Analysis of four toxic metals in a single rice seed by matrix solid phase dispersion -inductively coupled plasma mass spectrometry

Toxic metals in rice pose great risks to human health. Metal bioaccumulation in rice grains is a criterion of breeding. Rice breeding requires a sensitive method to determine metal content in single rice grains to assist the variety selection. In the present study, four toxic metals of arsenic (As), cadmium (Cd), chromium (Cr) and lead (Pb) in a single rice grain were determined by a simple and rapid method. The developed method is based on matrix solid phase dispersion using multi-wall carbon nanotubes (MWCNTs) as dispersing agent and analyzed by inductively coupled plasma mass spectrometry. The experimental parameters were systematically investigated. The limits of detection (LOD) were 5.0, 0.6, 10 and 2.1 ng g−1 for As, Cd, Cr, and Pb, respectively, with relative standard deviations (n = 6) of <7.7%, demonstrating the good sensitivity and precision of the method. The results of 30 real world rice samples analyzed by this method agreed well with those obtained by the standard microwave digestion. The amount of sample required was reduced approximately 100 fold in comparison with the microwave digestion. The method has a high application potential for other sample matrices and elements with high sensitivity and sample throughput.

selective MSPD sorbents such as multi-walled carbon nanotubes 18 , alumina 16 , C18 26 , silica 27 , and florisil 28 contain metal impurities which limit the application of MSPD in the extraction of metal elements. For example, multi-wall carbon nanotubes (MWCNTs) may contain up to 30 wt% of residual metal impurities (nickel, iron and etc.) 29 . Therefore, arsenic and mercury 30 were most suitable for elemental extraction with MSPD, but other metal elements were quite limited. Fortunately, the residual metal and impurities in MWCNTs can be purified by methods such as acid treatment 31 , microwave irradiation 32 , photochemical vapor generation 29 and plasma-thermal purification 33 . The lower matrix effect of purified MWCNTs enhanced the extraction and concentration of the target metal elements via MSPD.
The aim of this work was to investigate the potential of MSPD for the extraction of multiple metal elements in a single rice seed. Acid treatment was chosen for the purification of commercial MWCNTs, and washed MWCNTs were sequentially applied for the MSPD extraction of toxic metal elements (As, Cd, Cr and Pb) in a single rice seed. The proposed sample preparation method coupled with ICP-MS was validated and successfully applied to analysis of metal elements in rice samples.

Results and Discussion
Characterization of pretreated MWCNTs. Treated and untreated MWCNTs samples were first characterized by SEM (Fig. 1). There were some nano-scale metal particles presented in the untreated SEM image and these metal particles were encapsulated in the CNT central canal (Fig. 1a). However, these nano-scale metal particles were hardly found after purification, and were thought to be removed in the cleanup process (Fig. 1b). The purified MWCNTs did not show any damage and morphological changes such as length shortening, which meant that the pretreatment served as purification, but did not change or modify the W-MWCNTs characteristics. When the W-MWCNTs were ground with the rice sample, as demonstrated in Fig. 1c, the MWCNTs dispersed the rice sample completely. Most importantly, the MWCNTs generated numerous carbon nanofibers, which prevented the aggregation of the mixture. Therefore, MWCNTs were used as a good solid matrix to disperse the rice samples.
In order to further investigate the removal of the metal elements from the W-MWCNTs, the four heavy metal element impurities in MWCNTs were quantitatively determined before and after purification. Treated and  untreated MWCNTs (0.02 g) were digested by microwave digestion (power: 800 W; temperature: 160 °C; time: 20 min) with the concentrated nitric acid (5 mL) and hydrogen peroxide solution (1 mL). The purification treatment showed that approximately 85%, 76%, 96% and 97% of As, Cd, Cr and Pb were removed from MWCNTs, As

