Determination of leucine and isoleucine/allo-isoleucine by electrospray ionization-tandem mass spectrometry and partial least square regression: Application to saliva samples

Abstract

Herein we propose, for the first time, a rapid method based on flow injection analysis, electrospray ionization-tandem mass spectrometry (FIA-ESI-MS/MS) and multivariate calibration for the determination of l-leucine, l-isoleucine and L-allo-isoleucine in saliva. As far as we know, multivariate calibration has never been applied to the data from this non-separative approach. The possibilities of its use were explored and the results obtained were compared with the corresponding ones when using univariate calibration. Partial least square regression (PLS1) multivariate calibration models were built for each analyte by analyzing different saliva samples, and were subsequently applied to the analysis of another set of samples which had not been used in any calibration step. For Leu, the model worked satisfactorily with root mean square errors in the prediction step of 17%. This error can be considered acceptable and is common in methodologies that do not include a separation step. Results were compared with those obtained when univariate calibration was used, using the m/z transition 132.1 → 43.0 as the quantitation variable. In this case, the obtained results were not acceptable, with RMSEP of 236%, due to the fact that saliva samples contained another compound, different to the target analytes, which also shared the same transition. Ile and aIle have the same fragmentation patterns, so quantification of the sum of both compounds was performed, with RMSEP of 14% using a PLS1 model. Similar results were obtained when a univariate calibration model using the m/z transition 132.1 → 69.0 was employed. However, the use of this transition should be carefully examined when other compounds present in the matrix contribute to the analytical signal. The method increases sample throughput more than one order of magnitude compared to the corresponding LC-ESI-MS/MS method and is especially suitable as screening. When abnormally high or low concentrations of the analytes studied are obtained, the use of the method that includes separation is recommended to confirm the results.

Keywords: Electrospray ionization tandem mass spectrometry; Leucines; Multivariate calibration; Non-separative method; Saliva.