Evaluation of distributional homogeneity of pharmaceutical formulation using laser direct infrared imaging.

The distributional homogeneity of chemicals is a key parameter of solid pharmaceutical formulations. Indeed, it may affect the efficacy of the drug and consequently its safety. Chemical imaging offers a unique insight enabling the visualisation of the different constituents of a pharmaceutical tablet. It allows identifying ingredients poorly distributed offering the possibility to optimize the process parameters or to adapt characteristics of incoming raw materials to increase the final product quality. Among the available chemical imaging tools, Raman imaging is one of the most widely used since it offers a high spatial resolution with well-resolved peaks resulting in a high spectral specificity. However, Raman imaging suffers from sample autofluorescence and long acquisition times. Recently commercialised, laser direct infrared reflectance imaging (LDIR) is a quantum cascade laser (QCL) based imaging technique that offers the opportunity to rapidly analyse samples. In this study, a typical pharmaceutical formulation blend composed of two active pharmaceutical ingredients and three excipients was aliquoted at different mixing timepoints. The collected aliquots were tableted and analysed using both Raman and LDIR imaging. The distributional homogeneity indexes of one active ingredient image were then computed and compared. The results show that both techniques achieved similar conclusions. However, the analysis times were drastically different. While Raman imaging required a total analysis time of 4 h per tablet to obtain the distribution map of acetylsalicylic acid with a step size of 100 µm, it only took 7.5 min to achieve the same result with LDIR. The results obtained in the present study show that LDIR is a promising technique for the analysis of pharmaceutical formulations and that it could be a valuable tool when developing new pharmaceutical formulations.

Raman imaging as a new analytical tool for the quality control of the monitoring of osteogenic differentiation in forming 3D bone tissue.

In this study, adipose-derived stem cells (ASCs) are used to produce 3D bone grafts. The safety and the feasibility of using these bone grafts have been already showed and quality controls are already implemented. However, a cheaper, fast and non-destructive technique is required to monitor the osteogenic differentiation process. Here, the use of Raman imaging to monitor the synthesis of the extracellular matrix and its progressive mineralization occurring during the osteogenic differentiation process is investigated for the first time on a 3D in forming bone tissue. The attention was focused on Raman bands related to this matrix belonging to phosphate, phenylalanine and hydroxyproline, which are very distinctive and intense. The kinetic of the osteogenic differentiation process was first compared between a 2D and a 3D forming bone tissue. It was observed that the kinetics of the osteogenic differentiation process is slower in 3D in forming bone tissue. In a second step, an evaluation of the reliability of the Raman imaging method was performed including a study of the influence of the harvest biopsies position on the forming 3D bone tissue. The repeatability and the specificity of this method were also demonstrated. In a last step, several batches of ASCs were cultured and analyzed in 3D at different time points using Raman imaging. From the mean Raman spectra, mineral to matrix ratios (MTMR) were determined and used to evaluate the formation of mineral deposits accompanying the extracellular matrix synthesis which is indicative of an ongoing osteogenic differentiation process. These ratios peaked between the day 35 and 49. This observation was very interesting since it corresponds to the time at which the 3D bone grafts are used for the patient surgery. To conclude, Raman imaging allowed fast acquisition and time-resolved monitoring in vitro of the mineralization of extracellular matrix during osteogenic differentiation.

Quantitation of active pharmaceutical ingredient through the packaging using Raman handheld spectrophotometers: A comparison study.

Handheld Raman spectroscopy is actually booming. Recent devices improvements aim at addressing the usual Raman spectroscopy issues: fluorescence with shifted-excitation Raman difference spectroscopy (SERDS), poor sensitivity with surface enhanced Raman scattering (SERS) and information only about the sample surface with spatially offset Raman spectroscopy (SORS). While qualitative performances of handheld devices are generally well established, the quantitative analysis of pharmaceutical samples remains challenging. The aim of this study was to compare the quantitative performances of three commercially available handheld Raman spectroscopy devices. Two of them (TruScan and IDRaman mini) are equipped with a 785 nm laser wavelength and operate in a conventional backscattering mode. The IDRaman has the Orbital Raster Scanning (ORS) option to increase the analyzed surface. The third device (Resolve) operates with an 830 nm laser wavelength both in backscattering and in SORS modes. The comparative study was carried out on ibuprofen-mannitol-microcrystalline cellulose ternary mixtures. The concentration of ibuprofen ranged from 24 to 52% (w/w) while the proportions of the two excipients were varied to avoid cross-correlation as much as possible. Analyses were performed either directly through a glass vial or with the glass vial in an opaque polypropylene flask, using a validated FT-NIR spectroscopy method as a reference method. Chemometric analyses were carried out with the Partial Least Squares Regression (PLS-R) algorithm. The quantitative models were validated using the total error approach and the ICH Q2 (R1) guidelines with ±â€¯15% as acceptance limits.

