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See detailNew Methodology for the Development of Chromatographic Methods
Rozet, Eric ULg; Debrus, Benjamin ULg; Lebrun, Pierre ULg et al

Conference (2011, September 08)

As defined by ICH [1] and FDA, Quality by Design (QbD) stands for “a systematic approach to development that begins with predefined objectives and emphasizes product and process understanding and process ... [more ▼]

As defined by ICH [1] and FDA, Quality by Design (QbD) stands for “a systematic approach to development that begins with predefined objectives and emphasizes product and process understanding and process control, based on sound science and quality risk management”. A risk–based QbD–compliant approach is proposed for the robust development of analytical methods. This methodology based on Design of Experiments (DoE) to study the experimental domain models the retention times at the beginning, the apex and the end of each peak corresponding to the compounds of a mixture and uses the separation criterion (S) rather than the resolution (RS) as a Critical Quality Attribute. Stepwise multiple linear regressions are used to create the models. The estimated error is propagated from the modelled responses to the separation criterion (S) using Monte Carlo simulations in order to estimate the predictive distribution of the separation criterion (S) over the whole experimental domain. This allows finding ranges of operating conditions that will guarantee a satisfactory quality of the method in its future use. These ranges define the Design Space (DS) of the method. In chromatographic terms, the chromatograms processed at operating conditions within the DS will assuredly show high quality, with well separated peaks and short run time, for instance. This Design Space can thus be defined as the subspace, necessarily encompassed in the experimental domain (i.e. the knowledge space), within which the probability for the criterion to be higher than an advisedly selected threshold is higher than a minimum quality level. Precisely, the DS is defined as “the multidimensional combination and interaction of input variables (e.g., material attributes) and process parameters that have been demonstrated to provide assurance of quality” [1]. Therefore, this DS defines a region of operating conditions that provide prediction of assurance of quality rather than only quality as obtained with traditional mean response surface optimisation strategies. For instance, in the liquid chromatography there is a great difference in e.g. predicting a resolution (RS) higher than 1.5 vs. predicting that the probability for RS to be higher than 1.5 (i.e. P(RS> 1.5)) is high. The presentation of this global methodology will be illustrated for the robust optimisation and DS definition of several liquid chromatographic methods dedicated to the separation of different mixtures: pharmaceutical formulations, API and impurities/degradation products, plant extracts, separation of enantiomers, … References [1] International Conference on Harmonisation (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use, Topic Q8(R2): Pharmaceutical development, Geneva, 2009. Acknowledgements A research grant from the Belgium National Fund for Scientific Research (F.R.S-FNRS) to E. Rozet is gratefully acknowledged. [less ▲]

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See detailNEW TRENDS IN VALIDATION OF ANALYTICAL METHODS
Rozet, Eric ULg; Hubert, Philippe ULg

Conference (2011, September 08)

Analytical method validation is a mandatory step to evaluate the ability of developed methods to provide accurate results for their routine application in order to trust the critical decisions that will ... [more ▼]

Analytical method validation is a mandatory step to evaluate the ability of developed methods to provide accurate results for their routine application in order to trust the critical decisions that will be made with them. Even if several guidelines exist to help perform analytical method validations (ICH Q2R1 [1], USP <1225> [2], …) there is still the need to clarify the meaning and interpretation of analytical method validation criteria and methodology. Indeed, actually method validation is mostly realised as the traditional check list implementation of e.g. the ICH Q2R1 or USP <1225> method validation requirements. However, within the trend of Quality by Design [3], there is the need to switch from this traditional vision to an analytical method validation really adding value and providing a high level of assurance of analytical methods results reliability. Yet, different interpretations can be made of the validation guidelines as well as for the definitions of the validation criteria. This will lead to diverse experimental designs implemented to try fulfilling these criteria. Finally, different decision methodologies can also be interpreted from these guidelines. Therefore, the risk that a validated analytical method may be unfit for its future purpose will depend on a personal interpretation of these guidelines. The objective of this presentation is thus to show that analytical method validation should be planned and performed by first starting with the end in mind: what is the objective of the analytical methods under study? In such a way analytical method validation is coherent with the actual Quality by Design regulatory expectations. The risk of having validated an analytical method unfit for its purpose is strongly reduced as well as the risk of generating Out of Specification (OOS) results due to an unfit method. References [1] International Conference on Harmonisation (ICH) of Technical Requirements for registration of Pharmaceuticals for Human Use, Topic Q2 (R1): Validation of Analytical Procedures: Text and Methodology, Geneva, 2005. [2] USP 33 NF 28 S1, U.S. Pharmacopeia, 2007. USP–NF General Chapter <1225>. [3] International Conference on Harmonisation (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use, Topic Q8(R2): Pharmaceutical development, Geneva, 2009. [less ▲]

