Quantitative assays

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. 

Many pitfalls await the unwary. Here is a short list, compiled from more detailed considerations by Bunnett.8 One should properly identify the reactants. In particular, does each retain its integrity in the reaction medium A spectroscopic measurement may answer this. The identities of the products cannot be assumed, and both a qualitative identification and a quantitative assay are in order. Pure materials are a must—reagents, salts, buffers, and solvent must be of top quality. Careful purification is always worth one s time, since much more is lost if all the work needs repeating. The avoidance of trace impurities is not always easy. If data are irreproducible, this possibility must be considered. Reactions run in the absence of oxygen (air) may be in order, even if the reactants and products are air-stable. Doing a duplicate experiment, using a spent reaction solution from the first run as the reaction medium, may tell whether the products have an effect or if some trace impurity that altered the rate has been expended. 

Metabolic disorders of urea biosynthesis, while extremely rare, illustrate four important principles (1) Defects in any of several enzymes of a metabolic pathway enzyme can result in similar clinical signs and symptoms. (2) The identification of intermediates and of ancillary products that accumulate prior to a metabolic block provides insight into the reaction that is impaired. (3) Precise diagnosis requires quantitative assay of the activity of the enzyme thought to be defective. (4) Rational therapy must be based on an understanding of the underlying biochemical reactions in normal and impaired individuals. 

Several other methods have been demonstrated for determining the efficiency/. The most direct of these depends on a quantitative assay of the polymer for initiator fragments, which may then be compared with the amount of initiator decomposed. Evans, in the work to which previous reference was made on the polymerization of styrene by hydrogen peroxide and ferrous ions in aqueous solution, showed not only that each polymer molecule thus formed contained two hydroxyl groups, but also that the number of moles of hydroxyl groups found in the polymer was very nearly equivalent to the moles of peroxide decomposed. Since... 

The accuracy of any quantitative assay depends on the use of standards that have been thoroughly characterized by accepted and independent methods. Without careful preparation of standards, the reported values for samples will be systematically higher or lower than the true value. Chiron has devoted considerable effort to the development of gold standard preparations of RNA from HIV-1 and HCV and DNA from HBV for use in the bDNA assays. These standards have been made available to the U.S. Food and Drug Administration and the World Health Organization. 

Xue et al. [30] prepared a vaginal suppository formulation containing miconazole nitrate and determined its content by P-matrix ultraviolet spectrophotometry. The production of the suppository was finished with melting by the excipient of glyceryl esters fatty acid of artificial synthesis. Quantitative assay was conducted with a P-matrix ultraviolet spectrophotometer. The suppository was smooth and met the clinical requirement of vaginal disease treatment. The method of assay was accurate. 

It is well known that 1,10-phenanthrolines are highly active ironchelating agents. The parent compound itself has recently been shown to increase HIF-la levels in ocular tissue and to suppress 02-mediated epithelial cell proliferation when administered to mice [29]. A quantitative assay was developed to measure transcriptional potency of certain HIF stabilizers via an HRE-mediated (3-lactamase production in which the EC50 of 1,10-phenanthroline was measured to be approximately 8 pM. In addition, VEGF was dose-dependently produced in mouse embryonic fibroblasts by 1,10-phenanthroline with an EC50 of... 

In each of the aforementioned studies, qualitative IR spectroscopy was used. It is important to realize that IR is also quantitative in nature, and several quantitative IR assays for polymorphism have appeared in the literature. Sulfamethoxazole [35] exists in at least two polymorphic forms, which have been fully characterized. Distinctly different diffuse reflectance mid-IR spectra exist, permitting quantitation of one form within the other. When working with the diffuse reflectance IR technique, two critical factors must be kept in mind when developing a quantitative assay (1) the production of homogeneous calibration and validation samples, and (2) consistent particle size for all components, including subsequent samples for analysis. During the assay development for... 

This chapter outlines the chemical principles for luminescent detection of target DNA in hybridization and quantitative assays that utilize the above-mentioned chemiluminogenic and bioluminogenic reagents. 

PCR is a technique for in vitro amplification of DNA sequences that involves repeated cycles of denaturation, oligonucleotide annealing, and DNA polymerase extension [29], The amplified products following PCR cycles contain double-stranded DNA fragments of discrete length. These DNAs are copies of the template DNA that are bounded at the 5 -terminus by the oligonucleotide primer for the sequence extension with a heat-resistant DNA polymerase. In quantitative assays of PCR products, therefore, nonspecific products interfere with the assay. 

It is extremely important that the interaction of quinones with XO (Reaction (3)) is reversible that can lead to receiving erroneous results at the measurement of superoxide production by SOD-inhibitable cytochrome c reduction [28,29] (see also Chapter 27). Lusthof et al. [30] demonstrated that 2,5-bis(l-aziridinyl)-l,4-benzoquinones are directly reduced by XO. Interestingly at quinone concentrations greater than 25pmol I 1, quinones entirely suppressed one-electron reduction of dioxygen, and cytochrome c was completely reduced by the semiquinones formed. It is well known that cytochrome c and lucigenin are effective superoxide scavengers and due to that, these compounds are widely used in the quantitative assays of superoxide detection. Nonetheless, under certain experimental conditions they can be directly reduced by XO [31]. 

