Dispersive Raman Spectroscopy for Quantifying Amorphous Drug Content in Intact Tablets.

Process-induced inadvertent phase change of an active pharmaceutical ingredient in a drug product could impact chemical stability, physical stability, shelf life, and bioperformance. In this study, dispersive Raman spectroscopy is presented as an alternative method for the nondestructive, high-throughput, at-line quantification of amorphous conversion. A quantitative Raman method was developed using a multivariate partial least squares (PLS) regression calibration technique with solid-state nuclear magnetic resonance (ssNMR) spectroscopy as the reference method. Compositionally identical calibration tablets containing 20% w/w total MK-A drug in varying weight proportions (0%-50% w/w based on total MK-A) of amorphous and crystalline MK-A were compressed at 10-45 kN force. PLS predictions of amorphous content of tablets using Raman spectroscopy correlated well with ssNMR quantification. The predictive accuracy of this model led to a strong correlation (R2 = 0.987) with a root mean-squared error of prediction of 1.5% w/w amorphous MK-A in tablets up to 50% w/w amorphous conversion in compressive stress range of 60-320 MPa. Overall, these results suggest that dispersive Raman spectroscopy offers fast, sensitive, and high-throughput (<5 min/tablet) method for quantitating amorphous conversion.

In-line measurement of a drug substance via near infrared spectroscopy to ensure a robust crystallization process.

The crystallization of Etoricoxib, a polymorphic compound, has been optimized and controlled by seeding with the desired polymorph at a moderate supersaturation condition. To enhance the process robustness, near infrared spectroscopy (NIRS) has been evaluated as an inline measurement method for the concentration of Etoricoxib prior to seeding in the crystallization process. In this NIRS method, a spectral discriminant analysis based on principal component analysis (PCA) was established to detect the presence of solids produced by premature crystallization, or bubbles in the path of light. Once a spectrum was qualified as that of clear solution, concentration of Etoricoxib was calculated by a NIRS calibration model built with partial least squares (PLS) regression and with offline HPLC analysis as the reference method. This model was accurate with a standard error of cross validation (SECV) less than 1.2 mg/g Etoricoxib and a standard error of prediction (SEP) less than 1.7 mg/g over the concentration range from 50 to 170 mg/g, temperature range from 49 to 65 degrees C, and different sources of materials. In addition, all aspects of the offline HPLC method, especially the sampling procedure, were optimized to provide an accurate reference for NIRS calibration models. The application of this method at a pilot plant has demonstrated its capability of accurately measuring the process concentration of Etoricoxib as well as detecting the presence of solids produced by premature crystallization before seeding.

Mechanistic evidence for an alpha-oxoketene pathway in the formation of beta-ketoamides/esters via Meldrum's acid adducts.

A practical, one-pot process for the preparation of beta-keto amides via a three-component reaction, including Meldrum's acid, an amine, and a carboxylic acid, has been developed. Key to development of an efficient, high-yielding process was an in-depth understanding of the mechanism of the multistep process. Kinetic studies were carried out via online IR monitoring and subsequent principal component analysis which provided a means of profiling the concentration of both the anionic and free acid forms of the Meldrum's adduct 6 in real time. These studies, both in the presence and absence of nucleophiles, strongly suggest that formation of beta-keto amides from acyl Meldrum's acids occurs via alpha-oxoketene species 2 and rule out other possible reaction pathways proposed in the literature, such as via protonated alpha-oxoketene intermediates 3 or nucleophilic addition-elimination pathways.

Determination and differentiation of surface and bound water in drug substances by near infrared spectroscopy.

Near infrared spectroscopy (NIRS) was utilized to determine the water content during the drying of a drug substance with Karl Fisher titration as the reference measurement. NIRS calibration models were built with Partial Least Squares (PLS) regression based on spectral region of 1822-1948 nm for samples with 1-40% (w/w) water from five batches. A standard error of prediction (SEP) of 1.85% (w/w) was obtained from validation of the model with additional batches. A second NIRS calibration model using PLS was constructed in the region of 1-10% (w/w) water with samples from the same five calibration batches. This calibration model improved the accuracy of the prediction in the critical region around the end point of drying and provided a standard error of prediction 0.42% (w/w). In addition, direct spectral analyses with Principal Component Analysis (PCA) and peak ratios were applied to distinguish between surface (unbound) water and bound water incorporated into the crystal lattice of the drug substance. Direct spectral analysis indicated the existence of significant numerical and graphical differences between samples containing both surface and bound water, and those containing bound water only. Applying this method to monitor an actual drying process with the graphical assistance of spectral analysis, the drying process can be controlled, and the end point of drying clearly determined to ensure the desired hydrate form of the product. This study has demonstrated the in-line monitoring capability of NIRS to differentiate the surface and bound water simultaneously in addition to the determination of the water level.

Characterization and quantitation of aprepitant drug substance polymorphs by attenuated total reflectance fourier transform infrared spectroscopy.

