Discrimination and classification techniques applied on Mallotus and Phyllanthus high performance liquid chromatography fingerprints.

Mallotus and Phyllanthus genera, both containing several species commonly used as traditional medicines around the world, are the subjects of this discrimination and classification study. The objective of this study was to compare different discrimination and classification techniques to distinguish the two genera (Mallotus and Phyllanthus) on the one hand, and the six species (Mallotus apelta, Mallotus paniculatus, Phyllanthus emblica, Phyllanthus reticulatus, Phyllanthus urinaria L. and Phyllanthus amarus), on the other. Fingerprints of 36 samples from the 6 species were developed using reversed-phase high-performance liquid chromatography with ultraviolet detection (RP-HPLC-UV). After fingerprint data pretreatment, first an exploratory data analysis was performed using Principal Component Analysis (PCA), revealing two outlying samples, which were excluded from the calibration set used to develop the discrimination and classification models. Models were built by means of Linear Discriminant Analysis (LDA), Quadratic Discriminant Analysis (QDA), Classification and Regression Trees (CART) and Soft Independent Modeling of Class Analogy (SIMCA). Application of the models on the total data set (outliers included) confirmed a possible labeling issue for the outliers. LDA, QDA and CART, independently of the pretreatment, or SIMCA after "normalization and column centering (N_CC)" or after "Standard Normal Variate transformation and column centering (SNV_CC)" were found best to discriminate the two genera, while LDA after column centering (CC), N_CC or SNV_CC; QDA after SNV_CC; and SIMCA after N_CC or after SNV_CC best distinguished between the 6 species. As classification technique, SIMCA after N_CC or after SNV_CC results in the best overall sensitivity and specificity.

Accuracy profiles assessing the validity for routine use of high-performance thin-layer chromatographic assays for drug formulations.

The accuracy profile, based on total error, integrates several validation parameters, such as trueness, precision and linearity, providing one statistic which enables decision on the suitability of a method for its intended purpose. Two assay methods for formulations are validated using accuracy profiles as an alternative approach to classic method validation. It concerns high-performance thin-layer chromatography (HPTLC) methods, which initially were validated using the classic approach. The first method assayed sulfamethoxazole and trimethoprim, and the second lamivudine, stavudine and nevirapine. Both formulations are fixed-dose combination tablets. The resulting accuracy profiles showed that the 95% ß-expectation tolerance limits for all compounds fell well within the bias acceptance limits set at ±5%. This means that the two analytical thin-layer chromatographic methods are capable of making accurate results at the studied concentration ranges of each compound. Measurement uncertainties of every compound at each concentration level could also be determined from the accuracy profile data.

Multivariate data analysis to evaluate the fingerprint peaks responsible for the cytotoxic activity of Mallotus species.

The Mallotus genus comprises numerous species used as traditional medicines in oriental countries and provides scientists a broad basis in the search for pharmacologically active constituents. In this paper, the cytotoxicity of 39 Mallotus extracts, different in species, part of the plant used, origin, and harvest season, is evaluated combining cytotoxicity assays with fingerprint technology and data handling tools. At first, the antiproliferative activity of the plant extracts is analyzed both on a non-cancerous cell line (WI-38--human lung fibroblast) and on a cancerous cell line (HeLa human cervix carcinoma). The results are linked to a data set of high-performance liquid chromatographic fingerprint profiles of the samples using multivariate calibration techniques. The regression coefficients of the multivariate model are then evaluated to indicate those peaks potentially responsible for the cytotoxic activity of the Mallotus extracts. In a final step, the cytotoxic extracts are analyzed by HPLC-MS and the indicated peaks identified.

Optimization of a reversed-phase-high-performance thin-layer chromatography method for the separation of isoniazid, ethambutol, rifampicin and pyrazinamide in fixed-dose combination antituberculosis tablets.

