TY  - JOUR
TI  - High-Performance Liquid Chromatography Quantification of Glyphosate, Aminomethylphosphonic Acid, and Ascorbate in Culture Medium and Microalgal Cells
AU  - Ostera, Juan Manuel
AU  - Olmos, Valentina 
AU  - Puntarulo, Susana 
AU  - Bonetto, Julián G.
AU  - Malanga, Gabriela 
VL  - 15
IS  - 7
PY  - 2025
DA  - 2025/04/05
SP  - e5263
C1  - Bio-protocol 2025;15:e5263
DO  - 10.21769/BioProtoc.5263
UR  - https://doi.org/10.21769/BioProtoc.5263
AB  - Glyphosate (GLY) is a widely used herbicide that can induce oxidative stress in microalgae and other non-target organisms. The quantification of GLY in surface water is a difficult task, especially in trace-level concentrations, due to its high polarity and susceptibility to biotic and abiotic degradation. Several analytical methods have been developed for GLY quantification. Most of them use high-performance liquid chromatography (HPLC) coupled with detection by mass spectrometry (MS) and include a derivatization step to decrease the polarity of the herbicide to improve detection. This protocol describes an adaptation of an existing protocol for the quantification of GLY and its metabolite aminomethylphosphonic acid (AMPA) in a water-based microalgae culture medium using ultra-high-pressure liquid chromatography (UHPLC) coupled with fluorescence detection (FLR). The principal advantage of this protocol compared with other analytical methods that employ HPLC–MS is its low cost and accessibility since it does not require an MS detector nor radioactively labeled analytical standards. Ascorbic acid (AH-) is one of the most important hydrosoluble non-enzymatic antioxidants in eukaryotic cells and plays a key role in many metabolic pathways of critical importance in plants and algae. In this protocol, we also describe an adaptation of a previously published protocol to quantify AH- in blood samples to be used in microalgal cells exposed to GLY and GLY-based herbicides. The sample preparation procedure for this last protocol is fast, easy, and does not require expensive equipment. It uses an HPLC system coupled with an electrochemical detector (EC) for AH- quantification but may be adapted to be used with a UV-Vis detector.
KW  - Glyphosate
KW  - Aminomethylphosphonic acid
KW  - HPLC
KW  - Microalgae
KW  - Derivatization
KW  - Electrochemical detector
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False