Assessing the Detection Limit of a Minority Solid-State Form of a Pharmaceutical by 1 H Double-Quantum Magic-Angle Spinning Nuclear Magnetic Resonance Spectroscopy

The lower detection limit for 2 distinct crystalline phases by 1 H magic-angle spinning (MAS) solid-state nuclear magnetic resonance (NMR) is investigated for a minority amount of cimetidine (anhydrous polymorph A) in a physical mixture with the anhydrous HCl salt of cimetidine. Speci ﬁ cally, 2-dimensional 1 H double-quantum (DQ) MAS NMR spectra of polymorph A and the anhydrous HCl salt constitute ﬁ ngerprints for the presence of each of these solid forms. For solid-state NMR data recorded at a 1 H Larmor frequency of 850 MHz and a MAS frequency of 30 kHz on ~10 mg of sample, it is shown that, by following the pair of cross-peaks at a 1 H DQ frequency of 7.4 þ 11.6 ¼ 19.0 ppm that are unique to polymorph A, the level of detection for polymorph A in a physical mixture with the anhydrous HCl salt is a concentration of 1% w/w. © 2017 The Authors. Published by Elsevier Inc. on behalf of the American Pharmacists Association ® . This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)


Introduction
Salt formation is the most general and effective method for improving the aqueous solubility and dissolution rate of acidic and basic drugs.Currently nearly half of all active pharmaceutical ingredients (APIs) used in medication are in salt forms. 1 However, during various processing steps such as milling, compression, drying, and granulation, a salt can become unstable chemically and physically, such that it may transform to its free acid/base form or into other polymorphs or solvates.Many of the commonly used excipients in tablet formulations are acidic or basic and can, thus, change the local pH of a system.This can affect the stability of the salt and cause the transformation of the salt to the corresponding free acid/base form.The solid-state transformation from a salt to a free form will alter key properties of the API, such as solubility and dissolution behavior, leading to undesired changes in the bioavailability of the API. 2 In order to monitor the quality of pharmaceuticals, an effective and highly sensitive quantitation method is required.
A variety of techniques have been used to characterize the physicochemical properties of pharmaceuticals, 3 including powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC), and vibrational spectroscopies, notably IR and Raman.Although PXRD is considered the definitive test for the identification of a specific solid-state form, the effectiveness of PXRD for quantitative analysis of mixtures of solid phases can be diminished when the effects of preferred orientation are significant 4 ; however, if the crystal structures of all the solid forms present in the mixture are known, then quantitative analysis can be carried out by Rietveld refinement in which the effects of preferred orientation are taken rigorously into consideration.For example, Li et al. 5 have shown that the error associated with distinguishing polymorphs I and II of sulfamerazine by DSC or PXRD was ±3%, which is superior to that achieved by Raman spectroscopy.Siddiqui et al. 6 have also used PXRD (and solid-state nuclear magnetic resonance [NMR]) to quantify the amount of crystalline tacrolimus solid dispersions, 7 whereas Macfhionnghaile et al. 8 have investigated the effect of ball-milling and cryomilling on sulfamerazine using PXRD, IR, and near-IR spectroscopy.Using terahertz (THz) spectroscopy, 9 Strachan et al. 10 have shown that the concentration of carbamazepine form III in a physical mixture with form I can be detected down to a concentration of 1.5%.Moreover, Hisazumi et al. 11 have used THz spectroscopy to distinguish between anhydrous and hydrate forms of theophylline with an error of 3% in a pharmaceutical formulation.Recently, Thakral et al. 12 have used PXRD to follow salt disproportionation, in which the PXRD data were measured using a 2-dimensional area detectordusing this technique, conventional 1dimensional PXRD patterns that are free from the effects of preferred orientation can be obtained by appropriate integration of the 2-dimensional data.
4][15][16][17] Although the workhorse method is 13 C cross-polarization (CP) magic-angle spinning (MAS), the potential of applying 1 H fast MAS NMR is increasingly recognized for solid-state analysis.  In p0][51] Thus, a 2-dimensional 1 H DQ spectrum represents a "fingerprint" for a specific 3-dimensional packing arrangement adopted by an organic molecule, emphasizing the advantage over 1-dimensional 1 Ho r 13 C NMR spectra of spreading out into 2 dimensions.In this way, by using a high-resolution 1 HDQ combined rotation and multiple-pulse spectroscopy (CRAMPS) approach, 52 it has been shown how the presence of only the anhydrous form and not a hydrate form of an API in tablet formulation can be established. 19However, due to the spectral noise associated with the application of 1 H homonuclear decoupling in the CRAMPS approach, it is only possible to conclude that the hydrate form is absent within a detection limit of ~5% for the spectra presented in the study by Griffine ta l . 19An alternative approach is to use a combination of fast MAS and high magnetic field to give 1 HDQMAS spectra, where the resolution, although not as good as with the 1 H DQ CRAMPS method, 51 is sufficient to resolve distinct spectral features.For the specific case of the anhydrous HCl salt of cimetidine (the crystal structure of which has been determined recently 53 by a combined PXRD and NMR crystallography strategy) and the free form of cimetidine (anhydrous polymorph A), this article investigates the lower limit of detection of a minority solid-state form of an API, present in a physical mixture with another crystalline phase of the same API, using 1 H DQ MAS NMR spectroscopy.

