Predicting the photostability characteristics of active pharmaceutical ingredients using electron paramagnetic resonance spectroscopy.

OBJECTIVE: The purpose of this study was to determine if electron paramagnetic resonance (EPR) spectroscopy could be used to develop a quick method to predict the longer-term photostability characteristics of active pharmaceutical ingredients (APIs) for use in the early development phase. MATERIALS AND METHODS: EPR spectroscopy was used to study the photodegradation of nearly fifty different APIs in the solid state. RESULTS: Free radical formation was detected in all but three of the APIs studied and singlet oxygen formation was also detected in the presence of five of the APIs tested. The extent of free radical formation in each API after 10 minutes exposed to light was used to rank the APIs in terms of their stability characteristics and determine the probable risk of photodegradation of the API during International Conference on Harmonisation (ICH) compliant photostability testing. A correlation was obtained between the extent of free radical formation on exposure to light and the known level of photodegradation products formed during ICH compliant testing. CONCLUSION: The EPR methods were shown to have the potential to predict the ICH level of photodegradation for an API in the solid state using only a small amount of sample and after just 10 minutes exposed to light. This testing can be performed in a shorter time frame than ICH compliant testing and can potentially be used early in development to predict the photostability characteristics of an API.

Lean Raman imaging for rapid assessment of homogeneity in pharmaceutical formulations.

Solid dispersion formulations and drug in polymer matrices are increasingly being used by the pharmaceutical industry to enhance the solubility, or bio-availability, of active pharmaceutical ingredients (APIs). The degree of solubility or bio-availability enhancement, as well as properties such as chemical stability and physical characteristics, will be dependent on the homogeneity of the drug in polymer matrix. The use of Raman spectroscopy to assess homogeneity has traditionally been limited by the time required to acquire images from a statistically representative sample area. This may be overcome by employing a more rapid one-dimensional Raman line-mapping approach and using a statistical analysis to extract the critical information. This approach has been termed "lean" Raman imaging and allows a large area of sample to be probed in a relatively short space of time. This paper discusses the use of "lean" Raman imaging to assess two performance-indicating parameters of a drug in polymer formulation, sedimentation of the API within a capsule formulation and phase separation of the individual components. The development of a screening method, using Raman line mapping to allow rapid measurement of sedimentation of the API, is discussed. This method requires less than half an hour per capsule for data collection and processing. In addition, the development of a "lean" Raman mapping technique, using single line scans to assess drug and polymer domain sizes, is detailed. This technique employs a simple peak ratio approach coupled with statistical analysis to provide a measure of the degree of drug and polymer segregation without the need for acquisition of high pixel density images or multivariate analysis. The Raman mapping data is compared with both the dissolution profiles and processing parameters of the samples tested and a strong correlation is shown between formulation homogeneity and dissolution behavior.

Quantitative transmission Raman spectroscopy of pharmaceutical tablets and capsules.

Quantitative analysis of pharmaceutical formulations using the new approach of transmission Raman spectroscopy has been investigated. For comparison, measurements were also made in conventional backscatter mode. The experimental setup consisted of a Raman probe-based spectrometer with 785 nm excitation for measurements in backscatter mode. In transmission mode the same system was used to detect the Raman scattered light, while an external diode laser of the same type was used as excitation source. Quantitative partial least squares models were developed for both measurement modes. The results for tablets show that the prediction error for an independent test set was lower for the transmission measurements with a relative root mean square error of about 2.2% as compared with 2.9% for the backscatter mode. Furthermore, the models were simpler in the transmission case, for which only a single partial least squares (PLS) component was required to explain the variation. The main reason for the improvement using the transmission mode is a more representative sampling of the tablets compared with the backscatter mode. Capsules containing mixtures of pharmaceutical powders were also assessed by transmission only. The quantitative results for the capsules' contents were good, with a prediction error of 3.6% w/w for an independent test set. The advantage of transmission Raman over backscatter Raman spectroscopy has been demonstrated for quantitative analysis of pharmaceutical formulations, and the prospects for reliable, lean calibrations for pharmaceutical analysis is discussed.

Investigating the free radical trapping ability of NXY-059, S-PBN and PBN.

The spin trapping ability of the nitrones 2,4-disulphophenyl-N-tert-butyl nitrone (NXY-059), 2-sulphophenyl-N-tert-butyl nitrone (S-PBN) and alpha-phenyl-N-tert-butyl nitrone (PBN) for both hydroxyl and methanol radicals was investigated using electron paramagnetic resonance (EPR) spectroscopy. The radicals of interest were generated in situ in the spectrometer under constant flow conditions in the presence of each nitrone. The spin adducts formed were detected by EPR spectroscopy. This approach allowed for quantitative comparison of the EPR spectra of the spin adducts of each nitrone. The results obtained showed that NXY-059 trapped a greater number of hydroxyl and methanol radicals than the other two nitrones, under the conditions studied.