Development of a SERS strategy to overcome the nanoparticle stabilisation effect in serum-containing samples: Application to the quantification of dopamine in the culture medium of PC-12 cells.

The analysis of serum samples by surface-enhanced Raman spectroscopy (SERS) has gained ground over the last few years. However, the stabilisation of colloids by the proteins contained in these samples has restricted their use in common practice, unless antibodies or aptamers are used. Therefore, this work was dedicated to the development of a SERS methodology allowing the analysis of serum samples in a simple and easy-to-implement way. This approach was based on the pre-aggregation of the colloid with a salt solution. Gold nanoparticles (AuNPs) were used as the SERS substrate and, owing to its physiopathological importance, dopamine was chosen as a model to implement the SERS approach. The presence of this neurotransmitter could be determined in the concentration range 0.5-50 ppm (2.64-264 µM) in the culture medium of PC-12 cells, with a R2 of 0.9874, and at even lower concentrations (0.25 ppm, 1.32 µM) in another matrix containing fewer proteins. Moreover, the effect of calcium and potassium on the dopamine exocytosis from PC-12 cells was studied. Calcium was shown to have a predominant and dose-dependant effect. Finally, PC-12 cells were exposed to dexamethasone in order to increase their biosynthesis and release of dopamine. This increase was monitored with the developed SERS approach.

Critical review of surface-enhanced Raman spectroscopy applications in the pharmaceutical field.

Surface-enhanced Raman spectroscopy (SERS) is a sensitive analytical tool used in the pharmaceutical field in recent years. SERS keeps all the advantages of classical Raman spectroscopy while being is more sensitive allowing its use for the detection and the quantification of low-dose substances contained in pharmaceutical samples. However, the analytical performance of SERS is limited due to the difficulty to implement a quantitative methodology correctly validated. Nevertheless, some studies reported the development of SERS quantitative methods especially in pharmaceutical approaches. In this context, this review presents the main concepts of the SERS technique. The different steps that need to be applied to develop a SERS quantitative method are also deeply described. The last part of the present manuscript gives a critical overview of the different SERS pharmaceutical applications that were developed for a non-exhaustive list of pharmaceutical compounds with the aim to highlights the validation criteria for each application.

Development of an analytical method for crystalline content determination in amorphous solid dispersions produced by hot-melt extrusion using transmission Raman spectroscopy: A feasibility study.

The development of a quantitative method determining the crystalline percentage in an amorphous solid dispersion is of great interest in the pharmaceutical field. Indeed, the crystalline Active Pharmaceutical Ingredient transformation into its amorphous state is increasingly used as it enhances the solubility and bioavailability of Biopharmaceutical Classification System class II drugs. One way to produce amorphous solid dispersions is the Hot-Melt Extrusion (HME) process. This study reported the development and the comparison of the analytical performances of two techniques, based on backscattering and transmission Raman spectroscopy, determining the crystalline remaining content in amorphous solid dispersions produced by HME. Principal Component Analysis (PCA) and Partial Least Squares (PLS) regression were performed on preprocessed data and tended towards the same conclusions: for the backscattering Raman results, the use of the DuoScan™ mode improved the PCA and PLS results, due to a larger analyzed sampling volume. For the transmission Raman results, the determination of low crystalline percentages was possible and the best regression model was obtained using this technique. Indeed, the latter acquired spectra through the whole sample volume, in contrast with the previous surface analyses performed using the backscattering mode. This study consequently highlighted the importance of the analyzed sampling volume.

Global approach for the validation of an in-line Raman spectroscopic method to determine the API content in real-time during a hot-melt extrusion process.