In this study, we report the use of attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FT-IR) for the identification and quantitation of two polymorphs of Aprepitant, a substance P antagonist for chemotherapy-induced emesis. Mixtures of the polymorph pair were prepared by weight and ATR-FT-IR spectra of the powdered samples were obtained over the wavelength range of 700-1500 cm(-1). Significant spectral differences between the two polymorphs at 1140 cm(-1) show that ATR-FT-IR can provide definitive identification of the polymorphs. To investigate the feasibility of ATR-FT-IR for quantitation of polymorphic forms of Aprepitant, a calibration plot was constructed with known mixtures of the two polymorphs by plotting the peak ratio of the second derivative of absorbance spectra against the weight percent of form II in the polymorphic mixture. Using this novel approach, 3 wt % of one crystal form could be detected in mixtures of the two polymorphs. The accuracy of ATR-FT-IR in determining polymorph purity of the drug substance was tested by comparing the results with those obtained by X-ray powder diffractometry (XRPD). Indeed, polymorphic purity results obtained by ATR-FT-IR were found to be in good agreement with the predictions made by XRPD and compared favorably with actual values in the known mixtures. The present study clearly demonstrates the potential of ATR-FT-IR as a quick, easy, and inexpensive alternative to XRPD for the determination of polymorphic identity and purity of solid drug substances. The technique is ideally suited for polymorph analysis, because it is precise, accurate, and requires minimal sample preparation.

In situ moisture determination of a cytotoxic compound during process optimization.

A simple and safe prototype apparatus was designed and adapted for the in situ determination of the moisture content of a cytotoxic compound (9-fluorenylmethyl-protected doxorubicin-peptide conjugate, or Fm-DPC) by near-infrared absorbance spectroscopy during optimization of the chemical isolation procedure. The cytotoxic nature of the compound restricts one's ability to safely sample such drying processes for more traditional means of moisture determination for fear of hazardous solids dusting, hence in situ sampling approaches are of great importance. These concerns also exist for the process development laboratory, where despite the smaller scale of operations, the volume of experiments (hence cytotoxic samples) required to define a chemical process is often more significant. In this application, partial least squares regression was used with Karl Fischer volumetric titration analysis to generate a calibration model. Although pronounced differences in cake density were observed as a function of the buffer selected for the isolation process, the model still achieved a standard error of calibration of 0.63% w/w and a standard error of prediction of 0.99% (w/w). These results demonstrated the versatility of the prototype apparatus/data processing approach to model Fm-DPC drying under extremely variable conditions, as inherently expected during the investigational laboratory development of a chemical process.

Concentration determination of methyl magnesium chloride and other Grignard reagents by potentiometric titration with in-line characterization of reaction species by FTIR spectroscopy.

A potentiometric titration method for methyl magnesium chloride and other Grignard reagents based on the reaction with 2-butanol in THF has been developed and validated. The method employs a commercially available platinum electrode, using an electrolyte compatible with non-aqueous solvents. Well-defined titration curves were obtained, along with excellent method precision. The endpoint was precisely determined based on the first derivative of the titration curve. Different solvents such as THF, diethyl ether and methylene chloride provided similar results with regard to sharpness of the endpoint and method precision. The method was applied to a wide array of Grignard reagents including methyl magnesium bromide, ethyl magnesium chloride, propyl magnesium chloride, vinyl magnesium chloride, phenyl magnesium chloride, and benzyl magnesium chloride with similar precision and accuracy. Application of in-line FTIR was demonstrated for in situ monitoring of the titration reaction, allowing characterization of the reaction species. An authentic spectrum of the MeMgCl-THF complex was obtained using spectral subtraction and the vibrational absorbance bands were identified. FTIR also provided an alternative for detecting the titration endpoint, and the titration results so obtained, provided a cross-validation of the accuracy of the potentiometric titration.

Employment of on-line FT-IR spectroscopy to monitor the deprotection of a 9-fluorenylmethyl protected carboxylic acid peptide conjugate of doxorubicin.

A method for accurately determining the end-point, >98% conversion, of the deprotection reaction of a highly toxic 9-fluorenylmethyl (Fm) ester 1b to its corresponding carboxylate 1d in real time by FT-IR spectroscopy is reported. Advantages of this method over analysis by conventional chromatographic means include real time determination of the end-point of a reaction that is time sensitive to by-product formation, and elimination of sampling a highly toxic reaction mixture. The FT-IR method is based on monitoring, in real time, the disappearance of the Fm ester carbonyl band for 1b at 1737 cm(-1), during deprotection by piperidine, and calibration models were established by Partial Least Squares (PLS) regression analysis with high performance liquid chromatography (HPLC) as reference. The best calibration model was built with 5 PLS factors in the spectral range of 1780-1730 and 1551-1441 cm(-1) and resulted in a standard error of cross validation (SECV) of 0.63 mM 1b and a standard error of prediction (SEP) of 0.51 mM 1b in the range of 0-25 mM. This error of prediction is approximately 0.8% of the initial concentration of 1b and is well within our specifications of <2% initial concentration.