Quantitation of the large polypeptide glucagon by protein precipitation and LC/MS

✍ Scribed by David C. Delinsky; Kim T. Hill; Catherine A. White; Michael G. Bartlett

• Publisher: John Wiley and Sons
• Year: 2004
• Tongue: English
• Weight: 336 KB
• Volume: 18
• Category: Article
• ISSN: 0269-3879
• DOI: 10.1002/bmc.380

✦ Synopsis

Abstract

We present a method for the quantitation of glucagon from rat plasma by protein precipitation and LC/MS. No internal standard was used, as a labeled standard was not available and similar peptides did not show comparable extraction characteristics to glucagon. The LC system included a Keystone C~18~, 300 Å pore size column; a linear gradient was used with a mobile phase consisting of water and acetonitrile, each with 0.2% acetic acid and 0.02% trifluoroacetic acid. Glucagon was detected with the mass spectrometer in positive ion mode monitoring the 4+ charge state at m/z 871.7. The method had an approximated limit of detection of 1 ng/mL. The lower limit of quantitation (LLOQ) was 25 ng/mL (7.2 fmol/mL), which could be reduced with an appropriate internal standard. External calibration was used and calibration curves were found to be linear over the range from 25 to 1000 ng/mL (7.2 to 290 fmol/mL). The method showed a high degree of precision and accuracy both within and between runs at four validation points, including the LLOQ. Copyright © 2004 John Wiley & Sons, Ltd.