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Simultaneous speciation analysis of As, Cr and Se in the bioaccessible fraction for realistic risk assessment of food safety Nausheen Waheed Sadiq, and Diane Beauchemin Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03423 • Publication Date (Web): 18 Nov 2017 Downloaded from http://pubs.acs.org on November 19, 2017
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Analytical Chemistry
Simultaneous speciation analysis of As, Cr and Se in the bioaccessible fraction for realistic risk assessment of food safety Nausheen W. Sadiq† and Diane Beauchemin* Queen’s University, Department of Chemistry, 90 Bader Lane, Kingston, ON K7L3N6, Canada ABSTRACT: A simple and fast method was developed for risk assessment of As, Cr and Se in food, which is demonstrated here using three cooked and uncooked rice samples (basmati as well as organic white and brown rice). The bio-accessible fraction was first determined through on-line leaching of rice mini-columns (maintained at 37 ºC) sequentially with artificial saliva, gastric juice and intestinal juice while continuously monitoring potentially toxic elements (As, Cr and Se) by inductively coupled plasma mass spectrometry (ICPMS). Then, a new ion chromatography method with on-line detection by ICPMS was developed for the simultaneous speciation analysis of As, Cr and Se in the bio-accessible fraction to determine the portion of these elements that was actually toxic. Using gradient elution, four As species (As(III), As(V), monomethylarsonic acid and dimethylarsinic acid), two Cr species (Cr(III) and Cr(VI)) and two Se species (Se(IV) and Se(VI)) were separated within 12 min. The simultaneous speciation analysis of As, Cr and Se revealed that the simple act of cooking can convert all of the carcinogenic Cr(VI) to the safer Cr(III).
Risk assessment of food safety must be carried out to ensure the well-being of consumers. In general, this involves determination of the total concentration of elements in food. If the toxicity of elements strongly depends on their chemical form, then speciation analysis of food extracts may be performed. However, this does not take into account species transformation that may occur during digestion in the gastro-intestinal tract. Furthermore, dissolution in the gastro-intestinal tract must occur before absorption into the blood stream, where toxic effects may ensue. A more realistic approach to risk assessment should thus involve speciation analysis of the bioaccessible fraction from food. Arsenic, selenium and chromium are three elements that are safe in some forms or at some levels and are toxic in other forms or other levels because their toxicological and biological properties are speciation-dependent.1-5 They are often present in grains, such as rice,6, 7 as a result of contamination of irrigation water or soil, or naturally high levels in soil. The most toxic As species is As(III), followed closely by As(V), both of which are carcinogens, whereas monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) are less toxic but possible carcinogens.8, 9 The most toxic Cr species is Cr(VI), a carcinogen, 10 whereas Cr(III) has been considered a micronutrient although its essentiality has yet to be proven.11,12 Selenium is a micronutrient but in higher doses can pose a risk even if it is in a safe form.13 Both Se(IV) and Se(VI) are considered toxic species.14 For risk assessment of food safety, the As, Cr and Se species discussed in the preceding paragraph need to be determined. A survey of the literature revealed only a handful of publications on the simultaneous speciation analysis of As, Cr and Se.15-19 However, only As(III), As(V), Cr(III), Cr(V), Se(IV) and Se(VI) were considered because those are the prevalent forms of As, Cr and Se in natural and waste waters15,17,18 as well as ash,16 cement19 and soil leachates.16. A review on multielemental speciation analysis using liquid chromatography coupled to inductively coupled plasma mass spectrometry (ICPMS) predominantly focused on water and food sam-
ples.20 No method could be found for the simultaneous speciation analysis of As, Cr and Se in the bio-accessible fraction from food, which thus became the aim of this study. Although bio-availability, i.e., the fraction reaching the blood stream,21 should ideally be measured, only the fraction dissolved in the gastro-intestinal tract, i.e. bio-accessibility, is available for absorption into the circulatory system. Indeed, bio-availability is equal to bio-accessibility only in the worst case scenario.22 As a result, the measurement of bio-accessible species of potentially toxic elements can be used for a more realistic risk assessment than the speciation analysis of food. The food selected for this study is rice because several diets contain large amounts of rice.23,24 For instance, it is the main form of carbohydrates in the diet of those who have celiac disease.23 Furthermore, it is consumed worldwide by over half of the world’s population.8, 25 Although there may not be an elemental difference between rice that was organically grown or not,26, 27 there could be differences in bio-accessibility and in speciation. For this reason, this study investigates organically grown white and brown rice because they have never been examined in such a realistic risk assessment way. It also looks at basmati rice for the same reason. To this end, a method previously used for the speciation analysis of As in artificial saliva and gastric juice leachates by ion exchange chromatography (IEC) with detection by ICPMS28 was modified to enable the simultaneous speciation analysis of As, Cr and Se in these matrices so as to minimize the total analysis time. The leachates were obtained using a method previously developed,29, 30 involving sequential leaching of rice by artificial saliva, gastric juice and intestinal juice. The newly developed speciation analysis method was used to study the effect of cooking on speciation, as a previous study reported transformation of As(V) and DMA to As(III), the most toxic form.30 The effect of washing rice with clean water prior to cooking, which was reported to remove a significant amount of As,30-32 was also examined to see if this simple sample pretreatment, which can be easily done by consumers, could be used to reduce the concentration of toxic species.
