Determination of Underivatized Amino Acids in Yeast by LC-MS (QTOF) Following Microwave Assisted Acid Hydrolysis

Lilach Yishai Aviram, Analytical Chemistry Department , IIBR, Israel
Zoltan Mester, NRC, Ottawa, Canada

In this study we describe a simple, rapid and comprehensive method for micro scale sample preparation using microwave acid vapor hydrolysis/acid hydrolysis followed by quantitative amino acid analysis using liquid chromatography-mass spectrometry (Qtof) without derivatization.  The protein acid hydrolysis was performed in an open vessel microwave device using small amounts (0.5mg-2mg) of sample and 6M HCl. The vapor phase hydrolysis approach was compared to a condensed phase hydrolysis method. The microwave parameters including heating time, heating temperature and sample amount were optimized for 16 amino acids. The analysis of the amino acids was performed by UPLC–ESI-MS (QTOF) in positive ESI mode. Separation was achieved on a BEH C-18 column. Limits of detection and quantitation (LODs/LOQs), determined using standard solutions, were in the sub ppb range. These values are much lower than the actual concentrations of amino acids in the certified yeast reference material that was used to demonstrate the utility of both the acid vapor phase extraction in the open vessel microwave and the LC-Q-TOF analysis, without prior derivatization of the amino acids. The method was successfully applied for the determination of methionine and selenomethionine by isotope dilution and standard addition methods in the yeast.