Session: Session 5

Quantification of 20 essentials amino acids in cell’s culture fermentation without derivatization by high performance liquid chromatography coupled with MS detection (high resolution and triple quadrupole)

Amino acids are the fundamental building blocks of proteins and only 20 amino acids are protein genic. The object of the present work is to develop and validate a precise and reproducible method by liquid chromatography coupled with mass spectrometry to quantify amino acids, without derivatization, from microorganisms’s culture supernatant.

Assays were performed on 100 µL of sample from fermentor’s culture. Amino acids were separated by Atlantis Premier BEH Z-Hilic (Waters). Compounds were detected by high-resolution mass spectrometry using an Orbitrap Exploris 120 (Thermo Scientific). Labelled amino acids were used as internal standards for quantification. The validation of the method was based on the evaluation of selectivity, carry-over, linearity, repeatability, reproducibility, lower limits of detection and of quantification. The method was validated with a lower limit of 15 µM and a linearity between 15 and 400 µM. Precision ranged from 0.6% to 9.4%, and accuracy from 92.2% to 107%.

The robustness of the method was evaluated by using the two independent channels of the  Transcend TLX-2-system (Thermo Scientific). The use of this equipment allowed to increase the throughput of the method, with a single mass spectrometer:two samples can be analyzed in parallelthanks to bypass valves, fully managed by the software.

Moreover, the use of hydrophilic interaction technology in liquid chromatography allows separation of the isobaric pair, isoleucine and leucine. This technology, although presenting shiffing retention time issues, is very effective for these compounds. The overall robustness of the method is guaranteed by the use of the corresponding internal standard. High reproducibility of the method was confirmed by the analysis of samples processed with different dilution factors.

This method was successfully applied to several strains fermentation’s samples for the analysis of amino acids. This method was also successfully transferred to triple quadrupole detection.