However, to be a quantitative assay of superoxide detection, Reaction (1) had to be an exothermic reaction, i.e., the difference between the one-electron reduction potentials of reagents AE° = / °[02 /02] / °[A /A] must be <0. In this case the rate constants of Reaction (1) will be sufficiently high (10s—109 1 mol 1 s ). Among traditionally applied assays, three compounds satisfy this condition cytochrome c, lucigenin, and tetranitromethane (Table 32.1). 

A battery of different biochemical quantitative assays was applied to many different tissues and species. DNA damage and lipid peroxidation assays measure the direct impact of genotoxics and oxidant pollutants [16,17] whereas alteration of GSH levels in liver is a marker for oxidative stress [18]. Mercury and other heavy metals are known to induce metallothionein levels in different tissues although this effect is variable in different species and organs [19-22]. 

The complexation of neomycin with anionic dyes such as amaranth (F.D. C. Red No.2) is well knownl00,102 and has been made the basis of a quantitative assay for neomycinl°2,25. These complexes are again of the cation/anion type and their formation is dependant on the ionic strength of the solution. 

After mild hydrolysis, neomycin has been shown to give a positive Elson-Morgan reaction , a reaction characteristic of amino-sugarsl52 A method involving this reaction has been made the basis of a quantitative assay for neomycinl l which has been used to determine the neomycin content of fermentation broths. ... 

Using an acrylamide-gel as support, Coombe- 0 has demonstrated the quantitative assay of neomycin with a recovery of 96% in the presence of bacitracin and polymixin. In this case quantitation was achieved by densitometry after staining the gel with naphthalene black. 

Chinese Hamster CHO/Hgprt System. Chinese hamster ovary (CHO) cells have 21 or 22 chromosomes with one intact X chromosome and a large acrocentric marker chromosome (Natarajan and Obe, 1982). The use of these cells in mammalian mutation experiments was first reported by Hsie et al. (1975), and was refined into a quantitative assay for mutagenicity testing by O Neill. The performance of this system has been reviewed by the USA EPA Gene-Tox Program. The experimental procedure for this assay is similar to the V79/Hgprt system already described, and for more detailed descriptions the reader is referred to Li et al. (1987). 

A plethora of chemical reactions that are intimately associated with the quantitative analysis essentially belong to the class of reversible reactions. These reactions under certain prevailing experimental parameters are made to proceed to completion, whereas in certain other conditions they may even attain equilibrium before completion. In the latter instance, erroneous results may creep in with regard to the pharmaceutical substance under estimation. Hence, it has become absolutely necessary first to establish the appropriate conditions whereby the reactions must move forward to attain completion so as to achieve the ultimate objective in all quantitative assays. 

Optical purity can be quantitatively assayed by HPLC after reducing a sample to the amine with triphenylphosphine. A 50-mg sample was diluted with 10 1 THF water (1 mL in a screw cap vial) and treated with triphenylphosphine (190 mg). Gas evolution begins within 5 min once this subsides the reaction is sealed and placed in an oil bath at 50°C for 30 min. The mixture is diluted with HCIO4 (pH 1.0,1 mL) and washed with dichloromethane (2 x 1 mL). The acidic water phase contains the salt of the amine. A 200-pL sample was diluted to 1 mL and assayed by HPLC using a Crownpak CR(+) column (Diacel Chemical Industries) HPLC conditions aqueous pH 1.0 HCIO4, flow 0.5 mL/min, UV detection at 210 nm. The product had an enantiomeric excess of 96%, major enantiomer, Rt 3.4 min, and minor enantiomer, Rt 5.0 min. 

The fluorimetric methods often offer improved specificity and sensitivity over colorimetric procedures and the quantitative assays for the aromatic amino acids tyrosine and phenylalanine illustrate this point. 

The most commonly used format for quantitation assays is the sandwich assay format. Typically, a monoclonal antibody (MAb) is used to capture the product. It is then detected by another antibody, usually enzyme-labeled. A reference standard is used from which to compare the response of an unknown test sample. There is a relative increase in measured response (optical density) with increasing analyte concentration. Figure 11.2 is an example of a typical ELISA standard curve. 

It has already been stated that a suitable quantitative assay technique must be available to measure the reaction of interest and it is assumed that the experimenter has determined optimal reaction conditions for the enzyme of interest. All kinetic assay techniques assume that v is a variable and that [S] is known as such, preparation of substrate must be meticulous in terms of ensuring that concentrations are correct, and this in turn will rely upon factors such as good weighing and pipetting techniques with calibrated instruments capable of precise, accurate, and sufficiently sensitive measurement. 

Engvall E, Jonsson K, Perlmann P. 1971. Enzyme-linked immunosorbent assay. II. Quantitative assay of protein antigen, immunoglobulin G, by means of enzyme-labelled antigen and antibody-coated tubes. Biochim Biophys Acta 251 427-434. 

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