Validation and uncertainty estimation of analytical method for quantification of phytochelatins in aquatic plants by UPLC-MS

Phytochelatins (PCs) are peptides that play an important role in homeostasis and detoxification of heavy metal in plants. Furthermore, they have been proposed as earlier potential biomarkers of aquatic pollution by heavy metals. Nowadays, several researchers have reported on current methods for quantification of glutathione (GSH) and the PCs (phytochelatin 2, phytochelatin 3, phytochelatin 4) quantification in plants. However, no method has reported the uncertainty of the measurement, which helps to improve the accuracy and quality assurance in the PC quantification. In this work, a new methodology using ultra-high-performance liquid chromatography coupled to mass spectrometry (UPLC-MS) to measure with high precision and accuracy the PCs in aquatic plants, was validated. Selectivity, linearity, limit of detection, limit of quantification, precision, trueness and uncertainty estimation were examined as parts of the method validation. The described method shows excellent linearity in different ranges for all analytes with coefficients of determination higher than 0.99. The relative standard deviation for intra-day precision was 50%) and recovery (19-44%) were the most important contributors to the total uncertainty. The proposed method was applied to quantify GSH and PCs in the aquatic plants Lemna gibba L., Myriophyllum heterophyllum Michx., Arenaria paludicola and Hydrocotyle ranunculoides L. fil., showing statistical differences in the mass fraction of the analytes.