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Analytical Chemistry
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The method that is applied to rice here would be equally applicable to other types of food, as the separation conditions are governed by the gastro-intestinal fluids used for leaching.
EXPERIMENTAL SECTION Reagents. Artificial saliva was prepared by adding 6.8 g of KH2PO4 (ACS grade; Fisher Scientific, New Jersey, USA) and 77 mL of 0.2 mol/L NaOH (ACS grade; BioShop, Burlington, ON, Canada) to a volumetric flask followed by dilution to 1 L using doubly deionized water (DDW) (18.2 MΩ cm) while maintaining the pH at 6.5. All DDW used was purified using an Arium Pro UV|DI water purification system (Sartorius Stedim Biotech, Göttingen, Germany). Artificial gastric juice was prepared by adding 2.0 g of NaCl (ACS grade, BioShop, Burlington, ON, Canada), 3.2 g of pepsin (Sigma-Aldrich, Oakville, ON, Canada), 7.0 mL of sub-boiled HCl (ACS grade; Fisher Scientific, Ottawa, ON, Canada), and diluting to a final volume of 1 L using DDW in a volumetric flask. Artificial intestinal fluid was prepared by mixing 6.8 g of KH2PO4, 10 g of pancreatin (Sigma-Aldrich, St. Louis, MO, USA), 77 mL of 0.2 mol L-1 NaOH, diluting to a final volume of 1 L using DDW, with the pH maintained at 6.8. For the digestion of total rice and residues, sub-boiled HNO3 (ACS grade; Fisher Scientific, Ottawa, ON, Canada) and H2O2 (J.T. Baker, Phillipsburg, NJ, USA) were used. All HNO3 and HCl were purified using a DST-1000 sub-boiling distillation system (Savillex, Minnetonka, MN, USA). Elemental standard solutions were prepared from monoelemental solutions (1000 mg L-1) (SCP Science, Baie d’Urfé, QC, Canada). For speciation analysis, 1000 mg L-1 As standard stock solutions were made of each species from the following reagents: As(III) oxide with 0.2% NaOH (As(III)) (99.999%), As(V) oxide (As(V)) (99.9%) (all Alfa Aesar, Ward Hill, USA), cacodylic acid (DMA) (≥98%)(Sigma– Aldrich, St. Louis, MO, USA) and disodium methyl arsenate (MMA) (97.5%) (ChemService, West Chester, PA, USA). All stock solutions were then further diluted to 10 mg L-1 prior to being stored at 4°C in the dark. From these stock solutions, calibration standards were prepared daily. Similarly, solutions of individual species were prepared from sodium selenate (Se(VI)), sodium selenite (Se(IV)) (Sigma–Aldrich, St. Louis, MO, USA), Cr(VI) oxide (Cr(VI)) (VWR International, Graumanngasse, Vienna) and Cr(III) acetate hydroxide (Cr(III)) (Sigma–Aldrich, St. Louis, USA). Sub-boiled HNO3, methanol (Fisher Scientific, Ottawa, ON, Canada) and DDW were used to prepare the speciation analysis mobile phases. Samples of long grain organic brown and white rice as well as white long grain basmati rice were purchased from a local natural food store and were used as purchased. The organic brown and white rice originated from Arkansas and California respectively, while the basmati rice came from a rice-growing region in India. The rice (either brown or white) was cooked by adding 20 g of rice to 50 mL of boiling DDW and waiting until the rice absorbed all the water. Instrumentation. A Varian 820MS ICPMS instrument, equipped with a Peltier cooled Scott double-pass spray chamber and a collision reaction interface (CRI), was used (all Varian Inc., Mulgrave, Victoria, Australia) with a T2100 Burgener nebulizer (Burgener Research, Mississauga, ON, Canada). Speciation analysis was done using a DX600/BioLC liquid chromatography system equipped with a GS50 gradient pump, a 25-µL injection loop, an IonPac AG7 guard column, and an
