak intensities may not be proportional to the amount of each form in the sample.30 Thus it is generally assumed that spectra acquired with CP are not quantitative. Also, the instrumentation for performing SSNMR spectroscopy is several times more expensive than the above techniques. For

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quantitation studies relatively large samples (typically >100 mg) and acquisition times of several hours are required. A strong working knowledge of SSNMR spectroscopy is necessary to ensure that the data collection and analysis is performed correctly. The ability of SSNMR spectroscopy to quantify different crystalline and amorphous forms has previously been studied. Gao31 showed that delavirdine mesylate polymorphs could be quantitated with a limit of quantitation of 2–3%. In his work each relaxation rate constant was found to be the same for both forms, so that quantitation could be performed by direct integration of the resulting NMR spectra. Rohrs et al.32 utilized NMR spectroscopy to study the interaction between delavirdine mesylate and a disintegrant, croscarmellose sodium. The initial NMR spectrum and the NMR spectrum after exposure to 408C and 75% RH for 4 weeks were used to observe the changes that had occurred in the sample tablet. Spectral subtraction was used to observe the formation of delavirdine anhydrous free base. This change was also quantified with respect to time spent in the stability chamber, such that the maximum formation of the free base was 28% after 4 weeks, with minor additional increases (
5%) observed at 12 weeks. Suryanarayanan and Weidman33 used an internal standard for the quantitation of two hydrated forms of carbamazepine. Based on the relative intensities of the two peaks, the relative amounts of the carbamazepine forms could be calculated. Gustafsson et al.34 compared SSNMR spectroscopy with isothermal microcalorimetry for the determination of the amorphous component of lactose. This work showed that very low levels of amorphous content can be determined using SSNMR spectroscopy. However, the low detection limit was obtained by saturation of the response from the crystalline component during the NMR experiment, thereby decreasing the crystalline peak intensity relative to the amorphous peaks. Dramatic differences in the spin-lattice relaxation time (T1) are often observed between crystalline forms and amorphous forms because the higher molecular mobility in the amorphous form provides a mechanism for relaxation. Despite several studies that have demonstrated the ability of SSNMR spectroscopy to quantify mixtures of forms, there has been no study of the ability of this technique to directly quantify mixtures of polymorphic forms with different relaxation constants without the use of standards, nor has there been a study that describes the

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absolute quantitation of crystalline and amorphous forms. This paper presents a detailed study of the use of SSNMR spectroscopy to quantify polymorphic and amorphous forms of neotame. Neotame20 was chosen because it has multiple polymorphic forms and can readily be prepared in the amorphous state.21 The focus of this research is the absolute quantitation of three different forms of anhydrous neotame: crystalline polymorphs labeled Form A19 and Form G19,35,36 as found in the literature; and amorphous neotame. Quantitation data was obtained using high-resolution SSNMR spectroscopy with magic-angle spinning (MAS) and CP. The wt% of each form was determined directly from the NMR peak areas and compared to the ‘‘known’’ wt% by mass obtained during sample preparation. No calibration curves or correction terms were needed. In a mixture of two anhydrous polymorphic forms of neotame, it was possible to quantify the amount of each polymorph within 1– 2%. In mixtures of amorphous and crystalline forms of neotame, the amorphous content could also be determined within 5%. It was found that the crystalline standards used for preparing the mixtures were not pure crystalline forms, but rather a mixture of crystalline and amorphous forms.

EXPERIMENTAL Anhydrous Neotame Neotame monohydrate was obtained from the NutraSweet Company. Neotame anhydrate Form A was generated by heating the monohydrate to 658C in a water bath for 24 h under a nitrogen purge. Anhydrous Form G was generated by recrystallizing Form A in anhydrous acetonitrile. Amorphous neotame was prepared by heating neotame monohydrate to 958C in a vacuum oven (
103 Torr) for 2 h followed by quickly quenching the melt in liquid nitrogen. The anhydrous forms were kept at
0% relative humidity over Drierite, anhydrous calcium sulfate (The Drierite Company, Hammond, OH). Sample mixtures were prepared for SSNMR experiments by concurrently weighing out portions of each form in a single weighing pan to avoid errors due to transfer losses. The resulting mixture was then blended with a spatula. Completely uniform mixing was not necessary as the entire sample was analyzed in the NMR spectrometer.

Differential Scanning Calorimetry DSC experiments were performed using a PerkinElmer Pyris DSC using Perkin-Elmer software. Temperature scans from 50–1408C were performed at a heating rate of 208C/min. Approximately 2–4 mg of each sample were weighed in the DSC pan, which was crimped before the subsequent analysis. SSNMR Spectroscopy All 13C spectra were acquired on a Chemagnetics CMX-300 spectrometer using CP28 and MAS. Samples were packed into 7.5 mm zirconia rotors and spun at 3.5–4.0 kHz in a Chemagnetics probe outfitted with a PencilTM spinning module. Kel-F endcaps were used to hold the sample in the rotor. The variable-amplitude CP (VACP) experiment37 was used along with the total suppression of sidebands (TOSS) pulse sequence38 and two-pulse phase-modulated decoupling (TPPM).39 A 3.0 s pulse delay and a 1H 908 pulse width of 4.5 ms were used. The free induction decay contained 2048 data points acquired with a dwell time of 33.3 ms. To obtain a relatively high signal-to-noise ratio (SNR), 800–1024 transients were acquired. Spectra were externally referenced to tetramethylsilane using the methyl peak of hexamethylbenzene at 17.35 ppm. To measure the 1H T1r, the contact time was varied from 0.01 to 30 ms to accurately characterize the peak area versus contact time for samples of Form A, Form G, and amorphous neotame. Spectra of the mixtures of the different forms of neotame were acquired using a contact times from 2 to 10 ms. The CP relaxation rates were determined by fitting the data to the following biexponential equation:40
j

k 
M0 H exp T
exp TCH C
1H  Ið
Þ ¼ CH 1  TT1H where I(t) is the peak area for each contact time (t), M0 is the thermal equilibrium magnetization, gH and gC are the magnetogyric ratios for proton and carbon, respectively.

