Elemental Impurities in Pharmaceutical Excipients.

Control of elemental impurities in pharmaceutical materials is currently undergoing a transition from control based on concentrations in components of drug products to control based on permitted daily exposures in drug products. Within the pharmaceutical community, there is uncertainty regarding the impact of these changes on manufactures of drug products. This uncertainty is fueled in part by a lack of publically available information on elemental impurity levels in common pharmaceutical excipients. This paper summarizes a recent survey of elemental impurity levels in common pharmaceutical excipients as well as some drug substances. A widely applicable analytical procedure was developed and was shown to be suitable for analysis of elements that are subject to United States Pharmacopoeia Chapter <232> and International Conference on Harmonization's Q3D Guideline on Elemental Impurities. The procedure utilizes microwave-assisted digestion of pharmaceutical materials and inductively coupled plasma mass spectrometry for quantitative analysis of these elements. The procedure was applied to 190 samples from 31 different excipients and 15 samples from eight drug substances provided through the International Pharmaceutical Excipient Council of the Americas. The results of the survey indicate that, for the materials included in the study, relatively low levels of elemental impurities are present.

Rapid screening of anti-infective drug products for counterfeits using Raman spectral library-based correlation methods.

A new spectral library-based approach that is capable of screening a diverse set of finished drug products using only an active pharmaceutical ingredient spectral library is described in this paper. This approach obviates the need for a comprehensive drug product library, thereby streamlining the use of spectral library-based tests for anti-counterfeiting efforts, specifically to target finished drug products containing the wrong active ingredient or no active ingredient at all. Both laboratory-based and portable spectrometers are used in the study to demonstrate the usefulness and transferability of the spectral correlation method for field screening. The spectral correlation between the active pharmaceutical ingredient and finished drug product spectra is calculated using both full spectral analysis and targeted spectral regions analysis of six types of antimalarial, antibiotic and antiviral products. The spectral regions were determined using a moving window spectral correlation algorithm, and the use of specific spectral regions is shown to be crucial in screening finished drug products using only the active pharmaceutical ingredient spectrum. This comprehensive screening spectral correlation method is tested on seven different validation samples from different manufacturers as those used to develop the method, as well as simulated counterfeits which were prepared to mimic falsified drugs containing no active ingredient. The spectral correlation method is successful in correctly identifying 100% of the authentic products and simulated counterfeit samples tested.

Synthesis and detection of N-sulfonated oversulfated chondroitin sulfate in marketplace heparin.

N-sulfonated oversulfated chondroitin sulfate (NS-OSCS), recently reported as a potential threat to the heparin supply, was prepared along with its intermediate derivatives. All compounds were spiked into marketplace heparin and subjected to United States Pharmacopeia (USP) identification assays for heparin (proton nuclear magnetic resonance [(1)H NMR], chromatographic identity, % galactosamine [%GalN], anti-factor IIa potency, and anti-factor Xa/IIa ratio). The U.S. Food and Drug Administration (FDA) strong-anionic exchange high-performance liquid chromatography (SAX-HPLC) method resolved NS-OSCS from heparin and OSCS and had a limit of detection of 0.26% (w/w) NS-OSCS. The %GalN test was sensitive to the presence of NS-OSCS in heparin. Therefore, current USP heparin monograph tests (i.e., SAX-HPLC and %GalN) detect the presence of NS-OSCS in heparin.

Deep-Ultraviolet Resonance Raman (DUVRR) Spectroscopy of Therapeutic Monoclonal Antibodies Subjected to Thermal Stress.

The structural assessment of Rituximab, an IgG1 mAb, was investigated with deep-ultraviolet resonance Raman (DUVRR) spectroscopy. DUVRR spectroscopy was used to monitor the changes to the secondary structure of Rituximab under thermal stress. DUVRR spectra showed obvious changes from 22 to 72 °C. Specifically, changes in the amide I vibrational mode were assigned to an increase in unordered structure (random coil). Structural changes in samples heated to 72 °C were related to loss in drug potency via a complement dependent cytotoxicity (CDC) bioassay. The DUVRR spectroscopic method shows promise as a tool for the quality assessment of mAb drug products and would represent an improvement over current methodology in terms of analysis time and sample preparation. To determine the scope of the method, protein pharmaceuticals of different molecular weights (ranging from 4 to 143 kDa) and secondary structure (ß-sheet, α-helix and unordered structure) were analyzed. The model illustrated the method's sensitivity for the analysis of protein drug products of different secondary structure. Results show promise for DUVRR spectroscopy as a rapid screening tool of a variety of formulated protein pharmaceuticals.

