High sensitivity assays

Dean et al. [93] used a high performance liquid chromatographic method for the simultaneous determination of primaquine and carboxyprimaquine in plasma with electrochemical detection. After the addition of the internal standard, plasma was deproteinized by the addition of acetonitrile. Nitrogen-dried supernatants, resuspended in mobile phase were analyzed on a C8 reversed-phase column. Limits of detection for primaquine and carboxyprimaquine were 2 and 5 ng/mL with quantitation limits of 5 and 20 ng/mL, respectively. The assay sensitivity and specificity are sufficient to permit quantitation of the drug in plasma for pharmacokinetics following low dose (30 mg, base) oral administration of primaquine, typically used in the treatment of malaria and P. carinii pneumonia. 

From Analytical Chemists, a partial answer to this problem was the development and validation of new methods that permit an improvement in terms of productivity ( high-throughput ), sensitivity and selectivity, especially using very recent hyphenated analytical assays, such as HPLC-MS/MS, GC-MS/MS or further complex couplings, that can provide more complete information in a single analysis. 

For the determination of these compounds a binding inhibition immunoassay, consisting of the competitive immunoreaction of the unbound antibody present in an analyte-antibody mixture with the hapten derivative immobilized at the sensor surface, has been applied. With the aim of assuring the regeneration and reusability of the surface without denaturation of the immobilized molecule, the formation of an alkanethiol monolayer was carried out to provide covalent attachment of the ligand to the functionalized carbodiimide surface in a highly controlled way. For DDT, the assay sensitivity was evaluated in the 0.004 - 3545 pg/l range of pesticide concentration by the determination of the limit of detection 0.3 pg/1 and the I50 value 4.2 pg/1. 

Correlation Between Total Phenols Expressed as Percent Caffeic Acid Equivalents and Ozone Injury. Twelve cultivars of peanuts ranging from low to high ozone sensitivity were examined. Foliage of each of the cultivars was evaluated for content of total phenols before and after ozone treatment. Because of the relatively high concentrations of caffeic acid in peanut foliage, values are expressed as percent caffeic equivalents in the total phenol assay. Experimental results used in establishing the correlation between caffeic acid and ozone injury are shown in Table I. 

Compared with current LC-MS/MS methods, this approach increased assay sensitivity greatly (LOQ at low-pg/mL in plasma). The developed methods are also superior to some highly sensitive RIA methods, in that it offers significantly better selectivity and quantitative accuracy. 

The validity of any statement about the purity of a protein is directly linked to the quality of the analytical method used. The validation of immunoassay systems to detect protein impurities in rDNA pharmaceuticals must be achieved by careful production and characterization of the assay reagents. The studies presented here demonstrate that the blank run approach is reasonable for the isolation of reference materials and that high quality broad spectrum antisera can be produced to these mixtures. Significant improvements in assay sensitivity approaching the ppb level are attainable and should provide the methods to further improve product purity. 

Seissler J, Boms S, Wohlrab U, Morgenthaler NG, Mothes T, Boehm BO, et al. Antibodies to human recombinant tissue transglutaminase measured by radioligand assay Evidence for high diagnostic sensitivity for celiac disease. Horm Metab Res 1999 31 375-379. 

Analogous to the majority of dihydropyridines, nifedipine s, nitrendipine s, and nimodipine s chemical structures - a 2- or 3-nitrophenyl substituent in the 4-position combined with the dihydropyridine diester structure - results in a high response in electron capture detection (ECD), thus allowing high detection sensitivity and sufficient assay specificity towards endogenous compounds, metabolites or common co-medications (Muck et al. 1994). 

Luminescence detection has the advantage of very low background compared with fluorescent technologies, so assay sensitivity can be extremely high. 

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