Rapid and simple UPLC-MS/MS method for precise phytochelatin quantification in alga extracts.

Area of Science:

• Environmental Science
• Analytical Chemistry
• Biochemistry

Background:

• Quantitative phytochelatin (PC) analysis is challenging due to oxidation sensitivity, matrix effects, and lengthy sample preparation.
• Phytochelatins are crucial for heavy metal detoxification in plants and algae.

Purpose of the Study:

• To develop a rapid, simple, and sensitive method for precise determination of native PCs in crude extracts of Chlamydomonas reinhardtii.
• To quantify cadmium-induced PCs in C. reinhardtii using the developed method.

Main Methods:

• Coupling of ultra-performance liquid chromatography (UPLC) with electrospray ionization tandem mass spectrometry (ESI-MS/MS) in multi-reaction monitoring (MRM) mode.
• Optimized chromatographic conditions allowed baseline separation of 10 thiol peptides within 7 minutes.
• Achieved nanomolar detection limits in microliter sample volumes, corresponding to femtomole absolute detection limits.

Main Results:

• Quantified glutathione (GSH) and major cadmium-induced PCs (CysGSH, PC(2), PC(3), CysPC(2), CysPC(3)) in C. reinhardtii.
• Measured PC concentrations ranged from 15 to 198 nmol g(-1) fresh weight with high reproducibility.
• Determined the biological variation of PC synthesis across nine independent cultures to be approximately 13.7%.

Conclusions:

• The developed UPLC-ESI-MS/MS method provides a rapid and sensitive approach for accurate PC quantification in C. reinhardtii.
• This method facilitates a better understanding of heavy metal detoxification mechanisms in algae.
• The findings contribute to improved analytical techniques for thiol peptide analysis in biological samples.