Elsevier

Microchemical Journal

Volume 117, November 2014, Pages 122-126
Microchemical Journal

Investigation of arsenic species stability by HPLC-ICP-MS in plants stored under different conditions for 12 months

https://doi.org/10.1016/j.microc.2014.06.018Get rights and content

Highlights

  • As species stability was investigated in plants.

  • Effects of storage temperature, drying and grinding were investigated.

  • Lyophilization and cryogenic grinding were the best procedures for As species preservation.

Abstract

In studies of speciation, the conservation of species in their original forms is a very challenging issue, and the reliability of the results deeply depends on it. Preliminary steps of sample preparation and storage temperature may influence the conservation of species and extraction efficiency. Plant sample (Brachiaria brizantha Stapf. cv. Marandu) grown in As-containing soil was harvested and divided into four portions. The first portion was frozen-dried, milled with liquid N2 and stored at room temperature; the second portion was lyophilized and stored at 4 °C; and the remaining portions were kept at two storage temperatures (− 18 and − 80 °C) without any prior procedure. Aliquots of samples stored under different conditions were extracted every 2 months for a 12-month period. The total content of As was 2.30 ± 0.27 mg kg 1 of dried and ground sample and As species were determined by HPLC-ICP-MS. The extraction was more efficient in sample aliquots that were lyophilized and ground (87–90%) than those only stored under different temperatures (53–66%). Furthermore, only for lyophilized and ground samples dimethylarsenic acid (DMA) was extracted. Concerning the storage time under the two tested temperatures, no significant differences on species were found and they remained stable. Standard deviations for replicates of samples that were not frozen-dried and ground were higher, and this behavior can be explained by sample heterogeneity. For the As species here studied it can be concluded that lyophilization and cryogenic grinding strategies were the most suitable sample pretreatments for As speciation in plant tissue.

Introduction

Arsenic speciation in aquatic and terrestrial biota is an important topic, since plants are often the main form of potentially toxic element input into food chain, either directly by ingestion of edible plants or indirectly from animals (meat and milk) that eat leaves used for livestock fodder [1]. The determination of chemical species provides information about toxicity, availability and essentiality; however it requires a more complex procedure of analysis compared to total determination. In order to obtain reliable results, more rigorous analytical procedures taking into account all steps, such as sample preparation (drying, grinding, extraction) and storage (temperature, time, radiation), must be employed [2], [3], [4]. Additionally, to show that species conversion does not occur confers reliability to the results.

Usually, procedures employed in trace analysis involve drying and grinding of samples. The sample preparation for speciation is milder than for total content determination; thus, it is worth discussing whether the preliminary procedures also should be gentle. Some authors advised the direct analysis of fresh plant; however, this procedure is often difficult due to moisture content and sample heterogeneity [5]; others recommended the drying of the plant since it is believed that plant moisture can cause species conversion [6]. A paper has reported that milling is important to assess the species contained inside the cellular structure of the vegetable and also for providing greater homogeneity [7]. Furthermore, some studies focused on the storage temperature and divergences have been consistently found. Recent researches indicate that the plant should be kept at a low temperature, while others consider that the freezing/defrosting process of the sample led to species conversion and, therefore, they must be kept at room temperature [8], [9].

Another important point is the time that the sample remains in its original state. Most works about speciation analysis do not assess the sample stability on long-term storage, which is critical to determine the maximum storage time that still preserves the species. Samples of algae Cystoseira mediterranea Sauvageau were stored under different conditions for 45 days [9]. Bluemlein et al. [10] analyzed samples stored at − 80 °C and the concentration of arsenic–phytochelatin complexes, remained relatively constant during 21 days; on the other hand Raab et al. [11] noticed that long-term storage of plant at − 20 °C is not appropriate for conserving As–phytochelatin complexes.

High performance liquid chromatography–inductively coupled plasma mass spectrometry, HPLC-ICP-MS, is one of the most widely employed hyphenation techniques for speciation studies due to the simple coupling, high separation power of HPLC and suitable detection power of ICP-MS [12], [13].

The aim of this work was to evaluate the As species stability along 12 months with periodical determination of them (each two months), using HPLC-ICP-MS. Also, different storage temperatures and state of the sample, drying and grinding, were evaluated. The sample chosen Brachiaria brizantha Stapf cv. Marandu is a type of grass used in Brazilian cattle feed. In order to increase the As occurrence in plant leaves and thus ensuring species quantification, the plant was cultivated in a soil containing As(V) as Na2HAsO4·7H2O.

Section snippets

Reagents and standards

All glassware was immersed into 10% v v −1 HNO3 solution overnight before using and further thoroughly rinsed with distilled–deionized water (18.2  cm, Milli-Q, Millipore, Bedford, MA, USA). The standards used in the speciation studies were Na2HAsO4·7H2O (Vetec, Rio de Janeiro, RJ, Brazil), As2O3, CH4AsNa3·5H2O and C2H7AsO2 (Supelco, Bellefonte, PA, USA). Analytical grade methanol was also used for column cleaning (Tedia Company, Fair-field, OH, USA). The mobile phase, phosphate buffer, for

Results and discussion

The total As content was determined using the introduction of H2 gas through the skimmer cone at a flow rate of 80 mL min −1 to minimize polyatomic interferences. Therefore, total As content in B. brizantha adopted in all calculations of extraction efficiency was 2.30 ± 0.27 mg per kg of lyophilized and frozen-dried sample. The certified value for As in tomato leaves NIST 1573a is 0.112 ± 0.004 mg kg −1 and a concentration of 0.118 ± 0.008 mg kg −1 was determined, therefore evidencing the accuracy of the

Conclusions

It can be concluded that for As species evaluated in the present work, lyophilization and cryogenic grinding were the best sample preparation strategies. On the other hand, sample storage at low temperatures resulted in poor extraction efficiency. Furthermore, for each condition As species have remained relatively stable along 1-year period. The ratio As(III)/As(V) changed when comparing no pretreated (fresh plant) and pretreated sample (lyophilized and ground), indicating possible

Acknowledgments

The authors would like to thank the grants 2006/59083-9 and 2010/50238-5 São Paulo Research Foundation (FAPESP). We also acknowledge the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the grants – 300680/2008-2; 304557/2010-2 and 140375/2013-9 and for their financial support – 48077/2010-1.

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This article was presented as poster at the 17th Brazilian Meeting in Analytical Chemistry.

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