Experimental Section
Cimetidine and cimetidine hydrochloride were purchased from Sigma-Aldrich and used without further purification.The identification of the specific solid-state form was confirmed by PXRD as shown in the studies by Tatton et al. 33 and Watts et al. 53 Samples containing 10%, 5%, 1%, and 0.5% w/w polymorph A/anhydrous HCl salt were prepared by physically mixing appropriately weighed quantities of the 2 pure solid phases.
Solid-state NMR experiments were performed on a Bruker Avance III spectrometer operating at a 1 H Larmor frequency of 850.2 MHz (B 0 ¼ 20.0 T) using a Bruker triple-resonance probe, operating  1, while Tables 2 and 3 list the expected 1 H DQ peaks based on H$$$H proximities extracted from the geometry-optimized (CASTEP) crystal structures.The thick horizontal lines in (a) and (b) denote the pair of 1 H DQ peaks corresponding to the intramolecular proximity of the imidazole H2 (CH) and H3 (NH) protons, noting that for the anhydrous HCl salt this overlaps with the pair of 1 H DQ peaks corresponding to the intramolecular proximity of the imidazole H2 (CH) and H1 (NH þ ) protons.The base contour levels are at (a) 5.4%, (b) 11%, and (c) 2.6% with respect to the maximum peak height.(Note that a conference abstract has presented the spectra in this figure in a different format. 72) in double-resonance mode, supporting rotors of 2.5 mm outer diameter (corresponding to ~10 mg of sample).An MAS frequency of 30 kHz was used.The pulse sequence and coherence transfer pathway diagram for the 1 HD QM A S 49 experiment using back-toback recoupling 54,55 are shown in Figure 7 of the review by Brown and Spiess. 56A 16-step phase cycle was used to select Dp ¼ ±2onthe DQ excitation block and Dp ¼À 1onth efinal 90 pulse, where p is the coherence order.The 1 H9 0 pulse duration was 2.5 ms.In all experiments, 160 t 1 FIDs were recorded with a rotor-synchronized t 1 increment of 33.3 ms using the States time-proportional phase incrementation method to achieve sign discrimination in F 1 .For the anhydrous HCl salt of cimetidine and polymorph A of cimetidine, 16 transients were co-added with a recycle delay of 3 s.For the physical mixtures of polymorph A and the anhydrous HCl salt, a recycle delay of 9 s was used and 16 (10% and 5% w/w mixture samples), 48 (1% w/ w mixture sample), and 112 (0.5% w/w mixture sample) transients were co-added.The experimental times were thus 2 h (anhydrous cimetidine hydrochloride and cimetidine polymorph A), 6 h (10% and 5% w/w mixture samples), 19 h (1% w/w mixture sample), and 44 h (0.5% w/w mixture sample).
1 H chemical shifts are referenced with respect to neat TMS using adamantane as a secondary reference (1.85 ppm for 1 H). 57Experimental 1 H chemical shifts are stated to an accuracy of ±0.1 ppm.