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See detailA RAPID UHPLC-DAD-ESI-MSn METHOD FOR ANTHOCYANINS QUANTIFICATION FROM Euterpe oleracea FRUITS HARVESTED AT DIFFERENT TIMES
Dias, A.; Chataigné, G.; Rozet, Eric ULg et al

Poster (2011, June 09)

Euterpe oleracea is a palm tree widely distributed in northern South America. Its greatest occurrence and economic importance happens in the floodplains of the Amazonian delta. The fruits called açai are ... [more ▼]

Euterpe oleracea is a palm tree widely distributed in northern South America. Its greatest occurrence and economic importance happens in the floodplains of the Amazonian delta. The fruits called açai are an interesting source of different anthocyanins. Lately they have gained popularity in North America and in the European countries in the food industry and in the health sector due to their extremely high antioxidant capacity and potential anti-inflammatory activities [1]. Some studies have characterized chemically açaí pulps and have reported anthocyanin profiles which differ both qualitatively and quantitatively. Among other reasons, these differences may be associated with the stages of ripening of the fruits, since açai fruits are generally harvested in different maturation stages. The evaluation of the anthocyanin profile of açai fruits during different maturation stages is thus important for the post-harvest industry. In addition a rapid separation by UHPLC and an unambiguous identification by MSn are very useful for an effective quality control of the fruits. Thus, the aim of this study was to characterize the anthocyanin profiles of açai fruits at different stages of maturity. The fruits were harvested during the peak harvesting season, between July and October 2009, in the floodplains of the eastern Amazonian region (State of Pará, Brazil). A protocol of solid-liquid extraction of phenolic compounds was developed. Characterisation of the anthocyanins present in the fruits of Euterpe oleracea was conducted by UHPLC–DAD–ESI–MSn analysis, in positive ionization mode. All identified compounds was separated in 10 minutes of a total run time of 21 min instead of 55 min in the previously developed HPLC method. Six anthocyanins were identified in the extracts namely: cyaniding-3-glucoside, cyaniding-3-rutinoside, pelargonidin-3-glucoside, peonidin-3-glucoside, peonidin-3-rutinoside and cyanidin. The first two compounds were the major constituents in all maturity stages, with similar proportions, except for the first maturity stage for which the anthocyanins were under the limit of quantification. However, in the last maturity stage, cyanidin-3-glucoside became less abundant than cyanidin-3-rutinoside. On the other hand, cyanidin decreased with maturation. For the other compounds, proportions were similar along maturation. Hence, this work was important as it provides valid information on variation of anthocyanin profiles of açai fruits during maturation. This may contribute to the selection of an optimal maturity stage for harvesting as well as it will allow a rapid quality control of the fruits. [1]: Heinrich, M., et al., Açai (Euterpe oleracea Mart.)- A phytochemical and pharmacological assessment of the species’ health claims. Phytochem. Lett. (2010), doi: 10.1016/j.phytol.2010.11.005 [less ▲]

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See detailComment adéquatement déclarer valide ou non valide une méthode analytique ?
Rozet, Eric ULg; Hubert, Philippe ULg

Conference (2011, April 04)

Vu le rôle central que jouent les méthodes d’analyse dans l’ensemble des activités tant de l’industrie (bio)pharmaceutique, agro-alimentaire, chimique ou dans l’environnement, il est essentiel de les ... [more ▼]

