Rapid and simple UPLC-MS/MS method for precise phytochelatin quantification in alga extracts
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Quantitative phytochelatin (PC) analysis is, due to oxidation sensitivity of the PCs, matrix effects, and time consuming sample preparation, still a challenging analytical task. In this study, a rapid, simple, and sensitive method for precise determination of native PCs in crude extracts of the green alga Chlamydomonas reinhardtii was developed. Algae were exposed 48 h to 70 μM Cd. Coupling of ultra performance liquid chromatography and electrospray ionization tandem mass spectrometry with multi-reaction mode transitions for detection permitted the required short-time, high-resolution separation and detection specificity. Thus, under optimized chromatographic conditions, 10 thiol peptides were baseline-separated within 7 min. Relative detection limits in the nanomolar range in microliter sample volumes were achieved (corresponding to absolute detection limits at femtomol level). Next to glutathione (GSH), the most abundant cadmium-induced PCs in C. reinhardtii, namely CysGSH, PC2, PC3, CysPC2, and CysPC3, were quantified with high reproducibility at concentrations between 15 and 198 nmol g−1 fresh weight. The biological variation of PC synthesis of nine independently grown alga cultures was determined to be on average 13.7%.
KeywordsPhytochelatin quantification Chlamydomonas UPLC ESI-MS/MS
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