LC-MS/MS analysis of free amino acids

Foreword

There are countless publications about the analysis of amino acids (AA). In the course of the method setup and development, we tried classical pre-column derivatizations using fluorenylmethoxycarbonyl protecting (Fmoc) group for HPLC-PDA/FL analysis of AA however the column life was unacceptably short, a column could lose chromatographic resolving power suddenly after some 200-300 injections using the method and the column published and sold by Agilent. We tried Phenomenex EZ-Faast kits for derivatization and analysis of AA using GC-MS or LC-MS without fair results. Phenomenex will not tell you, but they use derivatization methods developed by Petr Hušek. And we tried several other methods however non of them provided a reliable quantitative solution because there exists a sample matrix in the real world and it is an unknown and unpredictable factor.

Mass spectrometry is a sensitive and distinctive analytical technique however absolute response in the MS analysis is susceptible to experimental parameters which cannot always be controlled, known, and counted. Therefore the best and the only solution for achieving accurate quantitative results in mass spectrometry is the use of isotopically labeled analogues (i.e. by using the isotope dilution method).

Today we can analyze up to 30 AA and by using 20 isotopically labeled AA the accuracy of results is significantly improved. 

Instrumental: QTRAP 6500+ mass spectrometer (Sciex) coupled with Nexera binary UPLC (Shimadzu). The mass spectrometer was operated in positive ESI scheduled MRM mode. Chromatographic separations were carried out using the HILIC-Z column (2.1×150 mm, 2.7µ particle size, Agilent) using a linear gradient of ACN/water (with 100 mM ammonium formate). The instrument was controlled and data were analyzed using Analyst and MultiQuant software respectively (Sciex).

LC-HRMS analysis of amino acids after derivatization

Instrument: The analysis is carried out using the LC-HRMS system which consists of Ultimate 3000 UPLC coupled with a Q-Exactive Plus mass spectrometer (Thermo Scientific). The mass spectrometer is operated upon positive ESI using “full scan” and “all ions fragmentation” acquisition modes at resolving power 70,000. The isobutyl carbamate derivatives of  AA are separated on reversed-phase Acclaim C18 column (Dionex) employing Acetonitrile/Water (with 0.1% acetic acid) linear gradient. The instrument was controlled and data were analyzed using Xcalibur and TraceFinder software respectively (Thermo Scientific).

Sample preparation: A sample is spiked with a mixture of isotopically labeled AA internal standards following derivatization with isobutyl chloroformate in the presence of borate buffer (pH ~11). After acidification with HCl (to pH 1-3) derivatives of AA are extracted with ethyl acetate which is evaporated following dissolution in acetonitrile/water. 

Some free amino acids may chelate with the steel parts of the LC system and elute as wide and tailing chromatographic peaks which result in reduced sensitivity. It is particularly true for sulfur-containing cysteine and cystine which usually are not detected in the LC-MS/MS analysis of free amino acids but are easily detected in LC-HRMS analysis after chemical derivatization.

Results and Conclusions

The use of isotopically labeled internal standards in mass spectrometry is essential for achieving accurate and reliable quantitative results. Validation studies and analyses carried out over a period of several years demonstrated excellent accuracy and reliability for those AAs that were quantified using isotopically labeled analogues and improved accuracy for other AA that were quantified using alternative IS.

This method was used in many studies, some results were published. 

The results of this application were published in:

"The Effect of Microbial Inoculum and Urea Supplements on Nutritive Value, Amino Acids Profile, Aerobic Stability and Digestibility of Wheat and Corn Silages", Animals, 2023, 13(13), 2197.

"The Goldilocks effect of respiration on canavanine tolerance in Saccharomyces cerevisiae", Current Genetics, October 2019, 65(8).