Vu le rôle central que jouent les méthodes d’analyse dans l’ensemble des activités tant de l’industrie (bio)pharmaceutique, agro-alimentaire, chimique ou dans l’environnement, il est essentiel de les valider. Les critères de validation ne sont pas une finalité et il est nécessaire de rappeler l’objectif d’une méthode analytique et de sa validation afin de planifier adéquatement cette étape de validation. Par ailleurs, les approches de décision habituelles qui se basent seulement sur des estimateurs ponctuelles ne répondent pas à l’objectif de la validation. Une stratégie alternative pour décider de la validité des méthodes est proposée utilisant les intervalles de tolérance : "ß-expectation tolerance interval". [less ▲]

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See detailPerformance of three HIV-1 DNA real-time PCRs for early diagnosis in infants and adults
Iserentant, G.; Masquelier, C.; Lambert, C. et al

Poster (2011, March)

Background Early pediatric HIV-1 diagnosis is a challenge in resource limited countries to identify HIV-infected newborns to provide early ART treatment. HIV DNA PCR testing is an accurate method for ... [more ▼]

Background Early pediatric HIV-1 diagnosis is a challenge in resource limited countries to identify HIV-infected newborns to provide early ART treatment. HIV DNA PCR testing is an accurate method for diagnosis in newborns and could be valuable for monitoring HIV infection in adults. We have evaluated the analytical, clinical performances and genotype inclusivity of 3 real-time Taqman PCRs targeting the ltr, int and env genes. Material and methods Total DNA was extracted from whole blood and Dried Blood Spots (DBS) using the NucleoSpin Blood kit (Macherey Nagel) and the Chelex resin. DNA amplification was performed using the Qiagen Multiplex PCR kit and detected on a 7300 Real time PCR system (Applied Biosystems). Method validation was realized as required by ISO 15189 standard. The accuracy profile methodology was applied. HIV-1 DNA of ACH2 cells (0, 25, 50, 100, 1000 and 5000 copies/PCR) mixed in whole blood were measured in 4 different series (2 PCR kits and 2 operators) of 5 independent DNA extraction of each standard. Linear and quadratic weighted and non weighted regression models were tested. Trueness, precision, limit of quantifications of the method and accuracy of the results were obtained for each model and compared. 93 whole blood samples of ART treated-patients with undetectable or low viraemia of 16 B and 77 non-B subtypes were assessed. Clinical evaluation of the assay was performed on 26 whole blood samples and 238 DBS from infants of Guinea Conakry determined HIV+ using the rapid Bioline HIV test Results The calibration model providing the most accurate results was the linear regression with an r² equal to 0.92 for ltr, 0.88 for int and 0.95 for env. Trueness, precision and accuracy were highly acceptable for the 3 sets of primers/probe not exceeding -0.23 log(copies/PCR) for bias and 0.5 log(copies) for intermediate precision standard deviation, respectively. The estimated lower limit of quantification was 41, 475 and 25 copies/PCR for ltr, int and env respectively. The accuracy profile of each gene showed that each future measurement using this assay has 80% probability to be within a limit of 0.5 log (copies/PCR) around the reference or true value of copies of each gene. 52/93 whole blood samples of ART-treated patients had 3 positive genes (Ct<40 cycles), 38/93 had two positive genes and 4/93 had only one positive gene (ltr). Among these patients, 3 were assigned as long term progressors and one was a subtype C strain. Clinical evaluation of the assay identified 1 HIV-1 infected newborn and 3 adults (at least 2 positive genes) for which HIV-1 infection was confirmed either by serology or viral load techniques as well as 96 HIV-1 infected infants using DBS. Conclusions This assay is suitable for detection of proviral HIV-1 DNA in infants and adults on whole blood and DBS samples. Despite an estimated lower limit of quantification higher than ltr and env, the int PCR was required to achieve a good genotype inclusivity. The method was fully validated as required by ISO 15189 and showed to provide sufficiently reliable results. [less ▲]

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See detailValidation, transfer and measurement uncertainty estimation of an HPLC-UV method for the quantification of artemisinin in hydro alcoholic extracts of Artemisia annua L.
ZIME DIAWARA, H.; GBAGUIDI, F.; Evrard, Brigitte ULg et al

in Journal of Pharmaceutical & Biomedical Analysis (2011), 56

Malaria is the world’s most important parasitic infection with 500 millions cases annually and almost 2 millions death per year. This disease is more present in Sub-Saharan Africa where 90% of the ... [more ▼]