This paper presents the development of a new RP-HPTLC method for the separation of pyrazinamide, isoniazid, rifampicin and ethambutol in a four fixed-dose combination (4 FDC) tablet formulation. It is a single method with two steps in which after plate development pyrazinamide, isoniazid and rifampicin are detected at an UV wavelength of 280 nm. Then ethambutol is derivatized and detected at a VIS wavelength of 450 nm. Methanol, ethanol and propan-1-ol were evaluated modifiers to form alcohol-water mobile phases. Systematic optimization of the composition of each alcohol in the mobile phase was carried out using the window diagramming concept to obtain the best separation. Examination of the Rf distribution of the separated compounds showed that separation of the compounds with the mobile phase containing ethanol at the optimal fraction was almost situated within the optimal Rf-values region of 0.20-0.80. Therefore, ethanol was selected as organic modifier and the optimal mobile phase composition was found to be ethanol, water, glacial acetic acid (>99% acetic acid) and 37% ammonia solution (70/30/5/1, v/v/v/v). The method is new, quick and cheap compared to the actual method in the International Pharmacopoeia for the assay of the 4 FDC tablets, which involves the use of two separate HPLC methods.

Potentially antioxidant compounds indicated from Mallotus and Phyllanthus species fingerprints.

The genera of Mallotus and Phyllanthus contain several species that are commonly used as traditional medicines in oriental countries. Some species show interesting pharmaceutical activities, such as an antioxidant activity. To produce clinically useful medicines or food supplements (nutraceuticals) from these herbs, the species should be identified and a thorough quality control should be implemented. Nowadays, the integration of chromatographic and chemometric approaches allows a high-throughput identification and activity prediction of medicinal plants. In this study, Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA) were applied and compared to distinguish Mallotus and Phyllanthus species. Moreover, peaks from their chromatographic fingerprints, which were responsible for their antioxidant activity were assigned. For the latter purpose, the relevant information was extracted from the chromatographic fingerprints using linear multivariate calibration techniques, i.e., Partial Least Squares (PLS) and Orthogonal Projections to Latent Structures (O-PLS). Results reveal that exploratory analysis using PCA shows somewhat diverging clustering tendencies between Mallotus and Phyllanthus samples than HCA. However, both approaches mainly confirm each other. Concerning the multivariate calibration techniques, both PLS and O-PLS models demonstrate good predictive abilities. By comparing the regression coefficients of the models with the chromatographic fingerprints, the peaks that are potentially responsible for the antioxidant activity of the extracts could be confirmed.

HPTLC methods to assay active ingredients in pharmaceutical formulations: a review of the method development and validation steps.

High-performance thin-layer chromatography (HPTLC) is still increasingly finding its way in pharmaceutical analysis in some parts of the world. With the advancements in the stationary phases and the introduction of densitometers as detection equipment, the technique achieves for given applications a precision and trueness comparable to high-performance liquid chromatography (HPLC). In this review, the literature is surveyed for developed and validated HPTLC methods to assay active ingredients in pharmaceutical formulations published in the period 2005-2011. Procedures and approaches for method development, validation and quantitative assays are compared with the standard ways of conducting them. Applications of HPTLC in some other areas are also briefly highlighted.

Potential antioxidant compounds in Mallotus species fingerprints. Part II: fingerprint alignment, data analysis and peak identification.

Some Mallotus species are commonly used as traditional medicine (TM) ingredients in Vietnam and China, but only a few are studied for their activities. In Part I, high-performance liquid chromatography (HPLC) fingerprints of 39 Mallotus samples (17 species) were developed and, because of the complexity of and the large differences between the samples, it was chosen to analyse the unaligned fingerprints. The peaks, potentially responsible for the antioxidant activity in given Mallotus species, were indicated by the regression coefficients from an orthogonal projections to latent structures (O-PLS) model. In the present study, an in depth discussion on the need for alignment of the Mallotus fingerprints for the indication of the potentially active compounds is made, as well as an experimental analysis and identification of the previously indicated peaks by HPLC-mass spectrometry (HPLC-MS). Additionally, to thoroughly study and discuss the alignment problem, the modelling and prediction of the antioxidant activity of green tea samples based on HPLC fingerprints were also considered.

Classification models for neocryptolepine derivatives as inhibitors of the ß-haematin formation.