RESULTS AND DISCUSSION Quantitation One challenge to studying polymorphic mixtures using SSNMR spectroscopy is peak overlap.

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Although the chemical shift of a particular carbon may vary by 10 ppm or more between polymorphs, this does not guarantee that each peak for the different forms in a mixture will be resolved. For reliable quantitation of a mixture of forms, the peaks must be adequately resolved to accurately measure the relative peak intensity and/or area. Figure 1 shows the 13C CPMAS NMR spectra of amorphous neotame and the neotame polymorphs labeled Form A and Form G. We chose to use the peak at 135.6 ppm for Form A, 138.6 ppm for Form G, and 138.0 ppm for the amorphous form. These peaks were previously assigned21 to carbon 7, the quaternary aromatic carbon in neotame (Fig. 2). Due to incomplete resolution of the carbon 7 peak of amorphous neotame from the carbon 7 peak of Forms A and G, these peak areas were determined by deconvolution. The use of TOSS can potentially cause problems in quantitation because TOSS spectra only show the centerband. There is no guarantee that the chemical shift anisotropy (CSA) pattern of a carbon in two polymorphs will be similar. Thus, the centerband of each CSA pattern may constitute a different fraction of the whole CSA pattern intensity. The best solution is to run the experiment at the highest spinning speed possible so that no spinning sidebands are observed, and the use of TOSS would be unnecessary. The disadvantage of faster spinning speeds is a reduction in SNR because a smaller rotor would be needed to spin faster. Alternatively, TOSS spectra acquired at

Figure 1. 13C CPMAS NMR spectra of various forms of anhydrous neotame. Asterisks identify the peak used for quantitation in each spectrum.

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Figure 2. Structure of neotame. Asterisk denotes carbon used for quantitation.

different spinning speeds (e.g., 2, 3, 4, and 5 kHz) should have the same relative intensity if the CSA patterns are identical. If the CSA patterns differ between polymorphs, then the relative signal ratios from the two polymorphs should change. If the ratios do change, then one could either calibrate the spectrometer with a 50/50 mixture to look for a spinning speed that produces a quantitative result, or sacrifice signal-to-noise to avoid differences in CSA tensors between polymorphs. For neotame, a 50/50 mixture produced the correct intensity ratio. As an additional check, if the CSA were different and the ratios in the TOSS spectra were not quantitative, the correlation curve would be nonlinear. In theory the peak area in an NMR spectrum is directly proportional to the number of nuclei that resonate at that particular frequency. However, if the spectrum is acquired using CP, then the peak areas will be proportional to the relaxation time constants TCH, 1H T1, and 1H T1r, as discussed below. By measuring each of these relaxation constants it is possible to determine the relative number of nuclei in different forms. All of the values must be determined experimentally for each sample. The 1H T1 value reflects how quickly the sample magnetization returns to equilibrium after a perturbation. To obtain quantitative results, all of the magnetization in the sample must return to >99% of its equilibrium value prior to the next perturbation. Therefore, the repetition delay between pulses must be
5 the longest 1H T1 value in the sample to avoid saturation. We found that a repetition time of 3 s was sufficient to avoid saturation for all forms of neotame studied here (data not shown). The 1H TCH and T1r values reflect how quickly the 13C magnetization grows and decays,

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Table 1. Relaxation Data for Neotame Samples Sample Form A Form G Amorphous

TCH ms

T1r ms

0.28  0.01 0.38  0.02 0.36  0.02

7.0  0.2 69  5 11.4  0.6

respectively, during CP (Tab. 1). These values must be determined so that the theoretical ‘‘initial’’ value of the magnetization can be determined (vide infra). Figure 3 shows a plot of the natural log of relative peak area for the three neotame forms versus contact time. The curve shapes are determined by the relaxation time constants TCH and 1H T1r (T1r(H)). The magnetization initially increases exponentially due to TCH. TCH relaxation constants for carbons in all forms were 0.3–0.4 ms. This result indicates that for contact times 2 ms >99% of the maximum magnetization possible has been transferred to the 13C nuclei. Note that a shorter contact time, for example, 1 ms, would result in a smaller difference between the relative peak intensities. However, at contact times <1 ms, differences in TCH values would affect the relative peak intensities. During magnetization transfer, the 1H magnetization decreases at a rate governed by T1r(H). If TCH  T1r(H), the contact time curve will reach a maximum
95–99% of possible signal and slowly decay according to T1r(H). This is the case for Form G, which has a T1r(H) of
70 ms. If the difference between TCH and T1r(H) is smaller, the maximum magnetization is substantially smaller. This is the case for both the amorphous

Figure 3. 13C CPMAS NMR contact time profile for various forms of neotame. The intensity scale is in arbitrary units.