Malaria is the world’s most important parasitic infection with 500 millions cases annually and almost 2 millions death per year. This disease is more present in Sub-Saharan Africa where 90% of the infections are found. Artemisinin and its semi synthetic derivatives (artemether, artesunate) have actually the most powerful activity on malaria, even in its complicated forms and resistance cases. Various methods have been proposed for detection and quantification of artemisinin in Artemisia annua L. by HPLC-UV, but the plant extracts used for this quantification were extracts obtained with organic solvents (toluene, petroleum ether, hexane …). To be able to use crude A. annua extracts prepared at low cost to formulate antipaludic drugs, we chose the use of a mixture of water and ethanol as solvent of extraction, but no adequate analytical method for this kind of extracts is published. The main objectives of this work were first to develop an analytical method for artemisinin quantification in hydro alcoholic extracts of A. annua. Second, this method had to be thoroughly validated by the research and development laboratory and, third, the transfer of this method to the routine laboratory had to be demonstrated. The final aim was to compare the estimation of measurement uncertainty obtained during the method validation with validation standards to measurement uncertainty estimates obtained during the method transfer study with real samples. The method was validated following the accuracy profile methodology and was found to be accurate in the concentration range of 10.0 to 54.0 µg/ml with CV <8%. Limit of detection and of quantification were 2.73 and 10.0 µg/ml, respectively. The method was then successfully transferred to a laboratory in Benin by showing that the quality of the results that it will generate during routine application of the method is sufficient. Finally, the measurement uncertainty of the method was estimated from the validation experiments as well as from the transfer study with authentic unspiked samples of A. annua. The comparison of these measurement uncertainty estimations showed that they were coherent. It confirmed thus that the estimation of measurement uncertainty from validation experiments predicts well the measurement uncertainty of real routine samples. This analytical method was thus shown to be convenient for routine analysis of hydro alcoholic extracts of A. annua in Benin. [less ▲]

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See detailDevelopment and validation of a ultra-high-performance liquid chromatography-UV method for the detection and quantification of erectile dysfunction drugs and some of their analogues found in counterfeit medicines
Sacré, Pierre-Yves ULg; Deconinck, Eric; Chiap, Patrice ULg et al

in Journal of Chromatography. A (2011), 1218

Pharmaceutical counterfeiting is a permanently growing problem. Control laboratories are constantly analysing counterfeit medicines. In industrialised countries, one of the main counterfeited class of ... [more ▼]

Pharmaceutical counterfeiting is a permanently growing problem. Control laboratories are constantly analysing counterfeit medicines. In industrialised countries, one of the main counterfeited class of medicines are erectile dysfunction drugs. This paper describes the development and validation of a fast method to detect and quantify the three authorised phosphodiesterase type 5 inhibitors and five analogues. The method is based on the use of a sub-2 microns polar-embedded column with a gradient using acetonitrile as organic modifier and 10 mM ammonium formate buffer (pH 3.5) as aqueous component of the mobile phase. The separation was achieved in less than 4.5 min. The method has also been compared to the registered HPLC method for the assay of Viagra® which was considered as the reference method. The method is also compatible with on-line coupling mass spectrometry and will significantly reduce analysis times and solvent consumption. [less ▲]

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See detailEvaluating the reliability of analytical results using a probability criterion: a Bayesian perspective
Rozet, Eric ULg; Govaerts, B.; Lebrun, Pierre ULg et al

in Analytica Chimica Acta (2011), 705

Methods validation is mandatory in order to assess the fitness of purpose of the developed analytical method. Of core importance at the end of the validation is the evaluation of the reliability of the ... [more ▼]