Raman imaging of pharmaceutical materials: refractive index effects on contrast at buried interfaces.

Resolution targets composed of bilayer polydimethylsiloxane (PDMS) devices with buried polyethylene glycol (PEG) channels have been fabricated using traditional photolithographic and micromolding techniques to develop resolution targets that mimic pharmaceutical materials. Raman chemical images of the resulting PEG-in-PDMS devices composed of varying parallel line widths were investigated by imaging the PEG lines through a thin overlayer of PDMS. Additionally, a scattering agent, Al2O3, was introduced at varying concentrations to each layer of the device to explore the effects of scattering materials on Raman images. Features in the resulting chemical images of the PEG lines suggest that reflection at the PEG/PDMS interface contributes to the Raman signal. A model based on geometric optics was developed to simulate the observed image functions of the targets. The results emphasize the influence of refractive index discontinuities at the PEG/PDMS interface on the apparent size and shape of the PEG features. Such findings have an impact on interpretation of Raman images of nonabsorbing, opaque pharmaceutical samples.

Qualitative screening for adulterants in weight-loss supplements by ion mobility spectrometry.

Ion mobility spectrometry (IMS) served as a rapid, qualitative screening tool for the analysis of adulterated weight-loss products. We have previously shown that sibutramine extracted into methanol from dietary supplements can be detected at low levels (2ng) using a portable IMS spectrometer, and have adapted a similar method for the analysis of additional weight-loss product adulterants. An FDA collaborative study helped to define the limits for fluoxetine with a limit of detection of 2ng. We also evaluated more readily available, less toxic extraction solvents and found isopropanol and water were comparable to methanol. Isopropanol was favored over water for two reasons: (1) water increases the analysis time and (2) aqueous solutions were more susceptible to pH change, which affected the detection of sibutramine. In addition to sibutamine and fluoxetine, we surveyed 11 weight-loss adulterants; bumetanide, fenfluramine, furosemide, orlistat, phenolphthalein, phentermine, phenytoin, rimonabant, sertraline and two sibutramine analogs, desmethylsibutramine and didesmethylsibutramine, using portable and benchtop ion mobility spectrometers. Out of these 13 active pharmaceutical ingredients (APIs), portable and benchtop ion mobility spectrometers were capable of screening products for 10 of these APIs. The developed procedure was applied to two weight-loss dietary supplements using both portable and benchtop instruments. One product contained didesmethylsibutramine while the other contained didesmethylsibutramine and phenolphthalein.

Selective melamine detection in multiple sample matrices with a portable Raman instrument using surface enhanced Raman spectroscopy-active gold nanoparticles.

Melamine adulteration of food and pharmaceutical products is a major concern and there is a growing need to protect the public from exposure to contaminated or adulterated products. One approach to reduce this threat is to develop a portable method for on-site rapid testing. We describe a universal and selective method for the detection of melamine in a variety of solid matrices at the 100-200 µg L(-1) level by surface enhanced Raman spectroscopy (SERS) with gold nanoparticles. With minimal sample preparation and the use of a portable Raman spectrometer, this work will lead to field-based screening for melamine adulteration. Citrate coated gold nanoparticles (Au NPs) were investigated for both colorimetric and Raman-based responses. Several non-hazardous solvents were evaluated in order to develop a melamine extraction procedure safe for field applications. Au NP agglomerates formed by the addition of isopropanol (IPA) prior to sample introduction enhanced the Raman signal for melamine and eliminated matrix interference for substrate formation. The melamine Raman signal resulted in a 10(5) enhancement through the use of Au NP agglomerates. To our knowledge, we have developed the first portable SERS method using Au NPs to selectively screen for the presence of melamine adulteration in a variety of food and pharmaceutical matrices, including milk powder, infant formula, lactose, povidone, whey protein, wheat bran and wheat gluten.