form and Form A which have a T1r(H) of
11 ms and
7 ms, respectively. For a contact time of 2 ms, the magnetization has already decreased by
25% for Form A, whereas for Form G the magnetization has decreased by
4%. This effect is shown in Figure 4, which is a spectrum of a 50/50 mixture of neotame Forms A and G acquired with a 2 ms contact time. Although approximately the same amount of neotame is present in each form, the relative intensities differ. The presence of other crystalline/amorphous forms should not affect the TCH and T1r(H) values for any of the forms. To determine the amount of each polymorph present in the mixture, spectra of each sample were acquired with contact times of 2, 4, 6, 8, and 10 ms. The shortest contact time was chosen to be >5 TCH. The natural log of the relative peak areas were plotted against the contact times as shown in Figure 5. Assuming a monoexponential T1r(H), which is reasonable for an organic compound such as neotame, the plot should be linear with a slope reflecting the T1r(H), and a y-intercept corresponding to the integrated area of the amount of the form present in the absence of T1r(H) relaxation. The percentage of Form A in the mixture is: 100  [exp(y-intercept(Form A))]/[exp((y-intercept (Form A)) þ exp(y-intercept(Form G))]. Table 2 lists quantitation data of mixtures of Form A and Form G. The (known) wt% by mass and (measured) wt% by SSNMR spectroscopy values in Table 2 agree well for all mixtures. The errors potentially arise from the SNR of the spectra and the assumption that the crystalline standards are pure. Figure 6 compares the wt% by mass to the wt% by SSNMR spectroscopy, and shows good agreement between the measured wt% by SSNMR and the known wt% by mass. The line should have a slope of unity and an intercept of zero if the wt%

Figure 4. 13C CPMAS NMR spectra of a mixture of 50/50 (w/w) neotame Form A and Form G.

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Figure 5. Plot of integrated intensities of carbon 7 from 13C CPMAS NMR spectra of a 50/50 (w/w) mixture for anhydrous neotame Form A and Form G versus contact time.

by SSNMR spectroscopy results agreed exactly with the wt% by mass. The NMR spectrum of a 50/50 mixture of amorphous neotame and Form G is shown in Figure 7. The carbon 7 peaks in the mixtures of the crystalline polymorphs and amorphous neotame are not well resolved. Thus deconvolution, rather than direct integration, was used to determine the relative peak areas. The amorphous component is easily identified and the spectrum can be deconvoluted into peak areas for the amorphous and crystalline components. Direct quantitation of the peak areas shown in Figure 7 for amorphous

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neotame and neotame Form G is not possible due to the differences in the CP dynamics. However, the CP dynamics for neotame Form A and amorphous neotame are the same. Tables 3 and 4 show the results of the quantitation of the mixtures of Form G and Form A with amorphous neotame, respectively. Figure 8 shows the corresponding plots of amorphous wt% by SSNMR versus amorphous wt% by mass. Unlike the data in Figure 6, the slope is no longer
1 nor is the intercept
0. This result arises from the presence of amorphous neotame in both crystalline forms. The data from the mixtures of amorphous and crystalline forms can be used to determine the amorphous content in the crystalline forms. Because a known amount of amorphous neotame was added to each mixture, the resulting yintercept corresponds to the amount of the amorphous impurity present in each of the crystalline forms. The data in Tables 3 and 4 were recalculated to reflect this amorphous impurity and are updated in Tables 5 and 6, respectively. The recalculation was performed by taking 0.865 times the mass of Form G (0.865 equals the intercept for the amorphous/Form G data from Figure 8 (13.5) subtracted from the total (100), then divided by 100) to determine the correct mass of Form G in the sample. For example, for the 70% amorphous sample in Table 3, the value of 0.1579 g for Form G was multiplied by 0.865 to determine the revised amount of Form G, which is 0.1367 g. The total sample mass is 0.4799 g, and Form G is now

Table 2. Data Showing Quantitation of Mixtures of Neotame Form A and Form G Form A G A G A G A G A G A G A G A G A G

Mass (g)

Wt% by Mass

Intercept

Relative Area

R2

Wt% by SSNMR

Diff (abs)

0.0478 0.3309 0.0759 0.2822 0.1034 0.2446 0.1273 0.1981 0.1961 0.1982 0.1954 0.1220 0.2413 0.1020 0.2966 0.0701 0.2961 0.0388

12.62 87.38 21.20 78.80 29.71 70.29 39.12 60.88 49.73 50.27 61.56 38.44 70.29 29.71 80.88 19.12 88.41 11.59

7.298 9.142 4.955 6.221 4.899 5.777 5.038 5.534 4.856 4.845 4.868 4.367 4.887 3.971 4.878 3.372 7.185 5.099

1478 9340 141.9 503.1 134.2 322.9 154.2 253.1 128.5 127.1 130.1 78.8 132.6 53.1 131.4 29.1 1320 164

0.9998 0.9899 0.9992 0.9906 0.9928 0.9682 0.9779 0.9956 0.9994 0.9954 0.9995 0.9996 0.9981 0.9476 0.9991 0.9995 0.9995 0.9166

13.7 86.3 22.0 78.0 29.4 70.6 37.9 62.1 50.3 49.7 62.3 37.7 71.4 28.6 81.8 18.2 89.0 11.0

1.0 0.8 0.4 1.3 0.5 0.7 1.1 1.0 0.5

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Figure 6. Plot of wt% by SSNMR spectroscopy versus wt% by mass of mixtures of crystalline polymorphs Form A and Form G.