Methods validation is mandatory in order to assess the fitness of purpose of the developed analytical method. Of core importance at the end of the validation is the evaluation of the reliability of the individual results that will be generated during the routine application of the method. Regulatory guidelines provide a general framework to assess the validity of a method, but none address the issue of results reliability. In this study, a Bayesian approach is proposed to address this concern. Results reliability is defined here as “the probability of an analytical method to provide analytical results within predefined acceptance limits around their reference or conventional true concentration values over a defined concentration range and under given environmental and operating conditions.” By providing the minimum reliability probability needed for the subsequent routine application of the method, as well as specifications or acceptance limits , the proposed Bayesian approach provides the effective probability of obtaining reliable future analytical results over the whole concentration range investigated. This is summarized in a single graph: the reliability profile. This Bayesian reliability profile is also compared to two frequentist approaches, the first one derived from the work of Dewé et al. [Dewé W., Govaerts B., Boulanger B., Rozet E., Chiap P., Hubert Ph., Chemometr. Intell. Lab. Syst. 85 (2007) 262-268] and the second proposed by Govaerts et al. [B. Govaerts, W. Dewé, M. Maumy, B. Boulanger, Qual. Reliab. Engng. Int. 24 (2008) 667-680]. Furthermore, to illustrate the applicability of the Bayesian reliability profile, this approach is also applied here to a bioanalytical method dedicated to the determination of ketoglutaric acid (KG) and hydroxymethylfurfural (HMF) in human plasma by SPE-HPLC-UV. [less ▲]

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See detailA multicentric evaluation of IDMS-traceable creatinine enzymatic assays
Pieroni, Laurence; DELANAYE, Pierre ULg; Boutten, Anne et al

in Clinica Chimica Acta (2011), 412

Chronic kidney disease definition is based on glomerular filtration rate (GFR) estimations which are derived from creatinine-based equations. The accuracy of GFR estimation is thus largely dependent of ... [more ▼]

Chronic kidney disease definition is based on glomerular filtration rate (GFR) estimations which are derived from creatinine-based equations. The accuracy of GFR estimation is thus largely dependent of those of serum creatinine assays. International recommendations highlight the need for traceable creatinine assays. The French Society of Clinical Biochemistry conducted a study for measuring accuracy of creatinine enzymatic methods. This evaluation involved 25 clinical laboratories. Creatinine was measured in serum pools ranging from 35.9±0.9 μmol/L to 174.5±3.1 μmol/L (IDMS determination) using 12 creatinine enzymatic methods. For all creatinine values greater than 74.4±1.4 μmol/L, the bias and imprecision did not exceed 5% and 5.9%, respectively. For the lowest value (35.9±0.9 μmol/L), the bias ranged from −1.8 to 9.9% (with one exception). At this level, the imprecision ranged from 1.9 to 7.8%. The true performances of the assays (couples of bias and relative standard deviation), were evaluated using Monte-Carlo simulations. Most of the assays fall within the maximum Total Error of 12% at all concentrations. This study demonstrates substantial improvements in the calibration, traceability and precision of the enzymatic methods, reaching the NKDEP recommendations. Moreover, most of these assays allowed accurate creatinine measurements for creatinine levels lower than 40 μmol/L. [less ▲]

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See detailModels to estimate overall analytical measurements uncertainty: assumptions, comparisons and applications
Rozet, Eric ULg; Rudaz, S.; Marini Djang'Eing'A, Roland ULg et al

in Analytica Chimica Acta (2011), 702

Evaluation of analytical results reliability is of core importance as crucial decisions are taken with them. From the various methodologies to evaluate the fitness of purpose of analytical methods ... [more ▼]

Evaluation of analytical results reliability is of core importance as crucial decisions are taken with them. From the various methodologies to evaluate the fitness of purpose of analytical methods, overall measurement uncertainty estimation is more and more applied. Overall measurement uncertainty allows to combine simultaneously the remaining systematic influences to the random sources of uncertainty and allows assessing the reliability of results generated by analytical methods. However there are various interpretations on how to estimate overall measurement uncertainty, and thus various models for estimating it. Each model together with its assumptions has great impacts on the risks to abusively declare that analytical methods are suitable for their intended purpose. This review paper aims at i) summarizing the various models used to estimate overall measurement uncertainty, ii) provide their pros and cons, iii) review the main areas of application and iv) as a conclusion provide some recommendations when evaluating overall measurement uncertainty. [less ▲]

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See detailInnovative high-performance liquid chromatography method development for the screening of 19 antimalarial drugs based on a generic approach, using design of experiments, independent component analysis and design space
Debrus, Benjamin ULg; Lebrun, Pierre ULg; Mbinze Kindenge, Jérémie et al

in Journal of Chromatography. A (2011), 1218

An innovative methodology based on design of experiments (DoE), independent component analysis (ICA) and design space (DS) was developed in previous works and was tested out with a mixture of 19 ... [more ▼]