Libraries, classifiers, and quantifiers: a comparison of chemometric methods for the analysis of Raman spectra of contaminated pharmaceutical materials.

In this study, pharmaceutical grade sorbitol was used as a model system for comparison of Raman based library spectral correlation methods with more sophisticated methods of chemometric data analysis. Both crystallizing sorbitol (CS) and non-crystallizing sorbitol (NCS) from several manufacturers were examined. The Raman spectrum of each sample was collected and identified by correlation with a spectral library that included the CS spectrum but not the NCS spectrum. The average hit quality index (HQI) for the measured NCS spectra and the library CS spectrum was 0.966 whereas the average HQI for the measured CS spectra was 0.991. Both HQIs exceeded the 0.950 threshold that is commonly used for material verification. To enhance the discrimination between CS and NCS, a CS/NCS classification model was constructed using soft independent modeling of class analogies (SIMCA). SIMCA was able to positively identify CS and NCS solutions with no misclassifications. When CS was adulterated with low levels (0-5%) of ethylene glycol (EG) and diethylene glycol (DEG), the HQI values of the measured spectra and the CS library spectrum were still above 0.950. When the CS SIMCA model was applied to adulterated CS spectra, it determined that CS samples with adulterant levels as low as 2% were outside of the CS class. A quantitative PLS model was also applied to EG adulterated CS and resulted in a detection limit of 0.9% for EG. The results obtained from these studies highlight the importance of selecting an appropriate data analysis process for the detection of low level adulterants in pharmaceutical raw materials using Raman spectroscopic screening methods.

Standardization of Raman spectra for transfer of spectral libraries across different instruments.

In this paper we evaluate methods for standardization of Raman spectra that are required to improve spectral correlation computations between spectra measured on different instruments. Five commercially-available 785 nm Raman spectrometers from different vendors were included in the study. These spectrometers have diverse specifications and performance levels and range in size from laboratory-based instruments to field-deployable portable and handheld platforms. Since each Raman spectrometer has different characteristics, spectra obtained on one instrument cannot readily be compared to a library acquired on a different instrument without performing various types of spectral corrections (standardization). We outline a procedure that combines previously established Raman shift and intensity correction protocols with a resolution matching step to facilitate the comparison of a centralized master library with spectra acquired on different geographically distributed Raman spectrometers. The standardization procedure is effective in reducing the inherent instrument-to-instrument variability so that spectra from different spectrometers can be compared and reliable results obtained using library-based spectral correlation methods. The findings have important implications for the ability to transfer Raman spectral libraries between instruments.

Quantitative evaluation of the sensitivity of library-based Raman spectral correlation methods.

Library-based Raman spectral correlation methods are widely used in surveillance applications in multiple areas including the pharmaceutical industry, where Raman spectroscopy is commonly used in verification screening of incoming raw materials. While these spectral correlation methods are rapid and require little or no sample preparation, their sensitivity to the presence of contaminants has not been adequately evaluated. This is particularly important when dealing with pharmaceutical excipients, which are susceptible to economically motivated adulteration by substances having similar physical/chemical/spectroscopic properties. We report a novel approach to evaluating the sensitivity of library-based Raman spectral correlation methods to contaminants in binary systems using a hit-quality index model. We examine three excipient/contaminant systems, glycerin/diethylene glycol, propylene glycol/diethylene glycol, and lactose/melamine and find that the sensitivity to contaminant for each system is 18%, 32%, and 4%, respectively. These levels are well-correlated to the minimum contaminant composition that can be detected by both verification and identification methods. Our studies indicate that the most important factor that determines the sensitivity of a spectral correlation measurement to the presence of contaminant is the relative Raman scattering cross section of the contaminant.

Multivariate calibration and instrument standardization for the rapid detection of diethylene glycol in glycerin by Raman spectroscopy.