0.1367/0.4799 ¼ 28.49%. The recalculation results of the amorphous and crystalline content present in the mixtures (plotted in Fig. 9) agree very well with the known wt% by mass. After this correction, the mixtures of Form G and amorphous neotame show a maximum deviation <2%, and the mixtures of Form A and amorphous neotame show a maximum deviation of
1%. Additional NMR experiments confirmed the presence of amorphous neotame in both Form A and Form G. For Form A, the peaks for the amorphous form and Form A were sufficiently resolved so that the amount of amorphous neotame could be determined using a standard CP experi-

Figure 7. (a) 13C CPMAS NMR spectra of a mixture of 50/50 (w/w) neotame Form G and amorphous neotame, and (b) the resulting carbon 7 peak areas from deconvolution of the two peaks.

ment (spectrum not shown). The amount of amorphous neotame (
20%) agreed very well with the y-intercept from the addition experiments shown in Figure 8. The carbon 7 peaks of Form G and amorphous neotame have approximately the same chemical shift. This similarity made it difficult to deconvolute the two peaks. An amorphous peak in a SSNMR spectrum can be 10 broader than a crystalline peak, thus 10% amorphous content could give a peak with
1% of the crystalline peak intensity. A difference NMR experiment was used to approximate the amorphous content in the Form G standard. Form G has a relatively long T1r(H), whereas amorphous neotame has a relatively short T1r(H). This difference can be utilized to acquire a SSNMR spectrum in which the signal intensity of the amorphous neotame is increased relative to that of Form G by collecting two acquisitions in one spectrum. One acquisition uses a short (>5 the TCH of either sample component) and one uses a long contact time. At the short contact time (2 ms here), the signal of each sample component (i.e., Form G and amorphous neotame) is near its maximum intensity because signal buildup due to TCH is over and signal decay due to T1r(H) has barely begun (see Fig. 3). At the longer contact time (8 ms here), more T1r(H) relaxation will have occurred, thus reducing all signal intensities. For sample components with short T1r(H), the signal intensity at the long contact time is significantly smaller than at the short contact time, whereas sample components with long T1r(H) retain most of their intensity. Furthermore, the signal at the long contact time is collected 1808 out of phase (i.e., inverted) with respect to the first. For sample components with long T1r(H), the final signal intensity will be very small because the two signals have nearly equal magnitudes with opposite phase. In practice the peak intensity of Form G decreases from 100% at 2 ms to 92% at 8 ms. Therefore, only 8% of the signal intensity from the 2 ms contact time signal remains. Likewise, the amorphous peak decreases from 100% to 60%, leaving 40% of its signal intensity from the 2 ms contact time signal. The relative peak area ratio of the amorphous neotame compared to Form G increased enough to allow the amorphous component to be observed and deconvoluted (Fig. 10). The resulting peak areas must be divided by the remaining relative signal intensity (40% for the amorphous neotame and 8% for Form G). The slopes of the two contact time profiles are known from previous NMR experiments on the mixtures

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Table 3. Data Showing Quantitation of Mixtures of Neotame Form G and Amorphous Neotame Form Amorphous G Amorphous G Amorphous G Amorphous G Amorphous G Amorphous G

Mass (g)

Wt% by Mass

Relative Area

R2

Wt% by SSNMR

Diff (abs)

0.0966 0.3847 0.1381 0.3748 0.1845 0.2646 0.2701 0.2587 0.3220 0.1579 0.3788 0.0419

20.07 79.93 26.93 73.07 41.08 58.92 51.08 48.92 67.10 32.90 90.04 9.96

119.3 252.8 137.2 242.2 124.5 136.6 121.4 94.1 121.5 52.5 131.5 12.2

0.9822 0.8831 0.9851 0.9726 0.9932 0.9453 0.9781 0.9601 0.9946 0.9588 0.9987 0.9229

32.1 67.9 36.2 63.8 47.7 52.3 56.3 43.7 69.8 30.2 91.5 8.5

12.0

of amorphous neotame and Form G, and a correction factor of 1.19 and 1.03 for amorphous neotame and Form G, respectively, can be used to account for the difference in intensities at a contact time of 2 ms. Data analysis indicates the presence of an amorphous component of
11.4%. The resulting amorphous content determined from the dual contact time experiment agrees quite well with the previously determined amorphous content of
13.5%, especially given that slight changes in relaxation time values can affect the values determined in the dual contact time experiment. DSC was also used to study mixtures of amorphous neotame with either Form A or Form G. The heats of fusion from the various mixtures were determined and plotted versus the wt% by mass of the crystalline polymorphs as shown in Figure 11. The resulting fits are linear (consistent with the quantitation data from the SSNMR spectra) but exhibit relatively large scatter. That scatter may be due to sampling error. Specifically, the degree of mixing required for reliable SSNMR analysis may have been insufficient for an aliquot

9.2 6.6 5.3 2.7 1.5

of only a few percent of the SSNMR sample to faithfully represent the whole sample composition. Since the samples were initially prepared for analysis using NMR spectroscopy, the degree of mixing was not sufficient for complete uniformity throughout the sample. The samples may have to be more thoroughly mixed or many more data points acquired for each mixture to average out the sampling error. The scatter does not reflect the ability of DSC to obtain quantitative data, it reflects the need for better mixing of the large samples used for NMR quantitation to compare data from these two techniques. Nevertheless, the linear fit is consistent with the quantitation data from the NMR spectra. Limit of Detection We also investigated the LOD of Form A in the presence of Form G using SSNMR spectroscopy. In general, the LOD is determined by two factors: the SNR of the spectrum, and the ability to resolve a small peak in the presence of a larger peak. The

Table 4. Data Showing Quantitation of Mixtures of Neotame Form A and Amorphous Neotame Form Amorphous A Amorphous A Amorphous A Amorphous A Amorphous A