H DQ MAS Solid-State NMR Spectra
In previous studies, 1-dimensional 13 C CP MAS NMR spectra, as well as the results of various heteronuclear dipolar experiments, have been presented for a free form of cimetidine (polymorph A), 58,59 whereas a 13 C CP MAS spectrum of the anhydrous HCl salt of cimetidine as well as a gauge-including projector-augmented wave (GIPAW) calculation 60,61 of the NMR parameters (for the reported crystal structure following geometry optimization) have been presented recently. 53Tatton et al. 33 have also presented 15 N-1 H spectral-editing and 14 N-1 H and 15 N-1 H correlation spectra for polymorph A of cimetidine, together with GIPAW calculations of the chemical shielding and electric field gradient tensors for the reported crystal structure 62 following geometry optimization.Note that distances stated in this paper are from the geometryoptimized crystal structures on which these GIPAW calculations were carried out (available as Supporting Information in studies by Tatton et al. 33 and Watts et al. 53 ).
Figures 1a and 1b present 1 H DQ MAS NMR spectra of (a) the anhydrous HCl salt of cimetidine and (b) polymorph A of cimetidine, recorded at a 1 H Larmor frequency of 850 MHz and an MAS frequency of 30 kHz.Because 1 H linewidths for the strongly dipolar-coupled network of protons in organic solids decrease with increasing MAS frequency, 63,64 narrower 1 H linewidths would be observed using the higher MAS frequencies of >60 kHz that can be achieved with rotors of smaller outer diameter (1.3 mm and less).However, the improved resolution would come at the cost of lower sensitivity associated with the considerably reduced sample volume (corresponding to sample mass ~1 mg or less).Enhanced resolution could also be achieved using the 1 H DQ CRAMPS approach (e.g., see Fig. 3 of the review by Brown 51 ) but, as noted in the Introduction, the disadvantage of using 1 H homonuclear decoupling is the observation of additional "noise" in the spectra which limits the lower detection of the minority phase to ~5%.For these reasons, it was decided that 30-kHz MAS at a 1 H Larmor frequency of 850 MHz provided the best compromise of sufficiently high-resolution and optimum sensitivity for 1 H DQ MAS NMR spectroscopy of the anhydrous HCl salt and polymorph A of cimetidinedsee the spectra presented in Figures 1a and 1b.
The 1 H DQ MAS NMR spectra presented in Figure 1 were recorded using 1 rotor period of back-to-back recoupling. 54,55Such 1 H DQ MAS NMR spectra probe DQ coherences between pairs of through-space dipolar coupled protons corresponding to a close (typically less than 3.5 Å) H$$$H distance, 49,51 with DQ peaks observed at the sum of the 2 single-quantum (SQ) frequencies.Table 1 presents an assignment of the experimentally observed 1 H chemical shifts for polymorph A and the anhydrous HCl salt of cimetidine based on GIPAW calculations (as reported previously). 33,53The significant change in the 1 H chemical shift of H3 reflects the change from an NH$$$N intermolecular hydrogen bond with N12 as the acceptor in polymorph A (involving uncharged donor and acceptor groups) to an NH þ $$$Cl À intermolecular hydrogen bond (involving charged donor and acceptor groups) in the anhydrous HCl salt.
Tables 2 and 3 list H$$$H proximities under 3.5 Å for the NH and imidazole CH protons and the corresponding 1 H DQ shifts for the anhydrous HCl salt and polymorph A of cimetidine, respectively.For the anhydrous HCl salt, the NH þ (H1) and NH (H3) imidazole 1 H chemical shifts overlap (at 15.0 ppm, see Table 1).Thus, in the 1 H DQ MAS spectrum in Figure 1a, the pair of 1 H DQ peaks at d DQ ¼ 15.0 þ 9.4 ¼ 24.4 ppm corresponds to an intramolecular proximity of 2.57 Å for the imidazole CH (H2) with both the NH þ (H1) and NH (H3) protonsdsee Table 2.We also note the very weak diagonal peak at d DQ ¼ 15.0 þ 15.0 ¼ 30.0 ppm in Figure 1a that corresponds to a 3.43 Å intermolecular proximity of the NH þ (H1) and NH (H3) protons (also see Table 2).As described in the study by Bradley et al., 65 to a first approximation, the relative intensity of distinct 1 H DQ peaks at the same 1 H single-quantum chemical shift is proportional to the ratio of the square of the dipolar coupling constants and hence is inversely proportional to ratio of H$$$H distances to the sixth powerdnote that (3.43/2.57) 6¼ 5.7.Other cross-peaks observed for the NH groups in the 1 H DQ MAS spectra in Figures 1a and 1b are due to intra-and intermolecular proximities to CH 2 and CH 3 protons (see Tables 2 and 3).
This study is focused on the pair of 1 H DQ peaks due to the intramolecular proximity of 2.57 and 2.55 Å for the imidazole H2 (CH) and H3 (NH) protons, whereby d DQ ¼ 15.0 þ 9.4 ¼ 24.4 ppm in Figure 1a for the anhydrous HCl salt and d DQ ¼ 11. 6 þ 7.4 ¼ 19.0 ppm in Figure 1b for polymorph A. The evident change in the positions of the pair of 1 H DQ peaks due to the intramolecular proximity of the imidazole H2 (CH) and H3 (NH) protons between the anhydrous HCl salt and polymorph A constitutes a "fingerprint" that is a diagnostic for the presence of either or both of the distinct solidstate forms.