Since the Food and Drug Administration (FDA) published a guidance based on the Process Analytical Technology (PAT) approach, real-time analyses during manufacturing processes are in real expansion. In this study, in-line Raman spectroscopic analyses were performed during a Hot-Melt Extrusion (HME) process to determine the Active Pharmaceutical Ingredient (API) content in real-time. The method was validated based on a univariate and a multivariate approach and the analytical performances of the obtained models were compared. Moreover, on one hand, in-line data were correlated with the real API concentration present in the sample quantified by a previously validated off-line confocal Raman microspectroscopic method. On the other hand, in-line data were also treated in function of the concentration based on the weighing of the components in the prepared mixture. The importance of developing quantitative methods based on the use of a reference method was thus highlighted. The method was validated according to the total error approach fixing the acceptance limits at ±15% and the α risk at ±5%. This method reaches the requirements of the European Pharmacopeia norms for the uniformity of content of single-dose preparations. The validation proves that future results will be in the acceptance limits with a previously defined probability. Finally, the in-line validated method was compared with the off-line one to demonstrate its ability to be used in routine analyses.

Monitoring of anatabine release by methyl jasmonate elicited BY-2 cells using surface-enhanced Raman scattering.

A new application of surface-enhanced Raman scattering (SERS) in the field of plant material analysis is proposed in this study. The aim was to monitor the release of anatabine by methyl jasmonate (MeJa) elicited Bright Yellow-2 (BY-2) cells. Gold nanoparticles (AuNps) were used as SERS substrate. The first step was to study the SERS activity of anatabine in a complex matrix comprising the culture medium and BY-2 cells. The second step was the calibration. This one was successfully performed directly in the culture medium in order to take into account the matrix effect, by spiking the medium with different concentrations of anatabine, leading to solutions ranging from 250 to 5000µgL(-1). A univariate analysis was performed, the intensity of a band situated at 1028cm(-1), related to anatabine, was plotted against the anatabine concentration. A linear relationship was observed with a R(2) of 0.9951. During the monitoring study, after the MeJa elicitation, samples were collected from the culture medium containing BY-2 cells at 0, 24h, 48h, 72h and 96h and were analysed using SERS. Finally, the amount of anatabine released in the culture medium was determined using the response function, reaching a plateau after 72h of 82µg of anatabine released/g of fresh weight (FW) MeJa elicited BY-2 cells.

A simple calibration approach based on film-casting for confocal Raman microscopy to support the development of a hot-melt extrusion process.

When developing a new formulation, the development, calibration and validation steps of analytical methods based on vibrational spectroscopy are time-consuming. For each new formulation, real samples must be produced and a "reference method" must be used in order to determine the Active Pharmaceutical Ingredient (API) content of each sample. To circumvent this issue, the paper presents a simple approach based on the film-casting technique used as a calibration tool in the framework of hot-melt extrusion process. Confocal Raman microscopic method was successfully validated for the determination of itraconazole content in film-casting samples. Then, hot-melt extrusion was carried out to produce real samples in order to confront the results obtained with confocal Raman microscopy and Ultra High Performance Liquid Chromatography (UHPLC). The agreement between both methods was demonstrated using a comparison study based on the Bland and Altman's plot.

A simple approach for ultrasensitive detection of bisphenols by multiplexed surface-enhanced Raman scattering.

Bisphenol A (BPA) is well known for its use in plastic manufacture and thermal paper production despite its risk of health toxicity as an endocrine disruptor in humans. Since the publication of new legislation regarding the use of BPA, manufacturers have begun to replace BPA with other phenolic molecules such as bisphenol F (BPF) and bisphenol B (BPB), but there are no guarantees regarding the health safety of these compounds at this time. In this context, a very simple, cheap and fast surface-enhanced Raman scattering (SERS) method was developed for the sensitive detection of these molecules in spiked tap water solutions. Silver nanoparticles were used as SERS substrates. An original strategy was employed to circumvent the issue of the affinity of bisphenols for metallic surfaces and the silver nanoparticles surface was functionalized using pyridine in order to improve again the sensitivity of the detection. Semi-quantitative detections were performed in tap water solutions at a concentrations range from 0.25 to 20 µg L(-1) for BPA and BPB and from 5 to 100 µg L(-1) for BPF. Moreover, a feasibility study for performing a multiplex-SERS detection of these molecules was also performed before successfully implementing the developed SERS method on real samples.