Mass (g)

Wt% by Mass

Relative Area

R2

Wt% by SSNMR

Diff (abs)

0.0656 0.4757 0.1505 0.3422 0.2583 0.2553 0.3540 0.1509 0.4545 0.0513

12.12 87.88 30.55 69.45 50.29 49.71 70.11 29.89 89.86 10.14

96.6 233.0 114.2 145.2 240.6 159.5 129.0 40.5 133.2 13.5

0.9717 0.9917 0.9696 0.9559 0.9937 0.9968 0.9904 0.9502 0.9964 0.8456

29.3 70.7 44.0 56.0 60.1 39.9 76.1 23.9 90.8 9.2

17.2 13.5 9.9 6.0 0.90

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Figure 8. Plot of amorphous wt% determined by NMR spectroscopy versus amorphous wt% by mass for mixtures of amorphous neotame with either Form A or Form G.

SNR can always be improved by signal averaging, but acquisitions times of >100 h are usually unrealistic. If the sample has very long 1H T1 values or is limited in quantity, the LOD may be determined by the SNR. If the peaks are poorly resolved in the spectrum, then the minor component may appear as a shoulder on the peak of the major component. This situation can significantly hinder accurate deconvolution. However, Forms A and G have a sufficient difference in chemical shift between the peaks of carbon 7 that resolution is possible at low levels of one form. Figure 12b shows the 13C CPMAS NMR spectrum of a mixture of 99.75% Form G and 0.25% Form A, and Figure 12a shows the spectrum of Form G. Form A is clearly present in the spectrum in Figure 12b. However, it is also present in the spectrum of the Form G standard (Fig. 12a). The

presence of Form A in the Form G standard is not surprising, because Form G is produced by recrystallization of Form A in acetonitrile. Figure 13 shows the deconvolution of the carbon 7 peaks of Form A for both the mixture and the Form G standard. Although the peak areas are consistent with the addition of 0.25% Form A to the Form G standard, the SNR of the spectrum of the Form G standard is too low for reliable quantitation. The error reported in both values represents the subjective opinion of the level at which the deconvolution did not accurately represent the spectrum baseline. Despite the limitations of the samples, it is possible to state that the LOD for Form A in Form G is at least 0.5%, and possibly significantly lower. The presence of significant proportions of amorphous and crystalline forms as impurities in the standards used to prepare the quantitation curve in Figure 6 prompted us to examine why the results for the mixtures of the two crystalline forms were so accurate. Because the peak areas were determined by integration, the presence of amorphous material in both standards made a comparable contribution to the peak areas of both Forms A and G, although the peak area of Form G may have been enhanced more than that of Form A due to differences in CP dynamics. Thus, the amorphous contribution to both peak areas can be approximately ignored. Think of the sample as being composed of Form G and Form A standards with purities of 86% and 80%, respectively. Also, calculations must account for the presence of
0.7% Form A impurity in Form G. Figure 14 shows the hypothetical results of various mixtures (with the linear fit obtained from the data in Figure 6 included for comparison). The

Table 5. Data Showing Quantitation of Mixtures of Neotame Form G and Amorphous Neotame After Correction for Amorphous Content Present in Form G Form Amorphous G Amorphous G Amorphous G Amorphous G Amorphous G Amorphous G

Mass (g)

Wt% by Mass

Relative Area

R2

Wt% by SSNMR

Diff (abs)

0.1483 0.333 0.1885 0.3244 0.2201 0.229 0.3049 0.2239 0.3422 0.1367 0.3844 0.0363

30.81 69.19 36.75 63.25 49.01 50.99 57.66 42.34 71.51 28.49 91.37 8.63

119.3 252.8 137.2 242.2 124.5 136.6 121.4 94.1 121.5 52.5 131.5 12.2

0.9822 0.8831 0.9851 0.9726 0.9932 0.9453 0.9781 0.9601 0.9946 0.9588 0.9987 0.9229

32.1 67.9 36.2 63.8 47.7 52.3 56.3 43.7 69.8 30.2 91.5 8.5

1.3

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0.6 1.3 1.3 1.7 0.1

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Table 6. Data Showing Quantitation of Mixtures of Neotame Form A and Amorphous Neotame After Correction for Amorphous Content Present in Form A Form Amorphous A Amorphous A Amorphous A Amorphous A Amorphous A

Mass (g)

Wt% by Mass

Relative Area

R2

Wt% by SSNMR

Diff (abs)

0.1599 0.3814 0.2184 0.2743 0.3089 0.2047 0.3839 0.1210 0.4647 0.0411

29.54 70.46 44.32 55.68 60.15 39.85 76.04 23.96 91.87 8.13

96.6 233.0 114.2 145.2 240.6 159.5 129.0 40.5 133.2 13.5

0.9717 0.9917 0.9696 0.9559 0.9937 0.9968 0.9904 0.9502 0.9964 0.8456

29.3 70.7 44.0 56.0 60.1 39.9 76.1 23.9 90.8 9.2

0.2

greatest potential error occurs at a 50/50 mixture, with the error becoming smaller at the extremes. The difference between the experimental points and the theoretical points for the mixture of 86% Form G, 80% Form A, and 0.7% Form A is very small, which is consistent with the data. There are several reasons why the presence of significant amorphous content was missed in Form A and Form G. For Form A, the initial neotame spectra were obtained with relatively few transients. While this was sufficient to obtain data suitable for quantitation of the crystalline forms, the amorphous form was ‘‘lost in the noise.’’ For Form A, where the amorphous peak is offset from the crystalline peak, obtaining a spectrum with high SNR was sufficient to observe the amorphous content. There are two reasons why we initially missed the amorphous content in Form G. First, overlap of the amorphous and crystalline peaks

Figure 9. Plot of amorphous wt% determined by NMR spectroscopy versus amorphous wt% by mass after correcting for amorphous content based on NMR spectroscopy.