Probing the Level of Detection of a Minority Phase in a Physical Mixture of 2 Distinct Crystalline Phases
To determine the level of detection by 1 H DQ MAS NMR of polymorph A of cimetidine in a physical mixture with the anhydrous HCl salt of cimetidine, spectra were recorded for samples comprising 10%, 5%, 1%, and 0.5% w/w mixtures of polymorph A/ anhydrous HCl salt.Specifically, Figure 1c presents a 1 H DQ MAS NMR spectrum of the 10% w/w mixture (note the lower base contour in Fig. 1c as compared to Figs. 1a and 1b).It is evident that a weak pair of 1 H DQ peaks at d DQ ¼ 11. 6 þ 7.4 ¼ 19.0 ppm due to   ppm, it is observed that the 1 H DQ peaks due to polymorph A are still evident above the noise level down to the 1% w/w mixture in Figure 2e.This conclusion is confirmed by the signal-to-noise analysis presented in Table 4 for the peaks at a 1 H DQ frequency of 19.0 ppm for the H2 and H3 Text and Methodology Q2(R1), section 6.2 states that "A signal-to-noise ratio between 3 or 2:1 is generally considered acceptable for estimating the detection limit." 66As such, the data in Table 4 establish 1% w/w as the level of detection using 1 H DQ MAS NMR at 30-kHz MAS and a 1 H Larmor frequency of 850 MHz for 2 crystalline phases of cimetidine.

Conclusions
Two distinct crystalline phases of cimetidine, polymorph A and the anhydrous HCl salt, are clearly distinguished using 1 H DQ MAS solid-state NMR spectroscopy by focusing on the pair of 1 HD Q peaks due to the intramolecular proximity (2.55 or 2.57 Å) of the neighboring H2 (CH) and H3 (NH) protons of the imidazole ring.Protonation at the N1 site in the anhydrous HCl salt as well as the fact that the 2 distinct solid-state forms have different intermolecular hydrogen-bonding arrangements leads to 1  Using this "fingerprint", solid-state NMR experiments carried out at a 1 H Larmor frequency of 850 MHz and an MAS frequency of 30 kHz have shown that polymorph A can be detected and quantified in a physical mixture with the anhydrous HCl salt down to a concentration of 1% w/w.Considering literature reports (see Introduction), this level of detection matches and in some cases improves upon that reported for other analytical techniques, for example, PXRD, DSC, Raman spectroscopy, and THz spectroscopy.In particular, we emphasize that distinguishing between 2 crystalline phases represents a different challenge to the identification of a minority amount of a crystalline phase in the presence of an amorphous phase.
We note that employing 1 H DQ MAS represents a complementary approach to solid-state NMR of other nuclei, notably 13 Cand 19 F. 67,68 Moreover, it has recently been shown that the sensitivity of solidstate NMR spectroscopy of moderately sized organic molecules can be significantly enhanced (e.g., by 5 times for paracetamol) by employing dynamic nuclear polarization, such that 13 Cr e f o c u s e d INADEQUATE spectra can be recorded at natural isotopic abundance in 16 h for sulfathiazole (which has a long T 1 relaxation time, necessitating a recycle delay of 30 s) 69 and 15 N-1 H correlation spectra can be obtained also at natural isotopic abundance for a pharmaceutical formulation. 70With the recent development of new dynamic nuclear polarization 1.3-mm MAS probe technology that allows faster MAS, 71 this approach seems promising for progressing to even lower levels of detection of minority solid-state forms by 1 H DQ MAS.This approach would also reduce the required experimental time (in this work, 19 h for the 2-dimensional spectrum of the 1% w/w sample), noting that experimental times are typically shorter for other techniques such as PXRD, Raman, and IR.Finally, as shown in our earlier work employing a 1 H DQ CRAMPS approach, 19 we note that the 1 H DQ solid-state NMR method presented here is readily applicable to tablet formulations with the shift of 1 HD Q peaks due to hydrogen-bonded protons to high ppm helping to avoid overlap with 1 H resonances of the excipients.

