Purity tests for

In the above approaches to choosing a TLC system, silica gel has been the sorbent of choice. This is due to the greater separation potential that silica gel has over reversed-phase sorbents. For purity testing, it is advisable to use both silica gel and reversed-phase sorbents. 

Egg phosphatidylcholine (PC), phosphatidic acid (PA), phos-phatidylglycerol (PG), phosphatidylserine (PS), DSPC, and Choi (for lipid sources and storage see Subheading 2.1 (1-3, and for purity testing, see Note 2). 

Spectroscopic methods for purity tests always remain secondary to traditional purity tests in terms of the inseparability by any separation technique (Schummer, 1997) (i) there is... 

This is usually a substance obtained from a supplier, but may be a substance produced by the user for purity testing, which is not a PS or a PRS and does not require information about its concentration. Such substances are in most cases decomposition products or intermediates in the synthesis process, and can be linked to a particular active substance. 

Dwell times for plates in a vaporization chamber can range from a few minutes for detection reactions to ca. 20 h for purity tests. They should be individually determined for each task and specified in a testing procedme. 

In the two TLC systems mentioned above any aglycons present lie at the solvent front. For this reason an additional chromatographic system has been developed for purity testing and stability investigations. This employs prewashed and activated HPTLC plates. 

In an earlier study, Balayiannis et al. [ 130] developed a capillary zone electrophoresis method for the analysis of a series of novel synthetic dideoxynucleoside analogs with potential anti-HIV activity. The method was readily applied for purity testing as well as to resolve cis and trans diastereomers. The purity and separation of diastereomers of such analogs are of great importance for further testing of their biological and pharmacological activity. 

For chromatographing protected, larger peptides (up to 10 amino acid units), Gttttmann [143] recommends silica gel or Alox layers and dimethyKormamide or acetic acid containing 5—10% water as solvents. Detection is with the chlorine-iodine reaction and jB/-values lie between 0.3 and 0.9. These are said to be not very reproducible but the method has been used successfully for purity testing. Schellenberg [144] recommends the solvents chloroform-acetone (90 + 10) and (80 + 20) cyclohexane-ethyl acetate (50 + 50) and chloroform-methanol (90 + 10). 

The simplest method for semiquantitative analysis by TLC is to develop a definite sample aliquot simultaneously with standards containing known weights of analyte. After detection, the weight of analyte in the sample is estimated by visual comparison of the size and intensity of the standard and sample zones. This method has accuracy and reproducibility in the 10-30% range, which is often adequate for the purpose intended. Visual compari,son works best if amounts near the detection limit are applied and if the sample is closely bracketed by the standards. Visual estimation is specified in different pharmacopoeias for purity testing of both drug active raw materials and formulated products (47). 

Developing a new method for purity test or assay-procedure it is necessary to prove its long-term suitability for its intended purpose, i.e. it should be validated. Standardization of validation procedure can be based on the guideline (81) of the International Conference on Harmonisation (ICH), where the different validation characteristics are clearly defined. Making a validation plan for the practice, one should take into account these definitions and the special features and error sources of the method tested. 

Due to the useful role of TLC when cost effectiveness is essential, TLC is widely used as a standard technique for rapid and accurate identification of the raw materials or finished products as well as for purity testing of raw materials and formulations in various pharmacopoeial prescriptions. 

In most cases, a different TLC system is used for identification of the substance than for purity testing. The reason is simple in the first case the main aim of the analysis is to differentiate between compounds belonging to the same type of compounds (group-type separation, separating compounds with similar but not the same structure), while in the second instance (purity testing) structurally closely related compounds can be separated. 

The official methods (pharmacopoeial methods) in this respect are different from the methods used in the industrial analysis. For example, in the United States Pharmacopoeia, HPLC is the primary method and TLC the second choice. Application of the methods used for purity testing in the British Pharmacopoeia is different TLC is the primary method, and HPLC is the second choice. But, both Pharmacopoeias are in agreement that conventional TLC is used without instrumentation. In the industrial pharmaceutical analysis, HPLC and instrumental TLC are more or less used equally and in many cases in conjunction. 

TLC is used for identification and purity testing of most of the steroid raw materials officially recommended by the various pharmacopoeias. In this field HPLC cannot compete with TLC. Most of the steroids used in formulations prescribed by recent editions of the USP are identified by TLC, but the purity of the products is usually either not controlled, or is tested by other methods. The approach of BP88 (also BP93) is different the purity of the formulated products is also checked by TLC. It is interesting that in several USP monographs, TLC alone is used for purity testing of the steroid raw materials. 

With only few exceptions, USP suggests the application of the same methods for identiHcation and purity testing of steroid raw materials. Again the approach of BP is different in nearly 50% of the applications different TLC methods are used for identification and purity testing. According to BP, the main aim of official pharmacopoeial TLC methods for the identification of steroid raw materials is to differentiate between various members of a group of steroids methods used for purity testing are developed for separation and semiquantitative estimation of related steroids, i.e., intermediates, byproducts, and decomposition products of a particular steroid. 

It is also worth mentioning that USP introduced a new principle for purity testing, namely a test for ordinary impurities, in which the same chromatographic system, i.e. gel with toluene-isopro-panol (9 1, v/v) is used for each steroid raw material. This chromatographic system serves for the evaluation of the impurity profile of the steroid investigated, and is not used for other analytical purposes nor for purity testing of formulated steroid products. 

The dimensions of the columns are selected in relation to the goal of the method development and the number of the relevant peaks that are to be expected. When the goal is a method purely for assay purposes, with a maximum sample throughput, one installs columns of length 50 mm into the thermostats. For purity testing and the goal of optimum resolution of aU peaks, then columns of length 250 mm should be used. If, however, one expects only a small number of peaks [n 7), it is recommended that the shorter columns are used. 

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