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

Biomedical Chromatography : BMC
David C DelinskyMichael G Bartlett

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 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.

References

May 5, 1995·Journal of Chromatography. B, Biomedical Applications·J S PartillaR B Rothman
Oct 6, 1995·Journal of Chromatography. B, Biomedical Applications·N KobayashiK Nakamura
Jul 20, 1999·Journal of Chromatography. B, Biomedical Sciences and Applications·M NiwaK Yamashita
Feb 24, 2001·Journal of Analytical Toxicology·S M DarbyM LeBeau

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Citations

May 22, 2008·American Journal of Physiology. Endocrinology and Metabolism·Julie A Harney, Robert L Rodgers
Dec 4, 2013·Bioanalysis·Veniamin N LapkoChris J Kafonek
Jul 22, 2005·Journal of Mass Spectrometry : JMS
Jul 17, 2021·Journal of Proteome Research·Rachel E ForemanRichard G Kay

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