Optimization of MSPD for metal elements. For optimization of the MSPD experimental conditions
used for extraction, the standard rice sample GBW10010 was used for sequential assessment of the heavy metals. The extraction efficiency was evaluated from a comparison of the obtained and certified values of heavy metals in GBW10010. MWCNTs were used as solid supports for heavy metal extraction in the rice samples, where the mass of the MWCNTs strongly affected the extraction efficiency of the analytes. In this work, the effect of the amount of MWCNTs on the extraction efficiency was first investigated. Figure 3a shows that in comparison with the no MWCNTs controls, the extraction efficiencies of all four target metals in the 0.02 g rice sample increased with the use of MWCNTs. Average recoveries of each analyte reached to a plateau in the range of 0.04-0.08 g of MWCNTs.
A lower amount of the MWCNTs did not produce efficient dispersion of the analytes. Therefore, a MWCNTs mass of 0.04 g was chosen for all subsequent extraction in this work.
The grinding time of MSPD for the analytes is another critical parameter. All recoveries of the analytes increased as the grinding time increased from 1 min to 4 min, followed by a plateau (Fig. 3b). The grinding time of 4 min was then applied in the subsequent experiments. It is noteworthy that in a preliminary experiment, two national standard rice samples were digested at 160 °C and 180 °C. The average recoveries of As at 160 °C were approximately 100%, but were only approximately 70% at 180 °C (data not shown), which indicated that the high digestion temperature resulted in the loss of As element. Therefore, the samples were ground at ambient temperature in the MSPD method rather than high temperature acid digestion in the traditional method. This is another important advantage of the MSPD method.
It is well known that aqua regia (HCl:HNO 3 , 3:1) has been used as an eluent for the extraction of heavy metals. Therefore, aqua regia was chosen as the eluent and the effects of aqua regia at various concentrations were also studied, as shown in Fig. 3c. The extraction efficiencies of the analytes were below 1% without using acid, and  Average ± standard deviation of three trials. b The numerical value in the bracket is for reference.
increased to 95% with the use of 1% or greater amounts of acid. Therefore, an eluent containing 1% (v/v) aqua regia was used for the remainder experiments.
Analytical performance. The proposed method was evaluated under the optimal conditions ( Table 1). The linear correlation coefficients of determination for calibration curves were better than 0.999 for all four metal elements at a trace concentration. The limit of detection (LOD), defined as the analyte concentration equivalent to three standard deviations of 11 measurements of a blank solution (HNO 3 , 2% v/v), was 5.0, 0.6, 10 and 2.1 ng g −1 for As, Cd, Cr and Pb, respectively. The precision of the proposed method was assessed by performing replicate  analyses of the certified reference material (CRM) samples. The precision of the method was expressed as relative standard deviations (RSDs, n = 6), and all was less than 7.7% for the tested metal elements.
Interference. The dispersion and extraction efficiency of the proposed method was limited by the capacity of the MWCNTs material. Interference may occur due to competition from other heavy metal elements. To evaluate the interference, the effects of several potential interfering metal elements were investigated. The standard rice sample (GBW10010) was used with the addition of a high concentration of interfering metal elements (Mg, Zn, Cu, Fe, Mn, Mo, Ce, Ni, and Se). The results showed no obvious interference observed from these target metal ions even at concentrations as high as 10 mg g −1 ( Table 2).

Sample analysis.
Since there were no rice seed samples with certified value of the metal elements, the accuracy of the proposed method was first evaluated by analysis of four CRMs (GBW10010, GBW10043, GBW10044, and GBW10045). The t-test showed that all the analytical results produced by the proposed method were not significantly different from the certified value at a 95% confidence level (Table 3). Further, the applicability of the method was compared with the standard microwave digestion method for metal elements. The 30 real world rice seed samples were analyzed by the developed MSPD extraction method (sample amount: about 0.02 g of a single seed) and microwave digestion (sample amount: 2 g). The results were not significantly different between the two methods (Fig. 4). However, the amount of sample required was reduced about 100 fold in comparison with the standard microwave digestion.

Conclusions
A simple and rapid solid sampling platform based on purified MWCNTs and assisted MSPD was developed for the simultaneous determination of trace amounts of As, Cd, Cr and Pb in a single rice seed sample. Compared with other methods for elemental metal analysis in rice samples, this method demonstrated several advantages such as allowing single seed analysis, use of less chemicals, lower energy consumption, simplicity and low cost. In future works, this method can be used to analyze other sample matrices and elements with high sensitivity and throughput.

Methods
Reagents. All chemicals were of analytical grade purity or greater. High purity 18.

Pretreatment of MWCNTs.
MWCNTs samples contain many residual metal impurities. Therefore, commercial MWCNTs were washed with HNO 3 and H 2 SO 4 to remove the metal impurities in the initial experiment as described in previous methods 31 . Briefly, commercial MWCNTs (2 g) were added to a reaction chamber together with acid solvent (300 mL, HNO 3 :H 2 SO 4 , 1:1), and then the reaction vessels were subjected to sonication for 6 h. After the reaction, the mixture was centrifuged at 15000 rpm for 5 min. After carefully removing the aqueous phase using a syringe, the solid MWCNTs phase was diluted and rinsed with ultrapure water until neutral, and then dried at 60 °C for future use. Scanning electron microscopy (SEM) was used to characterize the washed  MWCNTs (W-MWCNTs) before and after the treatment. In order to determine the removal efficiencies of metal elements from MWCNTs, ICP-MS was used to quantify the metal elements before and after the acid treatment.
Sample preparation. The MSPD procedure was performed for a single rice seed sample according to the schematic of the sample preparation illustrated in Fig. 5. A single rice seed (about 0.02 g) and W-MWCNTs (0.02 g) were weighed into an agate mortar and blended for around 5 min using an agate pestle to obtain a homogeneous mixture. This mixture was then quantitatively transferred into a 10 mL PTFE centrifuge tube, to which 1% HNO 3 :HCl (3:1, v/v, 2 mL) was added. After blending, the mixture was centrifuged at 15000 rpm for 2 min. Finally, the liquid phase was collected and subjected to ICP-MS analysis directly.
ICP-MS conditions. The NexION 300X ICP-MS (PerkinElmer, Inc., Shelton, CT) was used throughout this work. The optimized parameters and details of the instrumental settings were listed in Table 4. The water stream and metal sample solution were introduced into the system by a peristaltic pump at the flow rate of 0.5 mL min −1 . Rhodium (Rh, 25 μ g L −1 ) was added as an internal standard to minimize the instrumental signal fluctuation and matrix effects during the measurement of the samples.