This paper describes the construction of a QSAR model to relate the structures of various derivatives of neocryptolepine to their anti-malarial activities. QSAR classification models were build using Linear Discriminant Analysis (LDA), Quadratic Discriminant Analysis (QDA), Classification and Regression Trees (CART), Partial Least Squares-Discriminant Analysis (PLS-DA), Orthogonal Projections to Latent Structures-Discriminant Analysis (OPLS-DA), and Support Vector Machines for Classification (SVM-C), using four sets of molecular descriptors as explanatory variables. Prior to classification, the molecules were divided into a training and a test set using the duplex algorithm. The different classification models were compared regarding their predictive ability, simplicity, and interpretability. Both binary and multi-class classification models were constructed. For classification into three classes, CART and One-Against-One (OAO)-SVM-C were found to be the best predictive methods, while for classification into two classes, LDA, QDA and CART were.

Quality control of Citri reticulatae pericarpium: exploratory analysis and discrimination.

Extracts of Citri reticulatae pericarpium (PCR) are commonly used in the Traditional Chinese Medicine. The quality control of PCR is currently performed by single marker analysis, which can hardly describe the complexity of such natural samples. In this study, a fingerprint methodology for PCR based on high-performance liquid chromatography (HPLC) was developed and validated. A total of 69 fingerprints of authenticated PCR samples, commercial PCR samples, mixed peel samples, and other Citrus peels were recorded. Exploratory data analysis allowed optimizing the extraction procedure and detecting mixed peel samples. Once the optimizations were performed and the method validated, discrimination between the authentic PCR samples and all other samples was performed by p-Discriminant Partial Least Squares. The established model was able to differentiate between classes with a high reliability for each sample. Furthermore, evaluation of the score and loading plots of the model indicated nobiletin, tangeretin, naringin and hesperidin as important markers for the quality control of PCR.

Pressurized capillary electrochromatography in a screening for possible antioxidant molecules in Mallotus fingerprints: challenges, potentials and prospects.

Because of its eminent high resolution potential and minimal solvent consumption, pressurized capillary electrochromatography (pCEC) may offer an interesting alternative to HPLC for screening applications that need to resolve complex samples. In this paper, its potential was assessed in a screening of plant extracts from Mallotus species to indicate compounds with possible antioxidant activities by means of a PLS model built from their pCEC fingerprints. The main aim of this research was to find out whether pCEC can have an added value for this application. To get a complete overview of the techniques potential for this application, it was also assessed whether the technique can meet the requirements in terms of precision, sensitivity and column robustness. Encountered benefits and downsides were reported. Fingerprints with satisfactory sensitivity and precision could be obtained by concentrating the sample 5-fold and using optimized rinsing procedures, respectively. From the generated pCEC fingerprints of 39 Mallotus samples and their respective DPPH radical scavenging activity test results, a three-component PLS model was being built. The model proved good predictive abilities and easily allowed the indication of possible antioxidant compounds in the fingerprints. Despite its much higher peak capacity, the performance of pCEC to fingerprint the majority of the Mallotus extracts did not surpass that of a custom HPLC method. This was also reflected in its comparable power to indicate possible antioxidant compounds in the fingerprints after modeling. Because of its low detection sensitivity and modest column robustness, the benefit of the lower solvent consumption was partly paid-off by the current need for more system maintenance, also limiting the sample throughput. For the considered screening application, pCEC may suit as a viable but no preferred alternative technique.

Dissimilar chromatographic systems to indicate and identify antioxidants from Mallotus species.

The genus of Mallotus contains several species commonly used as traditional medicines in oriental countries. A data set containing 39 Mallotus samples, differing in species, cultivation conditions, harvest season and/or part of the plant was used to develop fingerprints on two dissimilar chromatographic systems. An exploratory analysis with principal component analysis (PCA) was performed on both data sets individually. The results were also combined to obtain additional information on the unknown samples included in the data set. Furthermore, the antioxidant activity of the samples was measured and modelled as a function of the fingerprints using the orthogonal projections to latent structures (O-PLS) technique. The regression coefficients of the models were studied to indicate the peaks potentially responsible for the antioxidant activity. The indicated peaks were analyzed and identified by HPLC coupled to mass spectrometry (HPLC-MS). Because of the complexity of biological samples, it was aspired to separate co-eluting components based on the significant difference in chromatographic selectivity on the dissimilar systems and consequently obtain additional, complementary information on the contribution of the individual components to the antioxidant activity. The results illustrate the potential use of dissimilar chromatographic systems. Several initially co-eluting compounds could be separated on the dissimilar system. The corresponding regression coefficients provided complementary information on the potential antioxidant activity of the separated compounds.

