Premium
Method development for the concentration determination of a protein in human plasma utilizing 96‐well solid‐phase extraction and liquid chromatography/tandem mass spectrometric detection
Author(s) -
Ji Qin C.,
Gage Eric M.,
Rodila Ramona,
Chang Min S.,
ElShourbagy Tawakol A.
Publication year - 2003
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.981
Subject(s) - chemistry , chromatography , analyte , extraction (chemistry) , solid phase extraction , standard curve , tandem mass spectrometry , mass spectrometry , analytical chemistry (journal) , liquid chromatography–mass spectrometry , quantitative analysis (chemistry) , tandem , detection limit , selected reaction monitoring , correlation coefficient , materials science , statistics , mathematics , composite material
Although liquid chromatography/tandem mass spectrometry (LC/MS/MS) technology has been widely used for quantitative analysis of small organic molecules, it has been a challenging task to quantitatively analyze protein samples utilizing this technology in biological matrices for pre‐clinical and clinical studies. Here we present our initial results in method development for the quantitative determination of rK5 protein concentrations in human plasma samples utilizing LC/MS/MS technology. A protein similar in structure to rK5, but with a slightly reduced molecular weight, was used as internal standard. A 96‐well solid‐phase extraction procedure was developed to effectively extract protein analytes from plasma samples. Quantitative analysis was obtained by a novel approach of protein monitoring that employed selective reaction monitoring (SRM). Even though mass spectrometry of the internal standard protein gave no fragment ions, SRM monitoring greatly reduced background interference. Using samples prepared in human plasma with sodium EDTA as anticoagulant, a correlation coefficient (r 2 ) of 0.9940 was obtained by producing a single standard curve with the injection of six rows of standards with a concentration range from 100 ng/mL to 10 μg/mL. The mean analytical recovery for these standards ranged from 91.5 to 103.6%. The CVs for individual standard levels ranged from 3.7 to 20.9%. The experiment was also repeated using samples prepared in human plasma with sodium heparin as anticoagulant, which produced a correlation coefficient (r 2 ) of 0.9952 obtained from a single standard curve with the injection of four rows of standards with a concentration range from 50 ng/mL to 10 μg/mL. The mean analytical recovery for the standards ranged from 96.2 to 104.6%. The CVs for individual standard levels ranged from 2.6 to 15.6%. Copyright © 2003 John Wiley & Sons, Ltd.