An innovative methodology based on design of experiments (DoE), independent component analysis (ICA) and design space (DS) was developed in previous works and was tested out with a mixture of 19 antimalarial drugs. This global LC method development methodology (i.e. DoE–ICA–DS) was used to optimize the separation of 19 antimalarial drugs to obtain a screening method. DoE–ICA–DS methodology is fully compliant with the current trend of quality by design. DoE was used to define the set of experiments to model the retention times at the beginning, the apex and the end of each peak. Furthermore, ICA was used to numerically separate coeluting peaks and estimate their unbiased retention times. Gradient time, temperature and pH were selected as the factors of a full factorial design. These retention times were modelled by stepwise multiple linear regressions. A recently introduced critical quality attribute, namely the separation criterion (S), was also used to assess the quality of separations rather than using the resolution. Furthermore, the resulting mathematical models were also studied from a chromatographic point of view to understand and investigate the chromatographic behaviour of each compound. Good adequacies were found between the mathematical models and the expected chromatographic behaviours predicted by chromatographic theory. Finally, focusing at quality risk management, the DS was computed as the multidimensional subspace where the probability for the separation criterion to lie in acceptance limits was higher than a defined quality level. The DS was computed propagating the prediction error from the modelled responses to the quality criterion using Monte Carlo simulations. DoE–ICA–DS allowed encountering optimal operating conditions to obtain a robust screening method for the 19 considered antimalarial drugs in the framework of the fight against counterfeit medicines. Moreover and only on the basis of the same data set, a dedicated method for the determination of three antimalarial compounds in a pharmaceutical formulation was optimized to demonstrate both the efficiency and flexibility of the methodology proposed in the present study. [less ▲]

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See detailNear infrared and Raman spectroscopy as Process Analytical Technology tools for the manufacturing of silicone-based drug reservoirs
Mantanus, Jérôme ULg; Rozet, Eric ULg; Van Butsele, K. et al

in Analytica Chimica Acta (2011), 699

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 ... [more ▼]

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 % 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. [less ▲]

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See detailApplication of a new optimization strategy for the separation of tertiary alkaloids extracted from Strychnos usambarensis leaves
Nistor, Iolanda ULg; Cao, Martine ULg; Debrus, Benjamin ULg et al

in Journal of Pharmaceutical & Biomedical Analysis (2011), 56

The HPLC separation of six alkaloids extracted from Strychnos usambarensis leaves has been developed and optimized by means of a powerful methodology for modelling chromatographic responses, based on ... [more ▼]

The HPLC separation of six alkaloids extracted from Strychnos usambarensis leaves has been developed and optimized by means of a powerful methodology for modelling chromatographic responses, based on three steps, i.e. design of experiments (DoE), independent component analysis (ICA) and design space (DS). This study was the first application of a new optimization strategy to a complex natural matrix. The compounds separated are the isomers isostrychnopentamine and strychnopentamine, 10-hydroxyusambarine and 11-hydroxyusambarine, also strychnophylline and strychnofoline. Three LC parameters have been optimized using a multifactorial design comprising 29 experiments that includes 2 center point replicates. The parameters were the percentage of organic modifiers used at the beginning of a gradient profile which consisted in different proportions of methanol (MeOH) and acetonitrile (MeCN), the gradient time to reach 70% of organic modifiers starting from the initial percentage and the percentage of MeCN found in the mobile phase. Subsequent to the experimental design application, predictive multilinear models were developed and used in order to provide optimal analytical conditions. The optimum assay conditions were: methanol/acetonitrile-sodium pentane sulfonate (pH 2.2; 7.5 mM) (33.4:66.6, v/v) at a mobile phase flow rate of 1mL/min during a 40.6 minutes gradient time. The initial organic phase contained 3.7% MeCN and 96.3% MeOH. The method showed good agreement between the experimental data and predictive value throughout the studied parameters space. Improvement of the analysis time and optimized separation for the compounds of interest was possible due to the original and powerful tools applied. Finally, this study permitted the acquisition of isomers profiles allowing the identification of the optimal collecting period of Strychnos usambarensis. [less ▲]

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