Development and validation of a normal-phase HPTLC method for the simultaneous analysis of lamivudine, stavudine and nevirapine in fixed-dose combination tablets.

This paper presents the development and validation of an improved method for the simultaneous analysis of lamivudine (LVD), stavudine (STV) and nevirapine (NVP) using high-performance thin-layer chromatography (HPTLC) with densitometric detection. Separation was performed on silica gel 60F(254) plates. The mobile phase is comprised of ethylacetate, methanol, toluene and concentrated ammonia (38.7:19.4:38.7:3.2, v:v:v:v). Detection wavelength was 254 nm. The R(f) values were 0.24±0.03, 0.38±0.04 and 0.69±0.04 (n=8) for LVD, STV and NVP, respectively. An F-test indicated that calibration graphs were adequately linear at the evaluated concentration ranges. The pooled %RSD for repeatability of the percentage amount recovered for LVD, STV and NVP were found to be 0.62, 0.54, and 0.79, and the pooled %RSD for time-different intermediate precision were 1.66, 1.27 and 1.21. The percentage recoveries for the trueness were 99.2%±1.5 for LVD, 98.6%±1.5 for STV and 99.3%±1.7 for NVP (n=3). Most factors evaluated in the robustness test were found to have an insignificant effect on the selected responses at 95% confidence level. This method was successfully used to analyze fixed-dose tablets samples of LVD, STV and NVP.

Influence of putrescine, cadaverine, spermidine or spermine on the formation of N-nitrosamine in heated cured pork meat.

The influence of biogenic amines (i.e. putrescine, cadaverine, spermidine and spermine) on the N-nitrosamine formation in heated cured lean meat was studied in the presence or absence of sodium nitrite and at different meat processing temperatures. Experimental evidence was produced using gas chromatography with thermal energy analysis detection (GC-TEA). Concentration of N-nitrosamines was modelled as a function of the temperature and the nitrite concentration for two situations, i.e. presence or absence of added biogenic amines to the meat. The significance of the influence of the changing parameters was evaluated by ANOVA (Analysis of Variance). It was found that higher processing temperatures and higher added amounts of sodium nitrite increase the yields of N-nitrosodimethylamine (NDMA) and N-nitrosopiperidine (NPIP). Spermidine and putrescine amplify the formation of NDMA, but spermine and cadeverine do not influence the formation of this N-nitrosamine. Spermidine and cadeverine cause a significant increase of NPIP. Beside N-nitrosopyrrolidine (NPYR) in some rare cases, no other volatile N-nitrosamines are detected.

Exploratory analysis of chromatographic fingerprints to distinguish rhizoma Chuanxiong and rhizoma Ligustici.

Identification and quality control of products of natural origin, used for preventive and therapeutical goals, is required by regulating authorities, as the World Health Organization. This study focuses on the identification and distinction of the rhizomes from two Chinese herbs, rhizoma Chuanxiong (from Ligusticum chuanxiong Hort.) and rhizoma Ligustici (from Ligusticum jeholense Nakai et Kitag), by chromatographic fingerprints. A second goal is using the fingerprints to assay ferulic acid, as its concentration provides an additional differentiation feature. Several extraction methods were tested, to obtain the highest number of peaks in the fingerprints. The best results were found using 76:19:5 (v/v/v) methanol/water/formic acid as solvent and extracting the pulverized material on a shaking bath for 15 min. Then fingerprint optimization was done. Most information about the herbs, i.e. the highest number of peaks, was observed on a Hypersil ODS column (250 mm × 4.6 mm ID, 5 µm), 1.0% acetic acid in the mobile phase and employing within 50 min linear gradient elution from 5:95 (v/v) to 95:5 (v/v) acetonitrile/water. The final fingerprints were able to distinguish rhizoma Chuanxiong and Ligustici, based on correlation coefficients combined with exploratory data analysis. The distinction was visualized using Principal Component Analysis, Projection Pursuit and Hierarchical Clustering Analysis techniques. Quantification of ferulic acid was possible in the fingerprints of both rhizomes. The time-different intermediate precisions of the fingerprints and of the ferulic acid quantification were shown to be acceptable.

