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A Strategy of Plasma Protein Quantitation by Selective Reaction Monitoring of an Intact Protein.
- Source :
-
Analytical Chemistry . 12/15/2003, Vol. 75 Issue 24, p7008-7014. 8p. - Publication Year :
- 2003
-
Abstract
- Immunoassays are used extensively in the quantitative analysis of proteins in plasma, urine, and other biological matrixes to support preclinical and clinical studies. Although immunoassays are both sensitive and rapid, difficulties during development of these assays are compounded by the need to have a specific antibody or antigen to the protein of interest. Furthermore, calibration curves of immunoassays are inherently nonlinear, and the technique often detects many structurally related components in addition to the analyte of interest. We have developed a novel strategy of analyzing protein concentrations in plasma by utilizing 96-well solid-phase extraction and LC-MS/MS detection of the intact protein. This strategy has been successfully applied in method development and assay validation of quantitatively analyzing protein rK5 concentrations in monkey plasma samples. Additional techniques such as precolumn regeneration and column heating were also incorporated into the assay. Total run time for each sample was ∼15 min. An LLOQ of 99.2 ng/ mL from a sample volume of 50 gaL, corresponding to only 380 fmol (3.97 ng) of the rK5 analyte being injected onto the analytical column (assuming 100% extraction recovery), was obtained. The validated linear dynamic range was between 99.2 and 52 920.0 ng/mL, with a correlalion coefficient (r[SUP]2) ranging from 0.9972 and 0.9994. The intraassay CV for this assay was between 0.6 and 3.8%, and the interassay CV was between 1.7 and 3.2%. Inter-assay mean accuracies were between 101.5 and 104.7%. The assay has proven rugged and specific and has been employed to generate data in support of preclinical studies. This strategy for rK5 assay could be used for the development of bioanalytical assays to provide preclinical and clinical support for other protein drug candidates and, furthermore, for the validation of biomarkers discovered from proteomic research. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 00032700
- Volume :
- 75
- Issue :
- 24
- Database :
- Academic Search Index
- Journal :
- Analytical Chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 12101562
- Full Text :
- https://doi.org/10.1021/ac034930n