The transfer of a multivariate calibration model for quantitative determination of diethylene glycol (DEG) contaminant in pharmaceutical-grade glycerin between five portable Raman spectrometers was accomplished using piecewise direct standardization (PDS). The calibration set was developed using a multi-range ternary mixture design with successively reduced impurity concentration ranges. It was found that optimal selection of calibration transfer standards using the Kennard-Stone algorithm also required application of the algorithm to multiple successively reduced impurity concentration ranges. Partial least squares (PLS) calibration models were developed using the calibration set measured independently on each of the five spectrometers. The performance of the models was evaluated based on the root mean square error of prediction (RMSEP), calculated using independent validation samples. An F-test showed that no statistical differences in the variances were observed between models developed on different instruments. Direct cross-instrument prediction without standardization was performed between a single primary instrument and each of the four secondary instruments to evaluate the robustness of the primary instrument calibration model. Significant increases in the RMSEP values for the secondary instruments were observed due to instrument variability. Application of piecewise direct standardization using the optimal calibration transfer subset resulted in the lowest values of RMSEP for the secondary instruments. Using the optimal calibration transfer subset, an optimized calibration model was developed using a subset of the original calibration set, resulting in a DEG detection limit of 0.32% across all five instruments.

Rapid limit tests for metal impurities in pharmaceutical materials by X-ray fluorescence spectroscopy using wavelet transform filtering.

We introduce a new method for analysis of X-ray fluorescence (XRF) spectra based on continuous wavelet transform filters, and the method is applied to the determination of toxic metals in pharmaceutical materials using hand-held XRF spectrometers. The method uses the continuous wavelet transform to filter the signal and noise components of the spectrum. We present a limit test that compares the wavelet domain signal-to-noise ratios at the energies of the elements of interest to an empirically determined signal-to-noise decision threshold. The limit test is advantageous because it does not require the user to measure calibration samples prior to measurement, though system suitability tests are still recommended. The limit test was evaluated in a collaborative study that involved five different hand-held XRF spectrometers used by multiple analysts in six separate laboratories across the United States. In total, more than 1200 measurements were performed. The detection limits estimated for arsenic, lead, mercury, and chromium were 8, 14, 20, and 150 µg/g, respectively.

Using a portable ion mobility spectrometer to screen dietary supplements for sibutramine.

In response to recent incidents of undeclared sibutramine, an appetite suppressant found in dietary supplements, we developed a method to detect sibutramine using hand-held ion mobility spectrometers with an analysis time of 15 s. Ion mobility spectrometry is a high-throughput and sensitive technique that has been used for illicit drug, explosive, volatile organic compound and chemical warfare detection. We evaluated a hand-held ion mobility spectrometer as a tool for the analysis of supplement extracts containing sibutramine. The overall instrumental limit of detection of five portable ion mobility spectrometers was 2 ng of sibutramine HCl. When sample extractions containing 30 ng/µl or greater of sibutramine were analyzed, saturation of the ionization chamber of the spectrometer occurred and the instrument required more than three cleaning cycles to remove the drug. Hence, supplement samples suspected of containing sibutramine should be prepared at concentrations of 2-20 ng/µl. To obtain this target concentration range for products containing unknown amounts of sibutramine, we provided a simple sample preparation procedure, allowing the U.S. Food and Drug Administration or other agencies to screen products using the portable ion mobility spectrometer.

Detection of diethylene glycol adulteration in propylene glycol--method validation through a multi-instrument collaborative study.

Four portable NIR instruments from the same manufacturer that were nominally identical were programmed with a PLS model for the detection of diethylene glycol (DEG) contamination in propylene glycol (PG)-water mixtures. The model was developed on one spectrometer and used on other units after a calibration transfer procedure that used piecewise direct standardization. Although quantitative results were produced, in practice the instrument interface was programmed to report in Pass/Fail mode. The Pass/Fail determinations were made within 10s and were based on a threshold that passed a blank sample with 95% confidence. The detection limit was then established as the concentration at which a sample would fail with 95% confidence. For a 1% DEG threshold one false negative (Type II) and eight false positive (Type I) errors were found in over 500 samples measured. A representative test set produced standard errors of less than 2%. Since the range of diethylene glycol for economically motivated adulteration (EMA) is expected to be above 1%, the sensitivity of field calibrated portable NIR instruments is sufficient to rapidly screen out potentially problematic materials. Following method development, the instruments were shipped to different sites around the country for a collaborative study with a fixed protocol to be carried out by different analysts. NIR spectra of replicate sets of calibration transfer, system suitability and test samples were all processed with the same chemometric model on multiple instruments to determine the overall analytical precision of the method. The combined results collected for all participants were statistically analyzed to determine a limit of detection (2.0% DEG) and limit of quantitation (6.5%) that can be expected for a method distributed to multiple field laboratories.