0.3 0.0 0.1 1.1

hinders identification of amorphous content in Form G. Furthermore, the line shape of the crystalline component is Lorentzian, thus it was very broad at the baseline. Detecting amorphous neotame in the presence of Form G will always be difficult. Second, the 1H T1 relaxation rates of crystalline and amorphous neotame are the same. It is unusual for the peaks to overlap, but it is also unusual for the amorphous and crystalline forms to have the same relaxation time. We chose neotame in part because it had a short 1H T1 relaxation rate. The 1H T1 of the crystalline form is usually significantly longer than the amorphous form for pharmaceuticals, which means that a saturation experiment could be used to selectively observe the amorphous component. A classic example is the quantitation of lactose, in which small amounts (<2%) of amorphous lactose were observed in the presence of crystalline lactose.34 One concern that arises when characterizing physical forms of solids is form conversion during measurement or as a result of the measurement conditions. The most likely cause of form conversion for stable forms during analysis by SSNMR spectroscopy is sample heating due to frictional heating of the rotor at high spinning speeds. For example, we have found with a 3.2 mm rotor that spinning at 20 kHz can produce a sample temperature as high as 708C.41 We have previously observed physical form conversions of neotame caused by frictional heating of the rotor spinning at 29 kHz.23 However, for a 7.5 mm rotor and at spinning speeds below 5 kHz as used in this study, we have not observed any significant sample temperature increase, nor have we observed conversion of neotame forms. Form conversion in the rotor is possible if the sample is very unstable and likely to convert. For example, amorphous

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Figure 12. 13C CPMAS NMR spectra of (a) anhydrous neotame Form G, (b) mixture of 99.75% anhydrous neotame Form G and 0.25% anhydrous neotame Form A. Spectra expanded 32.

We have previously discussed the quantitation of neotame anhydrate polymorphs using PXRD.43 In that study ‘‘pure’’ Form A and Form G were crystallized and the PXRD peak intensities measured. Mixtures of Form A and Form G were

prepared from the two standards, and the peak height/peak area were plotted against the wt% of Form A in the mixtures. The correlation between the wt% and the appropriate PXRD diffraction intensity was excellent. For these samples the presence of amorphous material would not affect the ability to quantify the forms, because a crystallinity <100% would result in a less intense peak for that form, but would still produce quantitative PXRD data. Indeed, initial attempts to quantify these two forms by SSNMR spectroscopy with the same materials used in the PXRD study produced deviations of
5% for a 1:1 mixture, indicating that one form contained significantly more amorphous material than the other form. Less than 10% amorphous content is difficult to detect with PXRD and DSC, and could easily have passed unnoticed. This problem highlights several differences between quantitation by

Figure 11. Plot of heat of fusion of crystalline with amorphous neotame versus wt% by mass.

Figure 13. Deconvolution of the 13C CPMAS NMR spectrum of neotame Form A (a) in Form G and (b) in a mixture of 99.75% Form G and 0.25% Form A.

Figure 10. (a) Dual contact time 13C CPMAS NMR spectrum acquired to enhance the signal from amorphous content relative to Form G. (b) Standard 13C CPMAS NMR spectrum of neotame Form G.

indomethacin produced by cryogrinding crystallizes in the rotor. Recrystallization of cryoground indomethacin has also been reported by Zografi and Crowley using PXRD.42 Comparison of PXRD and SSNMR Spectroscopy

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CONCLUSIONS

Figure 14. Plots of Form G wt% as would be determined by SSNMR spectroscopy versus Form G wt% based upon the mass of the Form G standard in sample. ^ Mixture of a hypothetical Form G standard composed of 86% Form G and 14% amorphous neotame and a hypothetical Form A standard that is 100% Form A. ~ Mixture of a hypothetical Form G standard composed of 86% Form G and 14% amorphous neotame and a hypothetical Form A standard that is 70% Form A and 30% amorphous neotame.  Theoretical results of the mixtures of the Form G and Form A standards accounting for their measured composition (Form G standard: 86% Form G, 13.3% amorphous neotame, 0.7% Form A; Form A standard: 80% Form A, 20% amorphous neotame). & Linear fit to the experimental data from Figure 6 for comparison.

PXRD or other techniques, which require pure forms for calibration, and quantitation using SSNMR spectroscopy. First, if the initial standards are impure, then the results obtained using these standards may be inaccurate. Determining the purity of the standard is extremely challenging, especially when the impurity (e.g., amorphous content) is undetectable in the analytical instrument. Second, if the matrix affects the peak area, as might happen in a formulation, the calibration curve becomes unreliable. Even techniques such as standard addition may not provide useful data if the line shape and peak widths differ between the formulation and the added material. Third, the requirement of pure standards limits the applicability of many techniques. As we reported previously, of the seven neotame polymorphs, only two could be prepared without polymorphic impurities, and were therefore amenable for quantitation by PXRD. The ability to quantify forms without the requirement of a standard gives SSNMR spectroscopy a significant advantage over other analytical techniques in studying mixtures of forms before the preparation of pure standards.