Evaluation of the influence of proline, hydroxyproline or pyrrolidine in the presence of sodium nitrite on N-nitrosamine formation when heating cured meat.

N-nitrosamines are meant to be probable or possible carcinogenic components, possibly formed out of a reaction between nitrite and N-containing substances such as amino acids and secondary amines. Nitrite is often used for processing meat products because of its colouring and antimicrobial properties. During this experimental setup, the influence of proline, hydroxyproline or pyrrolidine on N-nitrosamine formation in meat samples was evaluated. The N-nitrosamines concentrations were measured with gas chromatography-thermal energy analyzer. Only the concentrations of N-nitrosodimethylamine and N-nitrosopyrrolidine were found above the limit of detection in a number of tested experimental conditions. The concentration of these two N-nitrosamines was modelled as a function of temperature and nitrite concentration for different situations (presence or absence of added natural N-containing meat components). It could be concluded that proline and pyrrolidine promoted the formation of N-nitrosopyrrolidine. It could also be confirmed that the higher the temperature of the meat processing procedure and the higher the sodium nitrite amounts added, the higher were the yields of the respective N-nitrosamines.

Potential antioxidant compounds in Mallotus species fingerprints. Part I: Indication, using linear multivariate calibration techniques.

Some Mallotus species are used in traditional medicine in Vietnam and China. Some also show interesting activities, such as antioxidant and cytotoxic ones. Combining fingerprint technology with data-handling techniques allows indicating the peaks potentially responsible for given activities. In this study it is aspired to indicate from chromatographic fingerprints the peaks potentially responsible for the antioxidant activity of several Mallotus species. Relevant information was extracted using linear multivariate calibration techniques, both before and after alignment of the fingerprints with correlation optimized warping (COW). From the studied techniques, stepwise multiple linear regression is least recommended as it made an inadequate variable selection. Principal component regression theoretically can take largely varying variables uncorrelated to the antioxidant activity into account. However, in practice in the actual case study this problem was limited. These problems in principle do not occur using partial least squares (PLS) models. Of the tested PLS methods, orthogonal projections to latent structures was preferred because of its simplicity, reproducibility, reduced model complexity and improved interpretability of the regression coefficients, yielding a clearer view on the individual contribution of the compounds. Furthermore, reducing analysis times from 60min to 35 and 22.5min resulted in the same main compounds, indicated responsible for the antioxidant activity. Models built after alignment by COW did not result in additional information.

Development and validation of a normal-phase high-performance thin layer chromatographic method for the analysis of sulfamethoxazole and trimethoprim in co-trimoxazole tablets.

