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Pharmacokinetic and metabolism studies using uniformly stable isotope labeled proteins with HPLC/CRIMS detection
Author(s) -
Osborn Blaire L.,
Abramson Fred P.
Publication year - 1998
Publication title -
biopharmaceutics and drug disposition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.419
H-Index - 58
eISSN - 1099-081X
pISSN - 0142-2782
DOI - 10.1002/(sici)1099-081x(199810)19:7<439::aid-bdd124>3.0.co;2-b
Subject(s) - chromatography , high performance liquid chromatography , chemistry , analyte , pharmacokinetics , mass spectrometry , isotope , metabolism , stable isotope ratio , elution , biochemistry , pharmacology , biology , physics , quantum mechanics
We present a novel method for performing pharmacokinetic and metabolism studies on macromolecules that offers advantages over the existing techniques of radiolabeling, immunoassay or bioassays. Our strategy uses macromolecules with stable isotopes uniformly distributed throughout the structure. The stable isotope enrichment is detected using high performance liquid chromatography combined with chemical reaction interface mass spectrometry (HPLC/CRIMS). HPLC/CRIMS is a technique where analytes are first eluted from an HPLC column and then dissociated in a microwave reaction chamber. The dissociated analytes are oxidized using SO 2 and the resulting small molecules are detected by the mass spectrometer. The stable‐isotope labeled analyte is distinguished from the matrix carbon by monitoring the enrichment of 13 CO 2 . In order to demonstrate the feasibility of performing pharmacokinetic and metabolism studies using this technique, uniformly 13 C, 15 N‐labeled rat growth hormone was administered intravenously to rats and blood samples were collected. Raw plasma samples were analysed by HPLC/CRIMS. Growth hormone was detectable for 1 h following administration. The absolute amounts detected ranged from a high of 66 pmol to a low of 825 fmol in a 20 μL plasma sample. The data were modeled using PCNONLIN and were consistent with a one compartment model. The calculated half‐life was 7.7±0.7 min, with a clearance of 4.5±0.3 mL min −1 , values consistent with literature reports for growth hormone in rats. No circulating growth hormone metabolites were detected in the plasma. This paper demonstrates a novel technique for performing pharmacokinetic studies of proteins. The uniform labeling strategy also presents a viable comprehensive method for obtaining metabolism data on macromolecules. © 1998 John Wiley & Sons, Ltd.