Propagation of uncertainty in nasal spray in vitro performance models using Monte Carlo simulation: Part II. Error propagation during product performance modeling.

Monte Carlo simulations were applied to investigate the propagation of uncertainty in both input variables and response measurements on model prediction for nasal spray product performance design of experiment (DOE) models in the first part of this study, with an initial assumption that the models perfectly represent the relationship between input variables and the measured responses. In this article, we discard the initial assumption, and extended the Monte Carlo simulation study to examine the influence of both input variable variation and product performance measurement variation on the uncertainty in DOE model coefficients. The Monte Carlo simulations presented in this article illustrate the importance of careful error propagation during product performance modeling. Our results show that the error estimates based on Monte Carlo simulation result in smaller model coefficient standard deviations than those from regression methods. This suggests that the estimated standard deviations from regression may overestimate the uncertainties in the model coefficients. Monte Carlo simulations provide a simple software solution to understand the propagation of uncertainty in complex DOE models so that design space can be specified with statistically meaningful confidence levels.

Propagation of uncertainty in nasal spray in vitro performance models using Monte Carlo simulation: part I. Model prediction using Monte Carlo simulation.

Design of experiment (DOE) methodology can provide a complete evaluation of the influences of nasal spray activation and formulation properties on delivery performance which makes it a powerful tool for product design purposes. Product performance models are computed from complex expressions containing multiple factor terms and response terms. Uncertainty in the regression model can be propagated using Monte Carlo simulation. In this study, four input factors, actuation stroke length, actuation velocity, concentration of gelling agent, and concentration of surfactant were investigated for their influences on measured responses of spray pattern, plume width, droplet size distribution (DSD), and impaction force. Quadratic models were calculated and optimized using a Box-Behnken experimental design to describe the relationship between factors and responses. Assuming that the models perfectly represent the relationship between input variables and the measured responses, the propagation of uncertainty in both input variables and response measurements on model prediction was performed using Monte Carlo simulations. The Monte Carlo simulations presented in this article illustrate the propagation of uncertainty in model predictions. The most influential input variable variances on the product performance variance were identified, which could help prioritize input variables in terms of importance during continuous improvement of nasal spray product design. This work extends recent Monte Carlo simulations of process models to the realm of product development models.

Screening of heparin API by near infrared reflectance and Raman spectroscopy.

Near infrared (NIR) reflectance and laser Raman spectra for a set of 69 heparin powder samples obtained from several foreign and domestic suppliers were measured. Both the NIR and Raman spectra of individual heparin API powder samples were correlated with sample compositions determined from response corrected relative peak areas of the capillary electropherograms of the samples using a partial least squares (PLS) regression model. Twenty-eight sample spectra were used to develop PLS models for the three major sample components; heparin, oversulfated chondroitin sulfate (OSCS) and glycosaminoglycans (GAGs). The PLS models were then used to successfully predict the compositions of 41 additional heparin samples. The success of these rapid, nondestructive technologies to identify contamination of heparin with OSCS demonstrates the potential of spectroscopy and chemometrics for screening of processed raw materials. These technologies are meant for screening purposes and not meant to replace either of the methods (capillary electrophoresis and NMR) currently required by USP and FDA.

Detection of undeclared erectile dysfunction drugs and analogues in dietary supplements by ion mobility spectrometry.