Vibrational spectroscopy and microspectroscopy analyzing qualitatively and quantitatively pharmaceutical hot melt extrudates.

Since the last decade, more and more Active Pharmaceutical Ingredient (API) candidates have poor water solubility inducing low bioavailability. These molecules belong to the Biopharmaceutical Classification System (BCS) classes II and IV. Thanks to Hot-Melt Extrusion (HME), it is possible to incorporate these candidates in pharmaceutical solid forms. Indeed, HME increases the solubility and the bioavailability of these drugs by encompassing them in a polymeric carrier and by forming solid dispersions. Moreover, in 2004, the FDA's guidance initiative promoted the usefulness of Process Analytical Technology (PAT) tools when developing a manufacturing process. Indeed, the main objective when developing a new pharmaceutical process is the product quality throughout the production chain. The trend is to follow this parameter in real-time in order to react immediately when there is a bias. Vibrational spectroscopic techniques, NIR and Raman, are useful to analyze processes in-line. Moreover, off-line Raman microspectroscopy is more and more used when developing new pharmaceutical processes or when analyzing optimized ones by combining the advantages of Raman spectroscopy and imaging. It is an interesting tool for homogeneity and spatial distribution studies. This review treats about spectroscopic techniques analyzing a HME process, as well off-line as in-line, presenting their advantages and their complementarities.

Data processing of vibrational chemical imaging for pharmaceutical applications.

Vibrational spectroscopy (MIR, NIR and Raman) based hyperspectral imaging is one of the most powerful tools to analyze pharmaceutical preparation. Indeed, it combines the advantages of vibrational spectroscopy to imaging techniques and allows therefore the visualization of distribution of compounds or crystallization processes. However, these techniques provide a huge amount of data that must be processed to extract the relevant information. This review presents fundamental concepts of hyperspectral imaging, the basic theory of the most used chemometric tools used to pre-process, process and post-process the generated data. The last part of the present paper focuses on pharmaceutical applications of hyperspectral imaging and highlights the data processing approaches to enable the reader making the best choice among the different tools available.

Development of a quantitative approach using surface-enhanced Raman chemical imaging: first step for the determination of an impurity in a pharmaceutical model.

This publication reports, for the first time, the development of a quantitative approach using surface-enhanced Raman chemical imaging (SER-CI). A pharmaceutical model presented as tablets based on paracetamol, which is the most sold drug around the world, was used to develop this approach. 4-Aminophenol is the main impurity of paracetamol and is actively researched in pharmaceutical formulations because of its toxicity. As its concentration is generally very low (<0.1%, w/w), conventional Raman chemical imaging cannot be used. In this context, a SER-CI method was developed to quantify 4-aminophenol assessing a limit of quantification below its limit of specification of 1000 ppm. Citrate-reduced silver nanoparticles were used as SERS substrate and these nanoparticles were functionalized using 1-butanethiol. Different ways to cover the tablets surface by butanethiol-functionalized silver nanoparticles were tested and a homogeneity study of the silver nanoparticles covering was realized. This homogeneity study was performed in order to choose the best way to cover the surface of tablets by silver colloid. Afterwards, the optimization of the SER-CI approach was necessary and different spectral intensity normalizations were tested. Finally, a quantitative approach using SER-CI was developed enabling to quantify 4-aminophenol from 0.025% to 0.2% in paracetamol tablets. This quantitative approach was tested on two different series of tablets using different batches of silver nanoparticles.

Determination of 4-aminophenol in a pharmaceutical formulation using surface enhanced Raman scattering: from development to method validation.

