Degradation products quantification

The quantification of kinins in human tissues or body fluids has been limited due to the inherent difficulties in accurately measuring the concentration of ephemeral peptides. Today HPLC-based and RIA/capture-ELA measurements are established to determine kinins in human plasma, liquor or mine. Serine protease inhibitors need to be added to prevent rapid degradation of the kinins in vitro during sample preparation. Kinins and their degradation products have been studied in various biological milieus such as plasma/ serum, urine, joint fluids, kidney, lung and skeletal muscle [2]. Under normal conditions, the concentration of kinins in these compartments is extremely low for... 

Methods for Determining Parent Compounds and Degradation Products in Environmental Media. Analytical methods with the required sensitivity and accuracy are available for quantification of americium, both total and isotopic, in environmental matrices (see Table 7-2). Knowledge of the levels of americium in various environmental media, along with the appropriate modeling (see Chapters 3 and 5), can be used to evaluate potential human exposures through inhalation and ingestion pathways. 

Detection, identification and quantification of these compounds in aqueous solutions, even in the form of matrix-free standards, present the analyst with considerable challenges. Even today, the standardised analysis of surfactants is not performed by substance-specific methods, but by sum parameter analysis on spectrophotometric and titrimetric bases. These substance-class-specific determination methods are not only very insensitive, but also very unspecific and therefore can be influenced by interference from other compounds of similar structure. Additionally, these determination methods also often fail to provide information regarding primary degradation products, including those with only marginal modifications in the molecule, and strongly modified metabolites. 

In order to study simultaneously the behaviour of parent priority surfactants and their degradation products, it is essential to have accurate and sensitive analytical methods that enable the determination of the low concentrations generally occurring in the aquatic environment. As a result of an exhaustive review of the analytical methods used for the quantification within the framework of the three-year research project Priority surfactants and their toxic metabolites in wastewater effluents An integrated study (PRISTINE), it is concluded that the most appropriate procedure for this purpose is high-performance (HP) LC in reversed phase (RP), associated with preliminary techniques of concentration and purification by solid phase extraction (SPE). However, the complex mixtures of metabolites and a lack of reference standards currently limit the applicability of HPLC with UV- or fluorescence (FL-) detection methods. 

Impurity testing is pivotal in pharmaceutical development for establishing drug safety and quality. In this chapter, an overview of impnrity evaluations of drug substances and products by HPLC is presented from both the laboratory and regulatory standpoints. Concepts from the development of impurity profiles to the final establishment of public specifications are described. Useful strategies in the identification and quantification of impurities and degradation products are summarized with practical examples to illustrate impurity method development. 

Arnold, S.M., Hickey. W.J.. and Harris. R.F. Degradation of atrazine by Fenton s reagent condition optimization and product quantification. Environ. Sci. Technol, 29(8) 2083-2089,1995. 

The authors determined specificity using the known hydrolytic degradation products. The precision of spiked samples of these degradation products were determined and found to be acceptable (99.9 0.4%). Accuracy of the method was determined using spiked recoveries of piroxicam benzoate, and the recoveries were acceptable (99.1-100.5%). Assay precision n = 6, RSD = 0.4%) was in accord with recommended criteria [7]. Within-day precision was performed on two instruments on two separate days, and the overall intermediate precision was 1.0%. The method was linear over the expected analyte concentration range giving a regression line of 1 = 0.999. The detection (DL) and quantification levels (QL) were assessed, and the latter was determined as 0.185 pg/ml (ca. 0.04%). 

A method by Berger[12] utilized the stereoselective hydrolysis of diastereomeric peptides by leucine aminopeptidase. Z-L-Ala-D-Ala-OH was coupled to an all L-Ala peptide such as l-Ala-L-Ala-ONbz. Epimerization during coupling resulted in the formation of a small amount of L-Ala-L-Ala-L-Ala-L-Ala after deprotection, and since the peptidase has an absolute specificity for the all-L peptide, only the epimerized product was hydrolyzed. Quantification of the degradation products gave the extent of epimerization. These classical procedures, however, are specific to the particular coupling reaction under consideration and the results may not be fully applicable to all systems. Furthermore, they give no direct information about the rate of racemization. 

Quantitation limit is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. The quantification limit is a parameter of quantitative assays for low levels of compounds in sample matrices and is used particularly for the determination of impurities and/or degradation products. 

Quantification of the degradation products of raw-milk proteins by HPLC may furnish information on the shelf life of subsequently prepared UHT milk. Based on two earlier methods, Mottar et al. (127) applied HPLC to determine the specific proteolytic components that provide information concerning the presence and activity of gram-negative psychrotrophic bacteria. 

Lamouroux, C., Aychet, N., Lelievre, A., Jankowski, C.K., Moulin, C. 2004. High-performance liquid chromatography with electrospray ionization mass spectrometry and diode array detection in the identification and quantification of the degradation products of calix[4]arene crown-6 under radiolysis. Rapid Commun. Mass Spectrom. 18 1493-1503. 

For the quantification of the identified compounds, genuine reference compounds are needed, which are often not available for degradation products and metabolites. 

The most commonly used waste water treatment procedures are not sufficient to remove these dyes. Considerable efforts are therefore made to improve the efficiency of the physical, chemical and biological treatment procedures. However, in order to achieve this aim, reliable analytical methods for the identification and quantification of the azo dyes and their by- and degradation products are required. Since most of the dyes are non-volatile and thermally labile, LC-NMR and LC-MS are the methods of choice. 

The assessment of degradation in pharmaceutical products involves two aspects of analytical measurement. First, a selective analytical method must be available for accurate assay of the parent drug compound, in order to correctly measure any loss. Second, methodology should be in place for quantification of the degradation products formed. Ideally, when degradation occurs, the measured amount of parent drug lost should correlate well with the measured increase in degradation products. This correlation is referred to as mass balance )- More recently, the International Conference on Harmonization (ICH) has provided a definition of mass balance material balance as follows ... 

Inaccurate quantification of degradation product(s) due to differences in response factors. See Section VI. 

Extraction and Quantification of the Residue. When gas chro-matography is used as the method of quantification, extraction provides the most information of any of the residue analysis methods since both qualitative and quantitative information can be obtained. In this way any changes in ratios or the presence of degradation products in the residue can also be detected. 

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