An ion mobility spectrometry (IMS) method was developed to screen for the presence of undeclared synthetic erectile dysfunction (ED) drugs or drug analogues in herbal dietary supplements claiming to enhance male sexual performance. Ion mobility spectra of authenticated reference materials including three FDA approved drugs (sildenafil citrate, tadalafil, vardenafil hydrochloride trihydrate) and five previously identified synthetic analogues (methisosildenafil, homosildenafil, piperidenafil, thiosildenafil, thiomethisosildenafil) were measured to determine their reduced ion mobilities (K(0)). All eight compounds exhibited reduced mobilities between 0.8257 and 1.2876 cm(2)/(Vs). Twenty-six herbal products were then screened for the presence of these compounds, and 15 of the 26 products tested positive for the presence of ED drug or drug analogue adulterants based on their reduced ion mobilities. IMS results were compared against the results obtained from an independent LC/MS reference method for the identical samples. Herbal dietary supplements containing adulterants were classified with 100% accuracy and most of the adulterants were correctly identified by a comparison of the K(0) of the adulterant to the K(0) of the authenticated reference material. The results demonstrate that IMS is a viable method for screening herbal dietary supplements for the presence of ED drug or drug analogue adulterants.

Raman detected differential scanning calorimetry of polymorphic transformations in acetaminophen.

Acetaminophen is known to crystallize in three polymorphic forms. Thermally induced transformations between the crystalline forms and the super-cooled liquid have been observed by differential scanning calorimetry (DSC), but the assignment of calorimetric transitions to specific polymorphic transformations remains challenging, because the transition temperatures for several transformations are close to one another, and the characteristics of the observed transitions depend on experimental variables that are often poorly controlled. This paper demonstrates the simultaneous application of DSC and Raman microscopy for the observation of thermally driven transitions between polymorphs of pharmaceutical materials. Raman detected differential scanning calorimetry (RD-DSC) has been used to monitor the DSC thermograms of super-cooled liquid acetaminophen and confirms the assignment of two exothermic transitions to specific polymorphic transformations. Principal component analysis of the Raman spectra have been used to determine the number of independent components that participate in the phase transformations, and multivariate regression has been used to determine transition temperatures from the spectral data. The influence of the laser excitation source on measured DSC thermograms has also been investigated, and it has been demonstrated that a baseline shift occurs in RD-DSC when a polymorphic transformation occurs between crystalline and amorphous forms. RD-DSC has been used to examine the influence of sample aging and sample pan configuration on the observed polymorphic transformations, and both of these variables were found to influence the thermal behavior of the sample. The results demonstrate the advantage of simultaneous Raman spectroscopy and differential scanning calorimetry for the unambiguous assignment of thermally driven polymorphic transformations.

Assessment of the influence factors on in vitro testing of nasal sprays using Box-Behnken experimental design.

The purpose of the research was to investigate the influences of actuation parameters and formulation physical properties on nasal spray delivery performance using design of experiment (DOE) methodology. A 3-level, 4-factor Box-Behnken design with a total of 27 experimental runs was used in this study. Nine simulated aqueous formulations with different viscosities and surface tensions were prepared using carboxymethylcellulose sodium (CMC, gelling agent) and Tween80 (surfactant) each at three concentration levels. Four factors, actuation stroke length, actuation velocity, concentration of gelling agent, and concentration of surfactant were investigated for their influences on measured responses of shot weight, spray pattern, plume geometry and droplet size distribution (DSD). The models based on data from the DOE were then optimized by eliminating insignificant terms. Pfeiffer nasal spray pump units filled with the simulated formulations were used in the study. Nasal pump actuation stroke length exerts a strong, independent influence on shot weight, and also slightly affects spray pattern and plume geometry. Actuation velocity and concentration of gelling agent have significant effects on spray pattern, plume geometry and DSD, in a complicated manner through interaction terms. Concentration of surfactant has little, if any, influence on nasal spray characteristics. Results were fitted to quadratic models describing the inherent relationships between the four factors evaluated and nasal spray performance. The DOE study helped us to identify the source of variability in nasal spray product performance, and obtained better understanding in how to control the variability. Moreover, the quadratic models developed from the DOE study quantitatively describe the inherent relationships between the factors and nasal spray performance characteristics. With the assistance of the response surfaces developed from the DOE model, the time and labor in designing a nasal spray product to achieve desired product performance characteristics can be reduced.