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% β-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.

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.

Similarity assessment of complex chromatographic profiles of herbal medicinal products is important as a potential tool for their identification. Mathematical similarity parameters have the advantage to be more reliable than visual similarity evaluations of often subtle differences between the fingerprint profiles. In this paper, different similarity analysis (SA) parameters are applied on green-tea chromatographic fingerprint profiles in order to test their ability to identify (dis)similar tea samples. These parameters are either based on correlation or distance measurements. They are visualised in colour maps and evaluation plots. Correlation (r) and congruence (c) coefficients are shown to provide the same information about the similarity of samples. The standardised Euclidean distance (ds) reveals less information than the Euclidean distance (de), while Mahalanobis distances (dm) are unsuitable for the similarity assessment of chromatographic fingerprints. The adapted similarity score (ss*) combines the advantages of r (or c) and de. Similarity analysis based on correlation is useful if concentration differences between samples are not important, whereas SA based on distances also detects concentration differences well. The evaluation plots including statistical confidence limits for the plotted parameter are found suitable for the evaluation of new suspected samples during quality assurance. The ss* colour maps and evaluation plots are found to be the best tools (in comparison to the other studied parameters) for the distinction between deviating and genuine fingerprints. For all studied data sets it is confirmed that adequate data pre-treatment, such as aligning the chromatograms, prior to the similarity assessment, is essential. Furthermore, green-tea samples chromatographed on two dissimilar High-Performance Liquid Chromatography (HPLC) columns provided the same similarity assessment. Combining these complementary fingerprints did not improve the similarity analysis of the studied data set.

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.

This paper presents the development and validation of an improved method for the analysis of fluconazole using high-performance thinlayer chromatography (HPTLC) with densitometric detection. Separation was performed on silica gel 60 F254 plates. The mobile phase is comprised of ethyl acetate-methanol-ammonia-diaminoethane (85:10:5:0.5 v/v/v/v). Detection wavelength was 216 nm and the RF value obtained was 0.4 ± 0.03. An F-test indicated that the calibration graph was linear at the evaluated concentration range. The %RSD for repeatability and intermediate precision were 1.60 and 3.05, respectively. The mean percentage recovery for the trueness was 99.1 ± 2.1%. This method was successfully used to analyze the fluconazole content in marketed tablet samples.

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.

The development of a new drug substance is an expensive and time-consuming process. Therefore, the developers want to maximize the profit from the drug by patenting the concerned molecule as well as its synthesis pathway. In a later stage a faster or cheaper manufacturing process can be developed and patented. The aim of this study is to recognize paracetamol-containing drug formulations in relation to their synthesis pathways, in order to demonstrate the possibility to reveal fraudulently synthesized paracetamol. Since different synthesis pathways require different starting materials, solvents, reagents and catalysts and since they can produce different intermediates, it is expected that drug products originating from a different synthesis pathway will exhibit a different impurity profile. Therefore, in this study several paracetamol samples, synthesized in four different ways, are analyzed using trace-enrichment high-performance liquid chromatography (HPLC). The resulting chromatographic data were chemometrically treated in order to reveal clustering tendencies in the hope of distinguishing the different pathways. When performing principal component analysis (PCA) only 3 vaguely outlined clusters appeared. Projection pursuit (PP) was able to reveal 4 clusters and the samples with known synthesis pathway, except one, were classified in the different clusters. When hierarchical clustering and auto-associative multivariate regression trees (AAMRT) were applied, the samples of the four synthesis pathways could also be distinguished. AAMRT has an added value, since it can indicate the variables (peaks and thus also the impurities) that are responsible for the differences between the samples synthesized differently.

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.

Because of the increasing problem of drug counterfeiting and the potential danger related as well as the economic losses involved, the pharmaceutical industry and the regulatory instances are interested in the development of anti-counterfeiting and patent protection methodologies. In this paper, the evaluation of measured isotopic ratios by means of explorative chemometric techniques was performed to distinguish groups in two data sets containing samples of acetyl salicylic acid and ibuprofen, respectively. The samples in the data sets originated from different countries and manufacturers. For both compounds a clear distinction of groups of samples could be obtained. These groups could be explained based on the origin of the samples, both geographically as well as based on the manufacturer. Hypotheses were formulated concerning the synthetic pathways of the molecules and they were linked to the groups obtained with the chemometric tools.

4-aminophenol (4-AP) is the primary degradation product of paracetamol (PARA). According to the European Pharmacopoeia, 50 ppm 4-AP/PARA is the specification limit of 4-aminophenol in paracetamol drug substance. For drug products, often higher specification limits, such as 1000 ppm 4-AP/PARA are applied. This paper describes a fluorimetric method to quantify the low amount of this degradant (50 ppm) in a pharmaceutical preparation, i.e. in paracetamol tablets. The fluorimetric method was validated and the linearity, precision, trueness, range, limit of detection and limit of quantification were determined. They were found acceptable to assay the low amounts of 4-aminophenol in paracetamol tablets.

The impact of ChemoAC-a consortium of industry and the laboratory headed by Professor Massart at the Vrije Universiteit Brussel-on the advancement of Chemometrics is discussed by reviewing its 50 most important chemometric papers. Copyright (c) 2007 John Wiley & Sons, Ltd.

Herbs are used worldwide for preventive and therapeutic purposes. Therefore, identification and quality control of products of vegetal origin is required. Fingerprint chromatography is accepted by the World Health Organization as an identification and quality evaluation technique for medicinal herbs. In fingerprint development, the first step is to create general conditions maximizing the peak capacity. Four herbs, Liquorice, Cascara, Curcuma and Artichoke, were examined using different experimental conditions in order to propose a methodology to develop fingerprint chromatograms in high-performance liquid chromatography (HPLC) with ultra-violet (UV) and evaporative light scattering (ELS) detection. The methodology comprised a screening and an optimization phase. Monolithic C18 columns were used as stationary phases. Several organic modifiers were tested to define a gradient elution system with an optimal selectivity. From the screening phase, linear gradients of 5-60%, 5-54%, 5-95% and 5-95% acetonitrile for Liquorice, Cascara, Curcuma and Artichoke, respectively, were selected. The ELS detection did not provide additional information to the UV detection. The optimization phase selected the best experimental conditions for wavelength, column length, flow rate and slope of the gradient for the best modifier determined at the end of the screening. For the four examined herbs 254 nm as detection wavelength, 300 mm column length, 2 ml/min flow rate and a gradient time of 50 min were defined as the best conditions. If these latter unique conditions are confirmed for other case studies, the actual strategy even might be simplified by reducing the optimization stage.

The assay of a drug substance (DS) is one of the tests required to confirm the active pharmaceutical ingredient (API) quality at release. In the past, usually volumetric titration methods were performed, that were precise, but often non-specific. Nowadays specific chromatographic assay procedures are preferred. However, high performance liquid chromatographic (HPLC) methods, the way they are usually executed, tend to be less precise and have a larger total method variation compared to titration methods. The capabilities of fully validated titration and HPLC assay methods were determined and compared. It was studied which factors had the largest effects on the capability of chromatographic HPLC methods in order to improve their precision and precision-to-tolerance ratio. This was done using multiple Gage R&R (repeatability & reproducibility) studies and an experimental design approach. The investigations showed that it was feasible to define an HPLC method with a similar capability as the titration method. The most important factor determining the precision was demonstrated to be higher sample and reference material weights. When low weights are to be used, increasing the number of sample preparations and the number of reference solutions may enhance the method capability.