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

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.

Quantitative 1H NMR method for the routine spectroscopic determination of enantiomeric purity of active pharmaceutical ingredients fenfluramine, sertraline, and paroxetine.

Determining the enantiomeric purity of chiral therapeutic agents is important in the development of active pharmaceutical ingredients (API). A strategy for determining the enantiomeric purity of three APIs was developed using nuclear magnetic resonance (NMR) and the chiral solvating agent (CSA) 1,1-bi-2-naphthyl (1). While chiral chromatography is widely used to evaluate enantiomeric purity, it can sometimes suffer from tedious sample preparation obviating rapid measurements that are sometimes needed during the manufacture of such agents. The techniques described herein provide comparable enantiomeric purity results with those obtained with traditional chiral HPLC and other published methods for these compounds. Chiral analysis of standard samples of methylbenzylamine enantiomeric mixtures using 1 were found to be quantitative to approximately 1% minor enantiomer. Enantiomeric purity determination by NMR utilizing chiral solvating agents do not require special instrumental techniques, chemical derivatization or standards and is therefore ideally suited for rapid routine analysis. As a result, the technique demonstrated is commonly used in our laboratory as a complementary or alternative method to chiral HPLC or optical rotation measurements for routine determination of enantiomeric purity.

Solid-state NMR studies of pharmaceutical solids in polymer matrices.

Biodegradable drug-delivery systems can be formulated to release drug for hours to years and have been used for the controlled release of medications in animals and humans. An important consideration in developing a drug-delivery matrix is knowledge of the long-term stability of the form of the drug and matrix after formulation and any changes that might occur to the drug throughout the delivery process. Solid-state NMR spectroscopy is an effective technique for studying the state of both the drug and the matrix. Two systems that have been studied using solid-state NMR spectroscopy are presented. The first system studied involved bupivacaine, a local anesthetic compound, which was incorporated into microspheres composed of tristearin and encapsulated using a solid protein matrix. Solid-state 13C NMR spectroscopy was used to investigate the solid forms of bupivacaine in their bulk form or as incorporated into the tristearin/protein matrix. Bupivacaine free base and bupivacaine-HCl have very different solid-state NMR spectra, indicating that the molecules of these compounds pack in different crystal forms. In the tristearin matrix, the drug form could be determined at levels as low as 1:100 (w/w), and the form of bupivacaine was identified upon loading into the tristearin/protein matrix. In the second case, the possibility of using solid-state 13C NMR spectroscopy to characterize biomolecules lyophilized within polymer matrices is evaluated by studying uniformly 13C-labeled asparagine (Asn) in 1:250 (w/w) formulations with poly(vinyl pyrrolidone) (PVP) and poly(vinyl alcohol) (PVA). This work shows the capability of solid-state NMR spectroscopy to study interactions between the amino acid and the polymer matrix for synthetic peptides and peptidomimetics containing selective 13C labeling at the Asn residue.

Reaction of a peptide with polyvinylpyrrolidone in the solid state.

During stability studies at high temperature (70 degrees C) and low relative humidity ( approximately 0%), the recovery of an asparagine containing hexapeptide (VYPNGA) and its known deamidation products from solid polyvinylpyrrolidone (PVP) matrices was incomplete. To determine the causes of this mass loss, formulations were prepared by lyophilizing solutions containing PVP, glycerol, and the Asn-hexapeptide in pH 7.5 phosphate buffer, followed by storage at 70 degrees C and 0% relative humidity. Asn-hexapeptide loss was mono-exponential and reached a plateau at about 30% remaining. Total recovery of the peptide and its known deamidation products was approximately 30% of peptide load. Size exclusion chromatography with fluorescence detection indicated the formation of a PVP-peptide adduct that was stable in the presence of 6 M guanidine hydrochloride. Similar stability studies using N-acetyl phenylalanine, phenylalanine ethyl ester, and N-acetyl tyrosine ethyl ester demonstrated that the reaction involves the peptide N-terminus. The adduct was disrupted in the presence of carboxypeptidase-A, suggesting the formation of an amide bond between the peptide and PVP. (15)N solid-state nuclear magnetic resonance spectroscopy using (15)N-labeled valine as a model of the peptide N-terminus showed different populations of (15)N, suggesting that noncovalent peptide-polymer interactions precede amide bond formation.

Dehydration kinetics of neotame monohydrate.

The dehydration of neotame monohydrate was monitored at various temperatures by differential scanning calorimetry (DSC), thermogravimetry (TGA), hot-stage microscopy (HSM), powder X-ray diffractometry (PXRD), and (13)C solid-state nuclear magnetic resonance (SSNMR) spectroscopy. This work emphasizes kinetic analysis of isothermal TGA data by fitting to various solid-state reaction models and by model-free kinetic treatment. The dehydration of neotame monohydrate follows the kinetics of a two-dimensional phase boundary reaction (R2) at 40-50 degrees C with an activation energy of 75 +/- 9 kJ/mol, agreeing well with 60-80 kJ/mol from model-free kinetics. At a low heating rate in DSC and TGA, neotame monohydrate undergoes dehydration to produce anhydrate Form E, which then converts to anhydrate Form A, followed by the melting of A. Neotame monohydrate under dry nitrogen purge at 50 mL/min undergoes partial isothermal dehydration at 50 degrees C to produce neotame anhydrate Form A. When neotame monohydrate is heated very slowly from 50 to 65-70 degrees C over 24 h, pure Form A is obtained.

Neotame anhydrate polymorphs I: preparation and characterization.

PURPOSE: To prepare, characterize, and compare polymorphs of neotame anhydrate. METHODS: Neotame anhydrate polymorphs were prepared from amorphous or crystalline anhydrate by crystallization or suspension in various organic solvents, or by dehydration of neotame monohydrate. The following techniques were used for characterization: differential scanning calorimetry, thermogravimetry, hot-stage microscopy, powder X-ray diffractometry (PXRD), 13C solid-state nuclear magnetic resonance (SSNMR) spectroscopy, Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy, dynamic water vapor sorption/desorption, and density measurements. RESULTS: Seven polymorphs (Forms A-G) of neotame anhydrate were prepared and show different thermal properties and PXRD patterns. Two enantiotropically related pairs were identified: B and C; E and A. 13C SSNMR and FTIR spectroscopy clearly distinguish between Forms A, D, F, and G, which show similar needle-shaped morphology but distinct differences in dynamic water vapor sorption/desorption and density. The 13C SSNMR chemical shifts suggest conformational polymorphism. The stability in the presence of water vapor follows the rank order, G > A > D approximately = F, which resembles the rank orders of the molar volume and of the polarity of the solvents from which they crystallized. CONCLUSIONS: The neotame anhydrate polymorphs appear to show different molecular conformations. The less dense polymorphic structures crystallize from solvents of greater polarity and sorb water vapor less rapidly and less completely. Two enantiotropic pairs were discerned.