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.

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.

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.

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.

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.

The quality of essential antimicrobial and antimalarial drugs marketed in Rwanda and Tanzania: influence of tropical storage conditions on in vitro dissolution.

OBJECTIVE: The quality of 33 formulations of essential antimicrobial and antimalarial drugs (amoxicillin capsules, metronidazole tablets, sulfamethoxazole/trimethoprim tablets, quinine tablets and sulphadoxine/pyrimethamine tablets) marketed in Rwanda and Tanzania was assessed and the influence of tropical storage conditions on potency and in vitro dissolution investigated. METHODS: Drug content and in vitro dissolution were determined immediately after purchase and during 6-month storage under simulated tropical conditions (75% relative humidity, 40 degrees C) using the methods described in the USP 24 monographs on the drugs concerned. RESULTS AND DISCUSSION: At the time of purchase, the drug content of all the formulations was within the limits recommended by the USP 24, but after 6-month storage, the drug content of one sulfamethoxazole/trimethoprim and one quinine formulation were found to be substandard. Immediately after purchase, four formulations (three sulfamethoxazole/trimethoprim and one sulphadoxine/pyrimethamine combination) failed the USP 24 dissolution test. Except for three metronidazole and one quinine formulations, dissolution tests performed after 6 months of storage under simulated tropical conditions showed that drug release remained within the USP 24 recommended values. CONCLUSION: In both countries, essential drug formulations met pharmacopoeial potency requirements, but some had a poor in vitro drug release profiles. Some of the formulations tested were not stable upon storage under simulated tropical conditions.

Drug formulations intended for the global market should be tested for stability under tropical climatic conditions.

RATIONALE OBJECTIVE: The quality of drugs imported into developing countries having a tropical climate may be adversely affected if their formulations have not been optimized for stability under these conditions. The present study investigated the influence of tropical climate conditions (class IV: 40 degrees C, 75% relative humidity) on the drug content, in vitro dissolution and oral bioavailability of different formulations of two essential drugs marketed in Tanzania: diclofenac sodium and ciprofloxacin tablets. METHODS: Before and after 3 and 6 months storage under class IV conditions the drug content and in vitro dissolution were evaluated using United States Pharmacopoeia (USP) 24 methods. Following a randomized four-period cross-over study, the pharmacokinetic parameters of drug formulations stored for 3 months under class IV conditions were compared with those stored at ambient conditions. RESULTS: Drug content and drug release from all tested ciprofloxacin formulations were within USP-24 requirements and remained stable during storage at simulated tropical conditions. Oral bioavailability was also not influenced by tropical conditions. The dissolution rate of two diclofenac formulations (Diclo 50 manufactured by Camden and Dicloflame 50 manufactured by Intas) reduced significantly during storage under class IV conditions. After oral administration Camden tablets stored for 3 months under class IV conditions showed a reduction in C(max) (90% CI of C(max) ratio: 0.59 - 0.76). This reduction was smaller than expected based on the in vitro tests. CONCLUSIONS: Some drug formulations imported into Tanzania are not optimized for stability in a tropical climate. Manufacturers and regulatory authorities should pay more attention to the WHO recommendations for testing the stability of drugs under tropical climate conditions. Efforts should be made to improve the in vitro tests to better predict the bioavailability.

Principal component analysis of dissolution data with missing elements.

The use of principal component analysis (PCA) for incomplete dissolution data sets is examined. The PC space is constructed using a reference set and the test set is projected in that space. Several cases such as a reference set with missing data, an incomplete test set and both sets measured at different time points, are discussed using two examples: one simulation and one obtained from the pharmaceutical practice. From the many possibilities to deal with missing data, the expectation-maximization algorithm in combination with PCA was chosen. The influence on the similarity or f2 factor is examined too. The sampling with replacement or bootstrap technique, which can be used to obtain confidence limits, can also be used when missing data are present in one of the data sets.