Quantification of small therapeutic proteins in plasma by liquid chromatography-tandem mass spectrometry: application to an elastase inhibitor EPI-hNE4.
LC/ESI-MS/MS is a promising alternative to immunoassays in improving the analysis of recombinant therapeutic proteins in biological fluids for toxicity and pharmacokinetics purposes. To assess the sensitivity and validation issues associated with this technique, we use here as a model EPI-hNE4, a 56-amino acid recombinant protein, and demonstrate that a method based on tandem mass spectrometry combined with liquid chromatography and electrospray interface can reach sensitivity similar to that of ELISA but without its potential cross-reactivity. For this purpose, a triple quadrupole mass spectrometer operating in positive ion and single reaction monitoring mode with transition, m/z 1040 --> 1224.5, was used for selective peak detection. Particular issues related to the internal standard, i.e., elution and ionization patterns similar to the protein without stable isotope labeling, and to analytical interference due to endogenous binding antibodies were addressed. A limit of quantification in human or monkey plasma of 5 ng/mL was reached with a sample volume of 100 microL, corresponding to 40 fmol injected into the HPLC column. Intra- and interassay precision and accuracy were below 15%. No matrix effect was detected.
CEA, Service de Pharmacologie et d'Immunologie, 91191 Gif-sur-Yvette, France. becher@cea.fr
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Becher
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Pruvost
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Clement
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Tabet
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Ezan
D000818:Animals; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D000882:Haplorhini; D006801:Humans; D007553:Isotope Labeling; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors*; D010455:Peptides / Q000494:pharmacology*; D011480:Protease Inhibitors / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000097:blood*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods; D013997:Time Factors
D010455:Peptides; D011480:Protease Inhibitors; D011994:Recombinant Proteins; C512995:depelestat; D010196:Pancreatic Elastase