A surface enhanced Raman scattering (SERS) method able to quantify 4-aminophenol in a pharmaceutical formulation based on acetaminophen, also called paracetamol, was developed and, for the first time, successfully validated. In this context, silver nanoparticles were synthesized according to the method described by Lee-Meisel and used as SERS substrate. The repeatability of the silver colloid synthesis was tested using different methods to characterize the size and the zeta potential of silver nanoparticles freshly synthesized. To optimize the SERS samples preparation, a design of experiments implicating concentrations of citrate-reduced silver nanoparticles and aggregating agent was performed in order to maximize the Raman signal enhancement. Finally, an approach based on tolerance intervals and accuracy profiles was applied in order to thoroughly validate the method in a range of concentrations comprised from 3 to 15 µg mL(-1) using normalized band intensities. The standard addition method was selected as method calibration. Therefore, measurements were carried out on 4-aminophenol spiked solutions of the pharmaceutical formulation. Despite the well-known stability and reproducibility problems of SERS, the validation was performed using two operators and five batches of nanoparticles, one for each validation day.

Critical review of near-infrared spectroscopic methods validations in pharmaceutical applications.

Based on the large number of publications reported over the past five years, near-infrared spectroscopy (NIRS) is more and more considered an attractive and promising analytical tool regarding Process Analytical Technology and Green Chemistry. From the reviewed literature, few of these publications present a thoroughly validated NIRS method even if some guidelines have been published by different groups and regulatory authorities. However, as any analytical method, the validation of NIRS method is a mandatory step at the end of the development in order to give enough guarantees that each of the future results during routine use will be close enough to the true value. Besides the introduction of PAT concepts in the revised document of the European Pharmacopoeia (2.2.40) dealing with near-infrared spectroscopy recently published in Pharmeuropa, it agrees very well with this mandatory step. Indeed, the latter suggests to use similar analytical performance characteristics than those required for any analytical procedure based on acceptance criteria consistent with the intended use of the method. In this context, this review gives a comprehensive and critical overview of the methodologies applied to assess the validity of quantitative NIRS methods used in pharmaceutical applications.

Comparison of the quantitative performances and measurement uncertainty estimates obtained during method validation versus routine applications of a novel hydrophilic interaction chromatography method for the determination of cidofovir in human plasma.

Method validation is essential to ensure that an analytical method is fit for its intended purpose. Additionally, it is advisable to estimate measurement uncertainty in order to allow a correct interpretation of the results generated by analytical methods. Measurement uncertainty can be efficiently estimated during method validation as a top-down approach. However, method validation predictions of the quantitative performances of the assay and estimations of measurement uncertainty may be far away from the real performances obtained during the routine application of this assay. In this work, the predictions of the quantitative performances and measurement uncertainty estimations obtained from a method validation are compared to those obtained during routine applications of a bioanalytical method. For that purpose, a new hydrophilic interaction chromatography (HILIC) method was used. This method was developed for the determination of cidofovir, an antiviral drug, in human plasma. Cidofovir (CDV) is a highly polar molecule presenting three ionizable functions. Therefore, it is an interesting candidate for determination by HILIC mode. CDV is an acyclic cytidine monophosphate analog that has a broad antiviral spectrum and is currently undergoing evaluation in clinical trials as a topical agent for treatment of papillomavirus infections. The analytical conditions were optimized by means of design of experiments approach in order to obtain robust analytical conditions. These ones were absolutely necessary to enable the comparisons mentioned above. After a sample clean-up by means of solid phase extraction, the chromatographic analysis was performed on bare silica stationary phase using a mixture of acetonitrile-ammonium hydrogen carbonate (pH 7.0; 20mM) (72:28, v/v) as mobile phase. This newly developed bioanalytical method was then fully validated according to FDA (Food and Drug Administration) requirements using a total error approach that guaranteed that each future result will fall within acceptance limits of ±30% with a probability of 95% over a concentration range of 92.7-1020ng/mL. A routine application of the cidofovir determination in two pre-clinical trials demonstrated that the prediction made during the pre-study validation was consistent by retrospective analysis of the quality control (QC) samples. Finally, comparison of the measurement uncertainty estimations calculated from the method validation with those obtained from the routine application of the method was performed, stressing that the estimations obtained during method validation underestimated those obtained from routine applications and that the magnitude of this underestimation was function of the cidofovir concentration. Finally, this new HILIC method is reliable, easily applicable to routine analysis and transposable at low cost in other laboratories.

Near infrared and Raman spectroscopy as Process Analytical Technology tools for the manufacturing of silicone-based drug reservoirs.

