Can we trust mass spectrometry for determination of arsenic peptides in plants:: comparison of LC-ICP-MS and LC-ES-MS/ICP-MS with XANES/EXAFS in analysis of Thunbergia alata

The weakest step in the analytical procedure for speciation analysis is extraction from a biological material into an aqueous solution which undergoes HPLC separation and then simultaneous online detection by elemental and molecular mass spectrometry ( ICP - MS/ ES- MS). This paper describes a study to determine the speciation of arsenic and, in particular, the arsenite phytochelatin complexes in the root from an ornamental garden plant Thunbergia alata exposed to 1 mg As L-1 as arsenate. The approach of formic acid extraction followed by HPLC - ES- MS/ ICP - MS identified different As-III - PC complexes in the extract of this plant and made their quantification via sulfur ( m/ z 32) and arsenic ( m/ z 75) possible. Although sulfur sensitivity could be significantly increased when xenon was used as collision gas in ICP - qMS, or when HR- ICP - MS was used in medium resolution, the As: S ratio gave misleading results in the identification of As-III - PC complexes due to the relatively low resolution of the chromatography system in relation to the variety of As - peptides in plants. Hence only the parallel use of ES- MS/ ICP - MS was able to prove the occurrence of such arsenite phytochelatin complexes. Between 55 and 64% of the arsenic was bound to the sulfur of peptides mainly as As-III(PC2)(2), As-III(PC3) and As-III(PC4). XANES ( X- ray absorption near- edge spectroscopy) measurement, using the freshly exposed plant root directly, confirmed that most of the arsenic is trivalent and binds to S of peptides ( 53% As - S) while 38% occurred as arsenite and only 9% unchanged as arsenate. EXAFS data confirmed that As - S and As - O bonds occur in the plants. This study confirms, for the first time, that As - peptides can be extracted by formic acid and chromatographically separated on a reversed- phase column without significant decomposition or de- novo synthesis during the extraction step.