Solid-State NMR Spectroscopy of Pharmaceuticals

Solid-state NMR (SSNMR) spectroscopy is a useful tool for analyzing a variety of solid materials. SSCI has the capability of doing a variety of SSNMR techniques depending on the type of sample to be analyzed. The data acquisition parameters for these techniques must be modified appropriately for each different sample to obtain the best possible data.

Although any NMR active nucleus can be detected with the correct instrument settings, typically 13C is the most widely observed nucleus for SSNMR. The most common technique is 13C cross polarization magic angle spinning (CP/MAS) with high-power proton decoupling. The natural abundance of 13C (~1.1%) is sufficient to obtain a spectrum in a reasonable amount of time, especially using cross polarization enhancement from an abundant nucleus (usually 1H). The relatively low natural abundance of 13C has the advantage that there is little interference from 13C-13C dipolar coupling, which could cause significant peak broadening and would make 13C SSNMR much more difficult. The strong dipolar coupling between protons is the main reason that 1H SSNMR is very difficult to do except with special methods that still result in broad peaks. Some pharmaceuticals have fluorine atoms, which is potentially useful for 19F SSNMR. Although the 19F nucleus is at 100% natural abundance and provides good sensitivity, 19F SSNMR is quite difficult if there are more than a few closely spaced fluorines in the molecule of interest.

A good 13C CP/MAS spectrum requires adequate spinning speed, with the rotor positioned at the magic angle relative to the magnetic field, combined with efficient cross polarization, and effective high power proton decoupling. Modern NMR spectrometers and probes can rapidly spin a relatively small sample and still obtain good sensitivity if the experimental conditions are appropriately optimized.

SSCI has the following cGMP SSNMR capabilities:

  • 1D CP/MAS or MAS
  • 1D CP/MAS editing to differentiate C, CH, CH2, and CH3
  • 2D dipolar HETCOR
  • Modulated or CW decoupling
  • Ramped amplitude CP
  • Spinning sideband suppression (TOSS)
  • Sample spinning up to 18 kHz
  • Variable temperature control
  • Frequencies from 40-400 MHz (15N to 1H)
  • Minimum sample mass: ~5-10 mg
  • Typical sample mass: 40-50 mg (4 mm*); 80-110 mg (5 mm*); 200-250 mg (7 mm*)

* diameter of rotor used for the acquisition, the maximum spinning rates are: 4 mm -18 kHz, 5 mm – 15 kHz; 7 mm – 8 kHz

SSNMR Analytical Techniques

Many different solids can be analyzed by SSNMR. It can be used as a complementary technique to XRPD or may replace it for some types of analyses. Some of the most common applications of SSNMR for pharmaceutical compounds are listed below.

  • Characterization of polymorphs, solvates, salts, amorphous content, or co-crystals
  • Chemical identification
  • Analysis of formulations
  • Analysis of stereoisomers
  • Determine the number of molecules in the asymmetric unit
  • Chemical exchange analysis
  • Molecular motion analysis
  • Conformational/structural analysis

Analysis of Formulations

SSNMR spectroscopy is rather uniquely suited for the analysis of formulated materials. For the drug itself, the spectra can vary but they do mostly fall in the same regions due to the common functional groups. Most commonly the API will consist of aromatics, aliphatics, carboxylic acids, and heteroatoms. Excipients usually will not have any aromatics, aliphatics, or carboxylic acids, allowing for the easy identification of the active pharmaceutical ingredient (API) in the NMR spectrum.

This is not the case for XRPD. The diffraction lines can occur at any location, which can make component identification difficult in a formulated product. To further show this, several blends of ranitidine HCl (Alfa Aesar) and lactose monohydrate (USP) were prepared and analyzed by both XRPD and SSNMR spectroscopy. When using a standard scan rate of 1°/min, a detection limit of about 5% was achieved. Many of the diffraction lines overlapped further limiting the detection of small amounts of ranitidine HCl. Peak Overlap was not a limiting factor with the SSNMR experiments. Using this method for the analysis (8 hour experiment time), the peaks due to the ranitidine HCl are clearly resolved and a detection limit was approximated to be ~0.5%. An aspect that is interesting to note, is that the lactose peaks in the blends prepared were not as intense as they should be based on the percentage of lactose present. This was due to the NMR experiments being conducted with the optimized parameters for the ranitidine HCl (recycle delay and contact time). Generally with excipients, the relaxation time is much longer, for lactose, at 9.4 Tesla, the relaxation time is approximately 200 seconds, whereas the ranitidine HCl was 10 seconds. Because of this, the peaks due to the lactose became saturated over the course of the NMR experiment and did not add any signal to the lactose peaks. Additionally, this was only a two component blend, if more excipients were added, the detection limit by SSNMR spectroscopy would remain essentially the same since the excipient peaks will fall in the same region of the NMR spectrum, and if any inorganic excipients are used, they will not even be seen in the NMR spectrum. In the case of XRPD, this cannot be known.

