Quantitation of the large polypeptide glucagon by protein precipitation and LC/MS.
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 C18, 300 A 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.
Delinsky
David C
DC
Department of Pharmaceutical and Biomedical Sciences, The University of Georgia, Athens, GA 30602-2352, USA.
Hill
Kim T
KT
White
Catherine A
CA
Bartlett
Michael G
MG
D000595:Amino Acid Sequence; D000818:Animals; D011232:Chemical Precipitation; D002853:Chromatography, Liquid / Q000379:methods*; D005934:Glucagon / Q000097:blood* / Q000737:chemistry; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D051381:Rats; D017207:Rats, Sprague-Dawley; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D005934:Glucagon