Figure 1 .
Figure 1. 1 H (850 MHz) DQ MAS (30 kHz) spectra of (a) the anhydrous HCl salt of cimetidine, (b) polymorph A of cimetidine, and (c) a 10% w/w physical mixture of

Figure 2 .
Figure 2. Slices corresponding to a 1 H DQ frequency of 19.0 ppm, as extracted from 2dimensional 1 H (850 MHz) DQ MAS (30 kHz) spectra of (a) the anhydrous HCl salt of cimetidine, (b) polymorph A of cimetidine, and (c-f) physical mixtures of polymorph A and the anhydrous HCl salt with (c) 10%, (d) 5%, (e) 1%, and (f) 0.5% w/w of polymorph A.
H DQ peaks at d DQ ¼ 15.0 þ 9.4 ¼ 24.4 ppm for the anhydrous HCl salt and at d DQ ¼ 11. 6 þ 7.4 ¼ 19.0 ppm for polymorph A. By spreading out the peaks into 2 dimensions, the observation of 1 H DQ peaks at d DQ ¼ 11. 6 þ 7.4 ¼ 19.0 ppm is an unambiguous indicator of the presence of polymorph A.

Table 2 H
$$$H Proximities a (<3.5 Å) and Corresponding 1 H DQ Shifts (See Fig.1a) for the NH and Imidazole CH Protons in the Anhydrous HCl Salt of Cimetidine a Distances are stated for the geometry-optimized (CASTEP) crystal structure.

Table 1
Experimental a and GIPAW-Calculated b 1 H Chemical Shifts (in ppm) for the Anhydrous HCl Salt and Polymorph A of Cimetidine b Calculated isotropic chemical shifts are obtained from the calculated absolute shielding as d calc iso ¼ s ref À s cal , where s ref ¼ 30.0 ppm.c For CH 3 groups, the stated value of the calculated isotropic chemical shift is the average for the 3 protons.K. Maruyoshi et al. / Journal of Pharmaceutical Sciences xxx (2017) 1-6

Table 3 H
$$$H Proximities a (<3.5 Å) and Corresponding 1 H DQ Shifts (See Fig. 1b) for the NH and Imidazole CH Protons in the Geometry-Optimized (CASTEP) Crystal Structure of Polymorph A of Cimetidine a Distances are stated for the geometry-optimized (CASTEP) crystal structure.
1 H SQ resonances at 11.6 and 7.4 ppm.Following the guidance of the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, Validation of Analytical Procedures:

Table 4
Signal-to-Noise Ratios for the Peaks at a 1 H DQ Frequency of 19.0 ppm for the 1 HSQ H2 and H3 Resonances at 11.6 and 7.4 ppm (See Fig. 2) for Physical Mixtures of Polymorph A and the Anhydrous HCl Salt K. Maruyoshi et al. / Journal of Pharmaceutical Sciences xxx (2017) 1-6