Using near infrared (NIR) and Raman spectroscopy as PAT tools, 3 critical quality attributes of a silicone-based drug reservoir were studied. First, the Active Pharmaceutical Ingredient (API) homogeneity in the reservoir was evaluated using Raman spectroscopy (mapping): the API distribution within the industrial drug reservoirs was found to be homogeneous while API aggregates were detected in laboratory scale samples manufactured with a non optimal mixing process. Second, the crosslinking process of the reservoirs was monitored at different temperatures with NIR spectroscopy. Conformity tests and Principal Component Analysis (PCA) were performed on the collected data to find out the relation between the temperature and the time necessary to reach the crosslinking endpoints. An agreement was found between the conformity test results and the PCA results. Compared to the conformity test method, PCA had the advantage to discriminate the heating effect from the crosslinking effect occurring together during the monitored process. Therefore the 2 approaches were found to be complementary. Third, based on the HPLC reference method, a NIR model able to quantify the API in the drug reservoir was developed and thoroughly validated. Partial Least Squares (PLS) regression on the calibration set was performed to build prediction models of which the ability to quantify accurately was tested with the external validation set. The 1.2% Root Mean Squared Error of Prediction (RMSEP) of the NIR model indicated the global accuracy of the model. The accuracy profile based on tolerance intervals was used to generate a complete validation report. The 95% tolerance interval calculated on the validation results indicated that each future result will have a relative error below ±5% with a probability of at least 95%. In conclusion, 3 critical quality attributes of silicone-based drug reservoirs were quickly and efficiently evaluated by NIR and Raman spectroscopy.

Development and validation of a sensitive solid phase extraction/hydrophilic interaction liquid chromatography/mass spectrometry method for the accurate determination of glucosamine in dog plasma.

A sensitive and accurate LC/MS method was developed for the monitoring of glucosamine (GLcN) dog plasmatic concentration. In this scope, relatively low plasmatic concentrations of GLcN were expected, ranging from 50 to 1000 ng/mL. Liquid chromatography coupled to simple quadrupole mass spectrometry detection (LC/MS) was selected bringing the selectivity and the sensitivity needed for this application. Additionally, a solid phase extraction (SPE) step was performed to reduce matrix and ion suppression effects. Due to the ionisable character of the compound of interest, a mixed-mode strong cation exchange (Plexa PCX) disposable extraction cartridge (DEC) was selected. The separation was carried out on a Zorbax SB-CN column (5 microm, 4.6mm i.d. x 250 mm), considering hydrophilic interaction liquid chromatography (HILIC). Indeed, the mobile phase was made of methanol and 5mM ammonium hydrogen carbonate buffer at pH 7.5 (95/5, v/v). The detection was led at m/z ratios of 180.0 and 417.0, for GLcN and IS, respectively. Reliability of the results was demonstrated through the validation of the method using an approach based on the accuracy profile allowing managing the risk associated to the use of these methods in routine analysis: it is thus guaranteed that each future result will fall in the +/-30% acceptance limits with a probability of at least 90%. Successful application of the method to a preliminary pharmacokinetic study illustrated the usefulness of the method for pre-clinical studies.

Development and validation of a quantitative method for the selective determination of tin species in tin octoate by differential pulse polarography.

Tin octoate is used as a catalyst in the synthesis of polydimethylsiloxane (PDMS), a room temperature vulcanizing (RTV) silicone rubber. This rubber is largely used in the medical field due to its great biocompatibility. In this framework, a high-speed and costless analytical method for the determination of stannic ions, Sn(IV), in the presence of stannous ions, Sn(II), has been developed. The separation of these two ions was carried out using differential pulse polarography (DPP). For this purpose, the tin species contents in the catalyst is quantitatively extracted under inert condition to avoid any changes in the ratio Sn(IV)/Sn(II). Polarography showed well-shaped oxidation and reduction peaks respectively at -650 and -860mV for stannous ions. The peak of the stannic ion was well separated and appeared at -1210mV. Many parameters such as extraction process, extraction time, pH, chelating agents and polarographic conditions were optimized. We have also demonstrated that no oxidation of the stannous ions occurred during the sample preparation. The dosing range considered in this study extends between 10 and 40mug/mL, corresponding to 6.8% and 27.2% of the degradation product (Sn(IV)) in the catalyst, regarding to the sampling. Finally this method was successfully validated using the total error concept.