Physicochemical assays

After screening for toxicity, identification and/or quantification assays may need to be carried out if the screening method is not specific for the cyanobacterial toxin(s) under investigation. Suitable assays for these purposes include the physicochemical assays, HPLC, MS, and CE, and to some extent the immunoassays and protein phosphatase inhibition assays summarized in Section 2. 

A linear relationship should be evaluated across the range. Physicochemical assays are usually linear over a wide range of concentrations. 

More recent physicochemical assays have also confirmed that the thermal stability of penicillin G is pH-dependent (32). At 100 C, the half-life of penicillin G was given a value of 45 min in pure aqueous solutions, a value of 25 min at pH 5.5, and a value of 8 min at pH 8.2. In cooking oil heated at 180 C and 140 C, the half-life of penicillin G was estimated at 18 min and 45 min, respectively... 

For food labeling purposes, which require the actual amounts of vitamin A in the food rather than the nutritional value, data obtained by physicochemical assay are expressed on a weight basis. In plant-derived foods, the appropriate units are /3-carotene equivalents expressed in micrograms of /3-carotene. By definition, 1 /3-carotene equivalent is equal to 1 fig of all-trans-/3-carotene or 2 fig of other, largely animal-derived foods, the units are either micrograms of retinol or retinol equivalents (32). 

The manufacture of biological products, unlike that of pharmaceuticals, uses materials that present variability. The active substance is generally produced in small quantities and needs to be separated from complex mixtures containing several types of contaminants. Lots are generally small and quality control tests are usually based on biological techniques that present higher variability than physicochemical assays. The processes are also susceptible to microbial contamination. The in-process controls are of fundamental importance to detect quality deviations that cannot be assessed through assays performed only on the final products. 

This principle has been expanded to a dual electrode arrangement in which pH differences in the samples could be compensated (Durand et al., 1984). With a BuChE loading of 7.5 U/cm and under substrate saturation conditions the system was sensitive to micromolar inhibitor concentrations. The inhibition was markedly different with different pesticides. Such sensors are superior to physicochemical assays in that they detect the effectiveness of the inhibition. 

Many physicochemical assays are established to quantify the protein mass. It is determined by exploiting the extinction coefficient in optical density measurements or by colorimetric assays such as the Bradford, Lowry, bicinchoninic (BCA), and biuret assay [13, 14]. Albeit easy to perform, these colorimetric assays suffer from inaccuracies that are due to the use of inappropriate standards like bovine serum albumin. If relevant standards are not available, quantitative amino acid analysis [6], the (micro-)Kjeldahl nitrogen method [14, 15] or gravimetry as very accurate but time-consuming alternatives can be applied. 

Physicochemical assays, including spectrophotometric, fluorometric, chromatographic, enzymatic, immunological, and radiometric methods... 

Physicochemical properties requked include melting/boiling point, vapor pressure, solubiUty, and flammabiUty/explosion characteristics. The toxicological studies include acute toxicity tests, oral, inhalation, and dermal skin and eye kritation skin sensiti2ation subacute toxicity, oral, inhalation, and dermal and mutagenicity tests. In vitro reverse mutation assay (Ames test) on Salmonella typhimurium and/or E.scherichia coli and mammalian cytogenic test. In vivo mouse micronucleus test. 

The physicochemical properties of the reactants in an eiKyme-catalyzed reaction dictate the options for the assay of enzyme activity. Spectrophotometric assays exploit the abihty of a substrate or product to absorb hght. The reduced coenzymes NADH and NADPH, written as NAD(P)H, absorb hght at a wavelength of 340 run, whereas their oxidized forms NAD(P) do not (Figure 7—9). When NAD(P)+ is reduced, the absorbance at 340 run therefore increases in proportion to—and at a rate determined by—the quantity of NAD(P)H produced. Conversely, for a dehydrogenase that catalyzes the oxidation of NAD(P)H, a decrease in absorbance at 340 run will be observed. In each case, the rate of change in optical density at 340 nm will be proportionate to the quantity of enzyme present. 

Kansy, M., Senner, F., Gubemator, K. Physicochemical high throughput screening parallel artificial membrane permeation assay in the description of passive absorption processes. /. Med. Chem. 1998, 43, 1007-1010. 

As discussed above, it is important to try to optimize biological, physicochemical, and ADME properties in parallel. However, the data from all of these assays for the numerous compounds prepared by parallel synthesis make the interpretation of results challenging. The use of tools such as MVA helps in the effective utilization of all data in the optimization process. 

Although ADME assays are usually performed by analytical chemists, medicinal chemists—the molecule makers—need to have some understanding of the physicochemical processes in which the molecules participate. Peter Taylor [17] states ... 

In response to the concern expressed by the shellfish farmers operating in the Ebro River delta about the potential positive role of pesticides on the oyster and mussel mortalities observed in the area, our group, commissioned by and with the collaboration of the Catalan Water Agency (ACA), carried out a comprehensive study in which chemical and toxicity data were combined to assess potential toxic presures present in the delta. To this end, a combined approach scheme integrating the measurement of various general physicochemical parameters in water, quantitative chemical analysis of pesticides in water and biota, and ecotoxicity assays in water was applied to a series of samples collected at springtime (between mid-April and mid-June 2008) from six selected sites of the delta the two (northern and southern)... 

The next section describes the utilization of //PLC for different applications of interest in the pharmaceutical industry. The part discusses the instrumentation employed for these applications, followed by the results of detailed characterization studies. The next part focuses on particular applications, highlighting results from the high-throughput characterization of ADMET and physicochemical properties (e.g., solubility, purity, log P, drug release, etc.), separation-based assays (assay development and optimization, real-time enzyme kinetics, evaluation of substrate specificity, etc.), and sample preparation (e.g., high-throughput clean-up of complex samples prior to MS (FIA) analysis). 

---