title: A novel method for the simultaneous analysis of seven biothiols in rice (Oryza sativa L.) using hydrophilic interaction chromatography coupled with electrospray tandem mass spectrometry.
abstract: Analysis of biothiols is still problematic, due to their high polarity, oxidation sensitivity and time-consuming sample preparation. In this paper, a direct, rapid and sensitive method was developed for simultaneous quantification of unbound cysteine (Cys), glutathione (GSH) and phytochelatins (PCs) in rice leaf, stem and root samples by hydrophilic interaction chromatography coupled with electrospray tandem mass spectrometry (HILIC-MS/MS). Homogenized samples were extracted with water containing 50mM dithiothreitol, without derivatization and further clean-up, and the extracts were injected directly onto an Xbridge Amide-HILIC column (3.5μm, 150mm×2.1mm i.d.). The best chromatographic separation and MS sensitivity was achieved using a linear gradient elution with 10mM aqueous ammonium formate and acetonitrile as the mobile phase. In MS/MS mode the detection limit (S/N≥3) of seven biothiols was 3-105nM. Good linearities were observed (r>0.995) with linear dynamic range at least over three orders of magnitude. Recoveries for most analytes were within the range of 77-128%, with relative standard deviations less than 18.2%. The intra-day precision (n=7) was 6.1-11.7%, and the inter-day precision over 15 d (n=15) was 8.5-16.3% for all biothiols. The optimized HILIC-MS/MS method was applied to study the influence of different cadmium (Cd) concentrations (0, 1 and 50μM) on contents of Cys, GSH and PC2-6 in rice tissue. With increasing Cd concentrations in nutrient solutions, contents of PC2-4 in rice roots increased but contents of Cys and GSH decreased. Contents of PC2-4 in both rice leafs and stems increased markedly at high dose Cd (50μM) treatment compared with controls, compared with low Cd concentrations (1μM). However, both PC5 and PC6 were not detected throughout the stress experiment.
authors.affiliation: China National Rice Research Institute, Hangzhou 310006, China; Laboratory of Quality & Safety Risk Assessment for Rice (Hangzhou), Ministry of Agriculture, Hangzhou 310006, China.
authors.forename: Zhao-Yun
authors.initials: ZY
authors.lastname: Cao
authors.affiliation: Zhejiang Academy of Medical Sciences, Hangzhou 310007, China.
authors.forename: Li-Hua
authors.initials: LH
authors.lastname: Sun
authors.affiliation: China National Rice Research Institute, Hangzhou 310006, China; Laboratory of Quality & Safety Risk Assessment for Rice (Hangzhou), Ministry of Agriculture, Hangzhou 310006, China.
authors.forename: Ren-Xiang
authors.initials: RX
authors.lastname: Mou
authors.affiliation: China National Rice Research Institute, Hangzhou 310006, China; Laboratory of Quality & Safety Risk Assessment for Rice (Hangzhou), Ministry of Agriculture, Hangzhou 310006, China.
authors.forename: Rong
authors.initials: R
authors.lastname: Zhou
authors.affiliation: China National Rice Research Institute, Hangzhou 310006, China; Laboratory of Quality & Safety Risk Assessment for Rice (Hangzhou), Ministry of Agriculture, Hangzhou 310006, China.
authors.forename: Zhi-Wei
authors.initials: ZW
authors.lastname: Zhu
authors.affiliation: China National Rice Research Institute, Hangzhou 310006, China; Laboratory of Quality & Safety Risk Assessment for Rice (Hangzhou), Ministry of Agriculture, Hangzhou 310006, China. Electronic address: cmingxue@live.com.
authors.forename: Ming-Xue
authors.initials: MX
authors.lastname: Chen
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D003545:Cysteine / Q000032:analysis; D005978:Glutathione / Q000032:analysis; D057927:Hydrophobic and Hydrophilic Interactions; D012275:Oryza / Q000737:chemistry*; D054811:Phytochelatins / Q000737:chemistry; D018517:Plant Roots / Q000737:chemistry; D018547:Plant Stems / Q000737:chemistry; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D054811:Phytochelatins; D005978:Glutathione; D003545:Cysteine
keywords: Amide-HILIC; Biothiol quantification; Cd stress; ESI-MS/MS; Rice