An EPR, ENDOR and EIE study of gamma-irradiated poly (lactide-co-glycolide) polymers.

Gamma radiation of poly (lactide-co-glycolide) raw polymers and processed microspheres under vacuum and at 77 K results in the formation of a series of free radicals. The resulting powder electron paramagnetic resonance (EPR) spectrum contains a distribution of several different radicals, depending on the annealing temperature, and is therefore difficult to interpret. By utilising the selectivity of the electron nuclear DOuble resonance (ENDOR) and associated ENDOR induced EPR (EIE) techniques, a more direct approach for the deconvolution of the EPR spectrum can be achieved. Using this approach, the radiolytically induced CH3 *CHC(O)R- chain scission radical was identified at 120 K by simulation of the EIE spectrum. At elevated temperatures (250 K), this radical decays considerably and the more stable radicals -O*CHC(O)-, CH3 *C(OR)C(O)- and CH3 *C(OH)C(O)- predominate. This work demonstrates the utility of the EIE approach to supplement and aid the interpretation of powder EPR spectra of radicals in a polymer matrix.

Electron paramagnetic resonance spectroscopy studies of oxidative degradation of an active pharmaceutical ingredient and quantitative analysis of the organic radical intermediates using partial least-squares regression.

Electron paramagnetic resonance (EPR) spectroscopy was used to study the radical species formed during the oxidation of an active pharmaceutical ingredient in the solid state. It was found that the extent of radical generation correlated to the formation of an oxidative degradation product. Multifrequency EPR and electron nuclear double resonance spectroscopy gave additional information on the identity of the organic radical species involved in the oxidation process, and a mechanism was proposed for the degradation, involving the formation of both carbon-centered and peroxy radicals. The multivariate analysis technique of partial least-squares (PLS) regression was then used to determine the extent of oxidation of the active pharmaceutical ingredient from the EPR spectra. The suitability of this approach was demonstrated from its application to a series of standards. The conventional approach for the quantitative analysis of EPR spectra is to measure the peak height or to perform double integration of the spectral region containing the signal of interest. Both of these methods have intrinsic errors associated with them, particularly for weak EPR signals with a poor signal-to-noise ratio or a sloping background response. The results obtained showed that greatly improved quantitation was obtained using the PLS regression approach.

An EPR and ENDOR study of gamma- and beta-radiation sterilization in poly (lactide-co-glycolide) polymers and microspheres.

EPR/ENDOR spectroscopy was used to characterise the free radicals generated in a series of PLGA raw polymers and microspheres (with lactide:glycolide compositions of (75:25), (65:35) and (50:50)) after exposure to gamma (gamma-) and electron beam (beta-) irradiation at room temperature. Both sets of irradiated samples produced analogous EPR spectra, indicating that the type and distribution of free radicals generated by gamma-irradiation are similar to those generated by beta-irradiation. The radicals were identified by EPR simulations as the chain scission species -(CHO-(approximately 27% abundance),-C(CH(3))O- ( approximately 23% abundance) and the terminal-C(CH(3))-OR fragment (approximately 50% abundance), and these assignments were supported by the ENDOR analysis. The latter two radical species were demonstrated to originate from the lactide component of the PLGA polymer. Overall systematically higher radical concentrations were found as the lactide content of the PLGA raw polymer and microspheres increases (ie., 75:25 > 65:35 > 50:50) for both gamma- and beta-irradiation. However, while the relative concentrations of free radicals was similar in the raw polymer samples after exposure to gamma- or beta-irradiation, a substantial difference was found for the microsphere samples; an approximate doubling of the radical content was found in the gamma-irradiated PLGA microspheres compared to the identical beta-irradiated microspheres.

Electron magnetic resonance study of gamma-irradiated poly(lactide-co-glycolide) microspheres.

A series of poly(lactide-co-glycolide) samples of compositions ranging from (75:25) to (65:35) to (50% (65:35):50% (50:50)) were gamma-irradiated under ambient conditions in air. The irradiation doses used were 15, 20, 25, and 30 kGy. The generation of radicals resulting from the gamma-irradiation was confirmed using EPR. Two major radical species were observed and identified as centered at alkyl and alkyl peroxy groups. The indication from the X-band (9 GHz) frequencies showed that alkyl radicals gave rise to a quartet hyperfine pattern. However, measurements performed at higher W-band frequencies (90 GHz) showed that the X-band spectra are actually a composite profile arising from a series of overlapping individual resonances. Using combined EPR and ENDOR (Electron-Nuclear DOuble Resonance) measurements, an alkyl peroxy radical was identified. For increasing glycolide concentration from 75:25 to the 50:50 blend, there was a factor of 7 increase in the concentration of radicals A and B. Furthermore, both radical species were found to be stable for several weeks after storage at ambient temperature conditions. At elevated temperatures and humidities, radical stability decreased--the decay rate was estimated at approximately 3x10(-8) mol K(-1). The stability characteristics of the radicals under different conditions are attributed to changes in the morphology of the polymer.