Process impurities quantitation

If reference materials of the suspected impurities are available, the drug substance or finished drug product should be spiked at an appropriate level to demonstrate that the result is unaffected by the addition of the impurity. Figure 2 shows examples of individual chromatograms of the API and three known process impurities. As shown here, none of the three process impurities interfere with the API peak, although peaks for impurities A and C appear to overlap and could co-elute if both were present in the sample. This specificity may be acceptable if the method was designated as an assay method for the quantitation of the API. For a method intended to quantitate process impurities, the overlap of these two components would in most cases be unacceptable due to the inability of the method to accurately measure the two individual components. 

TLC methods can also be used for the determination of in-process impurities in drug substances. TLC methods are only semiquantitative when visual comparison of the size and intensity of spots is performed. Quantitative measurements are possible by means of densitometry or fluorescence measurements. The spots can also be carefully removed and dissolved in a suitable solvent for spectrophotometric measurement. 

Whereas individual components to be quantitated may be isolated to meet the 1.5% criteria outlined and subsequently quantitated, the more common approach is the use of separation procedures, coupled to suitable detection, to separate and monitor the individual analytes of interest. This is a particularly powerful approach because several of the requisite quantitative measurements can be made from a single analysis of the sample. Using optimized separation procedures, it is possible to monitor the API (for assay), organic synthetic process impurities, and degradation products during a single determination. Similarly, multiple residual solvents may be quantitated in a single analysis. 

Chromatographic analysis of folate compounds including methotrexate and other antifolates has been reviewed by McCormack and Newman (32). Process impurities in the reduced folate compound leucovorin calcium have been monitored using a TLC method with fluorescence detection (33). An overpressure layer HPTLC procedure has been used to improve the separation of folic acid from other water-soluble vitamins with good recovery and resolution. The method uses silica gel layers developed in butan-l-ol-pyridine-water (50 35 15) at a rate of 0.25 ml/min for baseline separation. Quantitation was achieved without derivatization (11). 

Lipopolysaccharides (LPS) or endotoxin may be introduced as the result of low level bacterial contamination of the cell culture or during the fill of the final drug product, and can be assessed by the Limulus amebocyte lysate (LAL) assay. Similarly, antifoaming agents that are introduced during cell culture are removed during purification, but verification of removal through quantitative HPLC assays is required. Lastly, purification process impurities such as debris from resin or... 

Contaminant by-products depend upon process routes to the product, so maximum impurity specifications may vary, eg, for CHA produced by aniline hydrogenation versus that made by cyclohexanol amination. Capillary column chromatography has improved resolution and quantitation of contaminants beyond the more fliUy described packed column methods (61) used historically to define specification standards. Wet chemical titrimetry for water by Kad Eisher or amine number by acid titration have changed Httle except for thein automation. Colorimetric methods remain based on APHA standards. 

In photoluminescence one measures physical and chemical properties of materials by using photons to induce excited electronic states in the material system and analyzing the optical emission as these states relax. Typically, light is directed onto the sample for excitation, and the emitted luminescence is collected by a lens and passed through an optical spectrometer onto a photodetector. The spectral distribution and time dependence of the emission are related to electronic transition probabilities within the sample, and can be used to provide qualitative and, sometimes, quantitative information about chemical composition, structure (bonding, disorder, interfaces, quantum wells), impurities, kinetic processes, and energy transfer. 

Process validation should be extended to those steps determined to be critical to the quality and purity of the enantiopure drug. Establishing impurity profiles is an important aspect of process validation. One should consider chemical purity, enantiomeric excess by quantitative assays for impurity profiles, physical characteristics such as particle size, polymorphic forms, moisture and solvent content, and homogeneity. In principle, the SMB process validation should provide conclusive evidence that the levels of contaminants (chemical impurities, enantioenrichment of unwanted enantiomer) is reduced as processing proceeds during the purification process. 

In a modern industrialised society the analytical chemist has a very important role to play. Thus most manufacturing industries rely upon both qualitative and quantitative chemical analysis to ensure that the raw materials used meet certain specifications, and also to check the quality of the final product. The examination of raw materials is carried out to ensure that there are no unusual substances present which might be deleterious to the manufacturing process or appear as a harmful impurity in the final product. Further, since the value of the raw material may be governed by the amount of the required ingredient which it contains, a quantitative analysis is performed to establish the proportion of the essential component this procedure is often referred to as assaying. The final manufactured product is subject to quality control to ensure that its essential components are present within a pre-determined range of composition, whilst impurities do not exceed certain specified limits. The semiconductor industry is an example of an industry whose very existence is dependent upon very accurate determination of substances present in extremely minute quantities. 

However, compared with the traditional analytical methods, the adoption of chromatographic methods represented a signihcant improvement in pharmaceutical analysis. This was because chromatographic methods had the advantages of method specihcity, the ability to separate and detect low-level impurities. Specihcity is especially important for methods intended for early-phase drug development when the chemical and physical properties of the active pharmaceutical ingredient (API) are not fully understood and the synthetic processes are not fully developed. Therefore the assurance of safety in clinical trials of an API relies heavily on the ability of analytical methods to detect and quantitate unknown impurities that may pose safety concerns. This task was not easily performed or simply could not be carried out by classic wet chemistry methods. Therefore, slowly, HPLC and GC established their places as the mainstream analytical methods in pharmaceutical analysis. 

The validation process begun in Phase I is extended during Phase II. In this phase, selectivity is investigated using various batches of drugs, available impurities, excipients, and samples from stability studies. Accuracy should be determined using at least three levels of concentration, and the intermediate precision and the quantitation limit should be tested. For quality assurance evaluation of the analysis results, control charts can be used, such as the Shewart-charts, the R-charts, or the Cusum-charts. In this phase, the analytical method is refined for routine use. 

---