Analysis of Stereoisomers by SSNMR (Enantiomers and Racemates)

Many pharmaceuticals and some common excipients are enantiomeric. One enantiomer of a drug is typically the most active form, which is a result of the stereochemistry of the physiological target. Most of the physical and chemical properties of enantiomers are identical except that each rotates the plane of polarized light in the opposite direction with equal magnitude. However, a mixture of equal amounts of each enantiomer produces a racemic mixture. The racemate is typically quite different than either enantiomer as can be seen for tartaric acid and mandelic acid. For both tartaric acid and mandelic acid, the 1H T1 relaxation times were significantly different for the enantiomers compared to the racemate. Interestingly, each pair of enantiomers also had much different relaxation times from each other, which was unexpected. Further work needs to be done to determine whether this difference is real or due to a paramagnetic impurity or possibly crystal defects. Tthere are two separate resonances for the carboxylic acid. This may indicate that each carbon in tartaric acid is different in the solid state or that there are two molecules per asymmetric unit in the crystal structure of the enantiomers and the racemate.

Analysis of Proteins by SSNMR

Protein pharmaceuticals are being developed more often to treat a wide variety of diseases, and particularly for some diseases that may not have effective small molecule treatments. Most proteins must be injected to bypass the digestive enzymes in the gastrointestinal tract. However, the solid-state form and stability of protein pharmaceuticals is important to characterize during the development process. Techniques such as XRPD, IR and Raman spectroscopies, SSNMR, and solid-state circular dichroism are potentially useful analytical procedures for crystalline or amorphous proteins.

All of the spectra are similar but distinctly different, which is expected because the proteins are essentially polymers of 20 different amino acid units connected by amide bonds. The insulin and lysozyme molecules are smaller (~6 and ~14 kDa, respectively) and supposedly crystalline, while the myoglobin and serum albumin are larger (~17 and ~67 kDa, respectively) and supposedly amorphous. The similar broad peaks in all of the spectra are mainly due to the rather wide chemical shift distribution for each type of resonance (e.g. carbonyl, aromatic, aliphatic, etc.). SSNMR of proteins can be even more useful for structural analysis if the protein is labeled with an NMR active nucleus such as 13C and 15N.

Quantification by SSNMR

SSNMR spectroscopy is typically used qualitatively for most applications. The spectral fingerprint of a solid sample is usually suitable for comparisons to spectra of other samples. Quantitative and semiquantitative SSNMR is less common because of the extensive time and effort required to obtain accurate data. However, careful experimental planning can yield information that is not possible to obtain by most other techniques. NMR is intrinsically a quantitative technique because the signals (resonances) obtained are directly proportional to the number of nuclei producing them. The main problem is being able to adjust the data acquisition conditions appropriately so that quantitative signals are produced in a reasonable amount of time. For CP/MAS SSNMR, it is important to account for cross polarization, relaxation, and decoupling effects, which cause distortions in the peak intensities. Either a calibration curve covering a useful quantitative range of the compound needs to be obtained, or the cross polarization and relaxation properties of each peak of interest must be determined. It is usually much more efficient to determine the response factor using a calibration curve and chemometrics to analyze the quantitative information.

Applications for Quantitative SSNMR:

  • Impurities
  • Polymorphs
  • Solvates
  • Salts
  • Co-crystals
  • Formulations
  • Reverse Engineering
  • Amorphous content

Requirements for Quantitative SSNMR:

  • Relatively sensitive nuclei (e.g. 19F, 31P, 13C)
  • Relaxation measurements (TCP, T1, T)
  • Internal standard (possibly)
  • Temperature control (possibly)
  • Replicates – validation
  • Baseline correction
  • Spectral deconvolution or integration
  • Calibration curve (usually)
  • Chemometrics – Principle Component Analysis

 

Table 1. Some differences between NMR of solids and liquids.

Characteristic Solids Liquids
Molecular motions

Peak widths

T1 relaxation (a)

T2 relaxation

CSA

Dipolar coupling

Sample spinning

Solvent

Slow

Broad

Longer

Shorter

Observed

Large

Static or fast (kHz)

None

Fast

Narrow

Shorter

Longer

Rarely observed

Small

Static or slow (Hz)

Deuterated

(a) Varies with temperature and rigidity of the solid.

 

Obtaining Liquid-like Spectra of Solids:

Rapid Magic Angle Sample Spinning – sample rotates at 54.74° vs. B0

  • Reduces CSA – spinning sidebands usually observed
  • Reduces dipolar coupling effects

High Power 1H Decoupling

  • Reduces dipolar coupling effects (1H-13C, 1H-1H, 13C-13C)

Cross-Polarization – 1H-13C (usually)

  • Increases 13C sensitivity
  • Reduces pulse delay time
2017-11-03T20:29:58+00:00
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