We have shown that 13C CPMAS NMR spectroscopy can be used to accurately quantify the amounts of forms of anhydrous neotame in a physical mixture without pure standards. The ratios of two crystalline forms in mixtures of anhydrous neotame, as calculated from the SSNMR data and from the weights used to prepare the mixtures, agreed within 1%. Mixtures of each crystalline form with amorphous neotame showed that each crystalline standard contained an amorphous impurity, and allowed us to accurately determine the amorphous content in the standard. The method of varying the ratio of amorphous standard to crystalline standard, which is a modified standard addition experiment, is highly advantageous when the peak intensities due to amorphous impurities are too low to be accurately determined by deconvolution or integration. The LOD of Form A in the presence of Form G was also studied, although the LOD could only be estimated because Form A is an impurity in Form G. Our results show that SSNMR spectroscopy is a very powerful technique for the analysis of mixtures of crystalline and amorphous forms in pharmaceutical solids even for forms with significantly different T1r relaxation times. For forms with similar TCH and T1r values, the spectra should require no correction to be quantitative provided that the spectra were acquired with a recycle delay at least 5 the longest 1H T1. Future directions of this research include applying this methodology to other pharmaceutical solids and to pharmaceutical formulations.

ACKNOWLEDGMENTS We thank the NutraSweet Company for providing the samples and for useful discussions. Financial support was provided by the University of Kansas, Johnson & Johnson Pharmaceutical Research and Development, the National Science Foundation (Grant CHE-0416214). We thank the United States Pharmacopeial Convention for a fellowship in drug standards for Zedong Dong. DHB was supported by a Postdoctoral Fellowship in Pharmaceutics from the PhRMA Foundation and by a research grant from Pfizer. EMG was supported by a Merck Predoctoral Fellowship in Pharmaceutical Chemistry.

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REFERENCES 1. Bugay DE. 1993. Solid-state nuclear magnetic resonance spectroscopy: theory and pharmaceutical applications. Pharm Res 10:317–327. 2. Brittain GH, Ed. 1995. Physical characterization of pharmaceutical solids. New York: Marcel Dekker. 3. Byrn S, Pfeiffer R, Ganey M, Hoiberg C, Poochikian G. 1995. Pharmaceutical solids: a strategic approach to regulatory considerations. Pharm Res 12:945–954. 4. Byrn SR, Pfeiffer RR, Stowell JG. 1999. Solid-state chemistry of drugs, 2nd edn., West Lafayette, Indiana: SSCI Inc. 5. Threlfall TL. 1995. Analysis of organic polymorphs: a review. Analyst 120:2435–2460. 6. Kuhnert-Braandstatter M, Reidman M. 1987. Thermal analytical and infrared spectroscopic investigations on polymorphic organic compounds. I. Mikrochim Acta 11:107–120. 7. Chemburkar SJ, Bauer J, Deming K, Spiwek H, Patel K, Morris J, Henry R, Spanton S, Dziki W, Porter W, Quick J, Baauer P, Donaubauer J, Naraynan BA, Soldani M, Riley D, McFarland K. 2000. Dealing with the impact of ritonavir polymorphs on late stages of bulk drug process development. Org Proc Res Dev 4:413–417. 8. Bugay DE. 2001. Characterization of the solidstate: spectroscopic techniques. Adv Drug Del Rev 48:43–65. 9. Stephenson GA, Forbes RA, Reutzel-Edens SM. 2001. Characterization of the solid-state: quantitative issues. Adv Drug Del Rev 48:67–90. 10. McGregor C, Saunders MH, Buckton G, Saklatvala RD. 2004. The use of high-speed differential scanning calorimetry (Hyper-DSC) to study the thermal properties of carbamazepine polymorphs. Thermochimica Acta 417:231–237. 11. Bauer H, Herkert T, Bartels M, Kovar K-A, Schwarz E, Schmidt PC. 2000. Investigations on polymorphism of mannitol/sorbitol mixtures after spray-drying using differential scanning calorimetry, X-ray diffraction and near-infrared spectroscopy. Pharmazeutische Industrie 62:231–235. 12. Yu L. 2001. Amorphous pharmaceutical solids: preparation, characterization and stabilization. Advanced Drug Delivery Reviews 48:27–42. 13. Bugay DE, Newman AW, Findlay WP. 1996. Quantitation of cefepime 2HCl dihydrate in cefepime 2HCl monohydrate by diffuse reflectance IR and powder X-ray diffraction techniques. J Pharm Biomed Anal 15:49–61. 14. Taylor LS, Zografi G. 1998. The quantitative analysis of crystallinity using FT-Raman spectroscopy. Pharm Res 15:755–761. 15. Campbell Roberts SN, Williams AC, Grimsey IM, Booth SW. 2002. Quantitative analysis of mannitol

