Quantification of phytochelatins and their metal(loid) complexes: critical assessment of current analytical methodology

Abstract

Whilst there are a variety of methods available for the quantification of biothiols in sample extracts, each has their own inherent advantages and limitations. The ease with which thiols readily oxidise not only hinders their quantification but also alters the speciation profile. The challenge faced by the analyst is not only to preserve the speciation of the sample, but also to select a method which allows the retrieval of the desired information. Given that sulfur is not a chromophore and that it cannot easily be monitored by ICP-MS, a number of direct and indirect methods have been developed for this purpose. In order to assess these methods, they are compared in the context of the measurement of arsenic-phytochelatin complexes in plant extracts. The inherent instability of such complexes, along with the instabilities of reduced glutathione and phytochelatin species,necessitates a rapid and sensitive analytical protocol. Whilst being a specific example, the points raised and discussed in this review will also be applicable to the quantification of biothiols and thiol-metal(loid) species in a wide range of systems other than just the analysis of arsenic-phytochelatin species in plant extracts.

Identification and quantification of phytochelatins in roots of rice to long-term exposure: evidence of individual role on arsenic accumulation and translocation

“…LMW, low molecular weight; HMW, high molecular weight; PCS, PC synthase; GCS, γ-glutamylcysteine synthetase; GS, glutathione synthetase; hPCS, homo-PC synthases; 1, As uptake by roots; 2, production of LMW PCs; 3, As–PCs complexes (As III +2PC 2 , As III +PC 3 , for example) stored in the vacuole; 4, transport to vacuoles; 5, release of PCs; 6, efflux of As up-taken; ?, unknown information; >, high concentration; <, low concentration; thicker dotted line, PC highly effective. Scheme elaborated according to descriptions from Klapheck et al (1994); Cobbett (2000); Chassaigne et al (2001); Cobbett and Goldsbrough (2002); Oven et al (2002); Meharg and Hartley-Whitacker (2002); Rey et al (2004); Vatamaniuk et al (2004); Song et al (2010); and Wood and Feldmann (2012).…”

Measurement of arsenic species in environmental, biological fluids and food samples by HPLC-ICPMS and HPLC-HG-AFS

“…Sánchez-Rodas and co-workers [73] The extraction and separation procedures that can be used to isolate, separate and measure lipid, water-soluble and residue -associated As species in various samples are outlined below. The choice of extraction solvent and mobile phase may also affect the integrity of As species, especially As complexes such as AsGSH [93]. Each step needs to be optimized and the selection of extractants and mobile phases for HPLC separation carefully considered.…”

“…Complexed As species in bile, urine and plants such as rice presents a challenge to speciate as they are easily denatured and difficult to chromatograph, because species are only stable for a short period, 1-2 hr, and easily dissociate on chromatographic columns unless a mobile phase with a low pH is used [71,93].…”

Metal species involved in long distance metal transport in plants

“…Nicotianamine and PSs have been analyzed, either using previous derivatization (Wada et al, 2007; Kakei et al, 2009; Schmidt et al, 2011) or by direct analysis (Xuan et al, 2007; Tsednee et al, 2012). In the case of PCs, analytical methods are also based on HPLC-MS techniques using either molecular detection (HPLC-ESI-MS and HPLC-ESI-MS/MS) or elemental detection (ICP-MS), and are often combined with derivatization (reviewed by Wood and Feldmann, 2012). For instance, N-(2-ferrocene-ethyl)maleimide is an electroactive pre-column tag that yields thiol-PCs conjugates, which can be separated and quantified by ESI-MS or ICP-MS detection, with detection limits for S at the nM level.…”