Pneumocystis carinii pneumonia (PCP) is often the ultimate mortal cause for immunocompromised individuals, such as HIV/AIDS patients. Currently, the most effective medicine for treatment and prophylaxis is co-trimoxazole, a synergistic combination of sulfamethoxazole (SMX) and trimethoprim (TMP). In order to ensure a continued availability of high quality co-trimoxazole tablets within resource-limited countries, Medicines Regulatory Authorities must perform quality control of these products. However, most pharmacopoeial methods are based on high-performance liquid chromatographic (HPLC) methods. Because of the lack of equipment, the Tanzania Food and Drugs Authority (TFDA) laboratory decided to develop and validate an alternative method of analysis based on the TLC technique with densitometric detection, for the routine quality control of co-trimoxazole tablets. SMX and TMP were separated on glass-backed silica gel 60 F(254) plates in a high-performance thin layer chromatograph (HPTLC). The mobile phase was comprised of toluene, ethylacetate and methanol (50:28.5:21.5, v:v:v). Detection wavelength was 254 nm. The R(f) values were 0.30 and 0.61 for TMP and SMX, respectively. This method was validated for linearity, precision, trueness, specificity and robustness. Cochran's criterion test indicated homoscedasticity of variances for the calibration data. The F-tests for lack-of-fit indicated that straight lines were adequate to describe the relationship between spot areas and concentrations for each compound. The percentage relative standard deviations for repeatability and time-different precisions were 0.98 and 1.32, and 0.83 and 1.64 for SMX and TMP, respectively. Percentage recovery values were 99.00%+/-1.83 and 99.66%+/-1.21 for SMX and TMP, respectively. The method was found to be robust and was then successfully applied to analyze co-trimoxazole tablet samples.

Potential antioxidant compounds in Mallotus species fingerprints. Part I: indication, using linear multivariate calibration techniques.

Some Mallotus species are used in traditional medicine in Vietnam and China. Some also show interesting activities, such as antioxidant and cytotoxic ones. Combining fingerprint technology with data-handling techniques allows indicating the peaks potentially responsible for given activities. In this study it is aspired to indicate from chromatographic fingerprints the peaks potentially responsible for the antioxidant activity of several Mallotus species. Relevant information was extracted using linear multivariate calibration techniques, both before and after alignment of the fingerprints with correlation optimized warping (COW). From the studied techniques, Stepwise Multiple Linear Regression is least recommended as it made an inadequate variable selection. Principal Component Regression theoretically can take largely varying variables uncorrelated to the antioxidant activity into account. However, in practice in the actual case study this problem was limited. These problems in principle do not occur using Partial Least Squares (PLS) models. Of the tested PLS methods, Orthogonal Projections to Latent Structures was preferred because of its simplicity, reproducibility, reduced model complexity and improved interpretability of the regression coefficients, yielding a clearer view on the individual contribution of the compounds. Furthermore, reducing analysis times from 60 min to 35 and 22.5 min resulted in the same main compounds, indicated responsible for the antioxidant activity. Models built after alignment by COW did not result in additional information.

Development of HPLC fingerprints for Mallotus species extracts and evaluation of the peaks responsible for their antioxidant activity.

Some Mallotus species are used in traditional medicine in Vietnam. To use certain species in Western medicines or as food supplements, they should be identified and quality control should be more strict, for instance, to avoid the erroneous switching of species. In species with interesting activities, the compounds responsible for them should be identified. For these identifications, HPLC fingerprint methodology can be used. In this paper, HPLC fingerprints of different lengths were developed for a number of Mallotus species. Secondly, a multivariate regression model was constructed to model the antioxidant activity of the Mallotus samples from the HPLC fingerprints with the aim to indicate peaks possibly responsible for this activity. For this purpose, after data pretreatment, the calibration technique partial least squares (PLS) was applied.

Critical analysis of the SCCNFP/SCCP safety assessment of cosmetic ingredients (2000-2006).

Over the years the Scientific Committee on Cosmetic products and Non-Food Products intended for consumers (SCCNFP) and its successor, the Scientific Committee on Consumer Products (SCCP) have assessed a substantial number of cosmetic ingredients, either present on the annexes of the cosmetic products directive or raising some concern for human health. The study of 185 SCC(NF)P opinions issued between 2000 and 2006, presented here, reveals that hair dyes and hair dye components clearly dominated the scene and that the percentage of positive opinions distinctly increased between 2003 and 2006. In order to explain the shift observed over time, the general content of the submissions as well as the perceived quality of the individual studies were related to the positive/negative outcomes of the SCC(NF)P opinions. Problem areas included identification of the compound under study and its physicochemistry, dermal absorption, mutagenicity/genotoxicity testing and exposure assessment. These studies, which form part of a standard ingredient dossier, were available, but sometimes of insufficient scientific quality as judged by the SCC(NF)P. To complete the general analysis some considerations from industry and the SCCP secretariat are mentioned.