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 12, DECEMBER 2005

polymorphs. FT-Raman spectroscopy. J Pharm Biomed Anal 28:1135–1147. Alexander L, Klug HP. 1948. Basic aspects of X-ray absorption in quantitative diffraction analysis of powder mixtures. Anal Chem 20:886–889. Iyengar SS, Phadnis NV, Suryanarayanan R. 2001. Quantitative analyses of complex pharmaceutical mixtures by the Rietveld method. Powder Diff 16: 20–24. Cao W, Bates S, Peck GE, Wildfong PLD, Qiu Z, Morris KR. 2002. Quantitative determination of polymorphic composition in intact compacts by parallel-beam X-ray powder diffractometry. J Pharm Biomed Anal 30:1111–1119. Dong Z, Padden BE, Salsbury JS, Munson EJ, Schroeder SA, Prakash I, Grant DJW. 2002. Neotame anhydrate polymorphs I: preparation and characterization. Pharm Res 19:330–336. Padden BE, Grant DJW, Munson EJ. 1999. Analysis of polymorphism in crystalline organic compounds using solid-state C-13 NMR spectroscopy and X-ray diffraction. Book of Abstracts, 218th ACS National Meeting, New Orleans, Aug 22–26:ANYL-069. Padden BE, Zell MT, Dong Z, Schroeder SA, Grant DJW, Munson EJ. 1999. Comparison of solid-state 13 C NMR spectroscopy and powder X-ray diffraction for analyzing mixtures of polymorphs of neotame. Anal Chem 71:3325–3331. Zell MT, Padden BE, Grant DJW, Chapeau M-C, Prakash I, Munson EJ. 1999. Two-dimensional high-speed CP/MAS NMR spectroscopy of polymorphs. 1. Uniformly 13C-labeled aspartame. J Am Chem Soc 121:1372–1378. Zell MT, Padden BE, Grant DJW, Schroeder SA, Wachholder KL, Prakash I, Munson EJ. 2000. Investigation of polymorphism in aspartame and neotame using solid-state NMR spectroscopy. Tetrahedron 56:6603–6616. Liang J, Ma Y, Chen B, Munson EJ, Davis HT, Binder D, Chang H-T, Abbas S, Hsu FL. 2001. Solvent modulated polymorphism of sodium stearate crystals studied by X-ray diffraction, solidstate NMR, and cryo-SEM. J Phys Chem B 105: 9653–9662. Chen LR, Padden BE, Vippagunta SR, Munson EJ, Grant DJW. 2000. Nuclear magnetic resonance and infrared spectroscopic analysis of nedocromil hydrates. Pharm Res 17:619–624. Bugay DE. 1995. Magnetic resonance spectrometry. Drugs and the Pharmaceutical Sciences 70:93– 125. Tishmack PA, Bugay DE, Byrn SR. 2003. Solid-state nuclear magnetic resonance spectroscopy-pharmaceutical applications. J Pharm Sci 92:441–474. Pines A, Gibby MG, Waugh JS. 1973. Protonenhanced NMR of dilute spins in solids. J Chem Phys 59:569–590.

NMR QUANTITATION OF SOLID FORMS

29. Stejskal EO, Schaefer J. 1977. Magic-angle spinning and polarization transfer in proton-enanced NMR. J Magn Res 28:105–112. 30. Harris RK. 1985. Quantitative aspects of highresolution solid-state nuclear magnetic resonance spectroscopy. Analyst 110:649–655. 31. Gao P. 1996. Determination of the composition of delavirdine mesylate polymorph and pseudopolymorph mixtures using 13C CP/MAS NMR. Pharm Res 13:1095–1104. 32. Rohrs BR, Thamann TJ, Gao P, Stelzer DJ, Bergren MS, Chao RS. 1999. Tablet dissolution affected by a moisture mediated solid-state interaction between drug and disintegrant. Pharm Res 16:1850–1856. 33. Suryanarayanan R, Wiedmann TS. 1990. Quantitation of the relative amounts of anhydrous carbamazepine (C15H12N2O) and carbamazepine dihydrate (C15H12N2O  2H2O) in a mixture by solid-state nuclear magnetic resonance (NMR). Pharm Res 7:184–187. 34. Gustafsson C, Lennholm H, Iversen T, Nystrom C. 1998. Comparison of solid-state NMR and isothermal microcalorimetry in the assessment of the amorphous component of lactose. Int J Pharm 174:243–252. 35. Dong Z, Munson EJ, Schroeder SA, Prakash I, Grant DJW. 2002. Conformational flexibility and hydrogen-bonding patterns of the neotame molecule in its various solid forms. J Pharm Sci 91: 2047– 2056. 36. Dong Z, Young VG, Jr., Sheth A, Munson EJ, Schroeder SA, Prakash I, Grant DJW. 2002.

37.

38.

39.

40.

41.

42.

43.

2605

Crystal structure of neotame anhydrate polymorph G. Pharm Res 19:1549–1553. Peersen OB, Wu X, Kustanovich I, Smith SO. 1993. Variable-amplitude cross-polarization MAS NMR. J Magn Res A 104:334–339. Dixon WT, Schaefer J, Sefcik MD, Stejskal EO, McKay RA. 1982. Total suppression of sidebands in CPMAS C-13 NMR. J Magn Res 49: 341–345. Bennett AE, Rienstra CM, Auger M, Lakshmi KV, Griffin RG. 1995. Heteronuclear decoupling in rotating solids. J Chem Phys 103:6951–6958. Alemany LB, Grant DM, Pugmire RJ, Alger TD, Zilm KW. 1983. Cross polarization and magic angle sample spinning NMR spectra of model organic compounds. 1. Highly protonated molecules. J Am Chem Soc 105:2133–2141. Isbester PK. 1999. Development of an isolated flow variable-temperature magic-angle spinning (MAS) nuclear magnetic resonance (NMR) probe for heterogeneous catalysis studies and high-temperature high-speed 19F MAS NMR techniques applied to fluoropolymers. Department of Chemistry, Minneapolis: University of Minnesota. Crowley KJ, Zografi G. 2002. Cryogenic grinding of indomethacin polymorphs and solvates: assessment of amorphous phase formation and amorphous phase physical stability. J Pharm Sci 91: 492–507. Dong Z, Munson EJ, Schroeder SA, Prakash I, Grant DJW. 2002. Neotame anhydrate polymorphs II: quantitation and relative physical stability. Pharm Res 19:1259–1264.

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