Validation of TLC Methods

Validation is defined as the formal determination of the suitabUity of a given analytical method for an intended apphcation, achieving this by determining the rehabUity of results obtained [144], The following extract from the paper Vahdation of Analytical Results in Pharmaceutical Analysis by Renger et al. serves as the basis of this Chapter. Briefly, based on differences in requirements and even in definitions of terms used in the United States and Europe, experts from these coimtries and from Japan, who have been attending meetings of the International Conference for Harmonization (ICH 1-1CH 3) since 1993, have prepared a scheme of imiform requirements for the vahdation of analytical results [145]. The framework of the present book would be exceeded if all the details and commentaries were discussed here. The interested reader is therefore referred to the hterature [144]. 

The four analytical determinations most commonly performed are, in brief  

Requirements that should be fulfilled by vahdation data are hsted in Table 24. 

Type of analytical procedure characteristics Identification Testing for impurities Quantitation Limit Assay dissolution (measurement only) content/ potency 

The various concepts used in the assessment of analytical results are defined below and methods for determining them are recommended [146]. 

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If TLC is used as an analytical method in quality control, the reproducibUity, i.e. lack of scatter (precision of both system and method), and also the accuracy of the analytical results must be determined. The GMP/GLP guidelines also require vahdation of the method, i.e. testing for linearity, selectivity, robustness and limits of detection and determination (see also Section 9.1 Validation of TLC Methods ). For method development, high demands are placed on the stationary phases of the chromatographic system ... 

Practical aspects of TLC method development comprise (i) searching for a suitable developing solvent (ii) optimising the visualisation and evaluation process and (iii) method validation. Table 4.34 lists the main features of HPTLC. 

TABLE 1 Summary of TLC Method Validation for Drug Substance ... 

The aim of the analysis should be kept in mind when analytical performance parameters are selected for the validation experiments. Table 5 lists the most important analytical performance parameters used for validation of TLC and HPLC methods. As is apparent from the data presented in Table 5, the parameters used for the two chromatographic techniques are not very different. The importance of each individual parameter used in the experiments to validate TLC or HPLC methods is, however, different, especially if the purpose of the analysis is considered. This is demonstrated in Table 5, where the primary analytical parameters are indicated as a function of the analytical aims. 

When a TLC method is validated, two of the tests that are done are (1) stability on plate and (2) stability in solution of the drug substance to determine how quickly a sample must be applied to the HPTLC plate and developed before degradation occurs, if it occurs at all. For example, five vials are prepared by placing 25 mg of drug substance in each vial, and labeling them as time 0,1,2, 3, and 4 h. The experiment begins... 

If tlie identity of the detected drug residue is still unknown after these tests have been performed, a TLC-bioautography procedure is then applied to isolate and tentatively identify the residues (64, 72). This method is not quantitative and only gives direction to the analyst as to what determinative/confirmatory method of analysis should be used. Following this tentative identification, presumptive positives are then quantified and confirmed by validated physicochemical methods. A TLC method (73) is applied for analyzing presumptive sulfonamide residues, a GC-ECD method (74) followed by a GC-MS NCI method (75) for analyzing chloramphenicol, and LC/UV methods (76,77) for analyzing -lactams and tetracyclines. 

A methodological approach for an effective and reliable quality control of Chinese star anise [I. verum Hook. F.) was developed and validated by Lederer et al. (2006). A combined method of TLC and HPLC-MS/MS was used for differentiation ofvarious Ulicium species, especially Chinese and Japanese star anise. Species can be distinguished by their TLC flavonoid pattern. A sensitive and selective HPLC/ESI-MS/MS method was developed for the detection and quantification of lower admixtures of I. anisatum and of further toxic Ulicium species at a low concentration range using the sesquiterpene, lactone anisatin, as a marker. This assay includes a solid-phase extraction clean-up procedure with a high recovery (> 90%). 

Szepesi, G. Some aspects of the validation of planar chromatographic methods in pharmaceutical analysis. I general principles and practical approaches. J. Planar Chromatogr.-Mod. TLC 1993, 6, 187-197. 

In this chapter the applications of TLC in a pharmaceutical environment such as method development and validation for drug substance and drug product, and impurity isolation and characterization by TLC are discussed. 

A validated TLC method is used to evaluate drug substance or drug product for release or stability purposes. Usually there are three kinds of impurities observed from a TLC analysis TLC-specified impurities, known impurities, and unknown impurities. 

The techniques have their uses for rapid and simple monitoring of mixtures to determine the approximate relative amounts of components. Preparative TLC is often useful to purify the product of a small-scale synthesis (e.g. 0.25 mm silica gel layers and elution of peptides with a 6 3 1 mixture of EtOAc MeOH water as the mobile phase, to isolate 4-10 mg of a peptide product). Attempts to make the method more sophisticated, to give reliable quantitative information, have been largely unsuccessful. Perhaps the simplification and wide availability of HPLC techniques have suppressed interest in furthering the role of TLC for analysis of mixtures of amino acids, but improved stationary phases have contributed to better reproducibility (HPTLC), and routine TLC monitoring to validate the purity of intermediates in peptide syntheses is widely used (Barlos et al., 1993). 

The first publications on the evaluation of thin-layer chromatograms by image processing appeared as early as the mid-1980s [118], These methods, also known as digital evaluation , were presented to the scientific world in the lectures of Prosek, who developed his own software for the purpose in 1991 [119], although only the enormous developments in the hardware (video cameras, computers, printers etc.) have made video evaluation of thin-layer chromatograms economically justifiable. Prosek presented his paper on the validation of quantitative TLC by video camera in 1997 [120]. 

In the TLC analysis of dry extracts prepared from medicinal plants, the sample preparation is performed in a different way from that prescribed in the monographs for the drugs in the pharmacopoeias. Also, there is no binder in the recommended solvent system, and in these cases a validation of the new in-house method is certainly necessary. 

Because the TLC plate is used only once, intermediate precision (reproducibility of the result from plate to plate) is particularly important for methods intended for routine use. To maximize precision the method must be robust. During validation or during method development the effects of the following... 

The AO AC standardized a lot of procedures based on TLC that were validated by collaborative studies and met the AO AC requirements regarding performance criteria. The AOAC standards provide detailed information on the apparatus, the reagents, the preparation of standards, and the sampling. A selection of these methods and the most recent ones are quoted in the standard procedures section. 

For obtaining reliable analysis results, the (high-performance) thin-layer chromatographic (TLC) method should he validated before using it as a quality control tool. The validation parameters that should he evaluated are stability of the analyte, specificity/selectivity, linearity, accuracy, precision, range, detection limit, quantification limit, and robustness/ruggedness. 

The reaUstic standard deviations in TLC or HPTLC analyses are 0.2% on multiple scanning of one spot, 0.8-1.5% on multiple spotting and analysis of the same sample solution, and 1.5-2.0% on multiple analysis of the same sample. As a general mle, the standard deviation of a method should be lower than 1/6 of the specification range, " or the RSD value should not be more than 2% [10 pof validation purposes, precision is determined... 

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