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IonPac AS7 (25-cm long, 4-mm diameter) anion exchange column (all Dionex, Oakville, ON, Canada). Polyether ether ketone tubing (0.17-mm internal diameter) connected the analytical column to the nebulizer. During on-line leaching and speciation analysis, data acquisition was done in time-resolved mode with five points per peak and one scan per replicate. For the analysis of residues, data acquisition was in steady-state mode with a 10-s integration time. Multivariate Optimization of CRI Conditions. Multivariate optimization of the nebulizer gas flow rate, sampling depth, radio frequency (RF) power to the plasma, argon sheath gas flow rate and CRI skimmer gas flow rate was conducted using Minitab 16. Table 1 summarizes the resulting conditions as well as the separations conditions. The CRI H2 gas flow rate was optimized while monitoring the ratio of the 40 Ar35Cl+ interference signal (mass-to-charge ratio (m/z)=75) to the signal of Ge+ (m/z=74). Ge was used as surrogate analyte due to its closeness in m/z to that of As. On-line mixing of gastric juice and Ge solution (5 µg L-1) was conducted using a Y connector. The signal ratio of m/z=75 over m/z=74 was continuously monitored while increasing the CRI H2 gas flow rate. The H2 flow rate corresponding to the minimum 40 Ar35Cl+/74Ge+ response ratio was selected. While this addition significantly decreased the analyte signal, it was required in order to obtain accurate results for the bulk analysis of gastric juice leachates, due to the high chloride ion concentration that induced 40Ar35Cl+ polyatomic interference on the only As isotope. On-line Leaching Method. 0.2-0.25 g of rice was rolled in quartz wool and packed into a PTFE tube (8-cm long, 3/15-in outer diameter, 1/8-in inner diameter), with a quartz wool plug at each end to prevent the escape of particles. A mini-column containing only quartz wool was also used as a blank. All quartz wool was soaked overnight in 10% (v/v) HNO3, rinsed with DDW and dried prior to use. The artificial saliva, gastric juice, intestinal fluid, mini-columns and standards were all maintained at 37 ⁰C (human physiological temperature) using a thermostatically controlled water bath. Saliva, then gastric juice, followed by intestinal fluid, were sequentially pumped through the mini-column to the nebulizer of the ICPMS instrument using the instrument’s peristaltic pump. The leaching time was set to 5 min for each reagent, as no additional analyte was then released. Analytes were monitored in time-resolved mode to obtain real-time leaching data. External calibration was performed using matrix-matched standard solutions and Table 1. ICPMS and IEC Operating Conditions Parameter Ar plasma gas flow rate Ar auxiliary gas flow rate Sample uptake rate Plasma RF power Ar sheath gas flow rate Ar aerosol carrier gas flow rate H2 CRI skimmer gas flow rate Sampling depth (mm) Dwell time (ms)
Monitored ions IEC column temperature Mobile phase flow rate Mobile phase A Mobile phase B Mobile phase C Gradient elution program
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Optimal setting (range tested) 18.0 L min-1 1.75 L min-1 1.0 mL min-1 1.44 (1.3-1.5) kW 0.04 (0-0.10) L min-1 1.1 (0.7-1.2) L min-1 65 (0-100) mL min-1 6.0 (5-7) mm 10 ms (total analysis) 80 ms (As), 250 ms (Se), 500 ms (Cr) (speciation analysis) 52,53 Cr+, 75As+, 77, 78Se+ 20 °C 1.35 mL min-1 0.5 mM HNO3, 1% MeOH 50 mM HNO3, 1% MeOH 0.8 M HNO3 100% A, 3 min; 100% B, 2.5 min; 100% C, 10 min
blank, which were injected through a 100-µL injection loop, using a valve (Model 5020, Rheodyne, Cotati, CA) connected to a universal automatic actuator (Anachem Ltd., Luton, England), into a carrier of the same leaching reagent (i.e. artificial saliva, gastric juice or intestinal juice). A five-point matrixmatched calibration based on peak area was used for analysis of the leachates. Mass Balance. To verify mass balance, residues were digested with 2.5 mL of sub-boiled HNO3 and 1 mL of H2O2 heated to 50 °C for 1 hour. The solution was then diluted to 20 mL with DDW. Total digestions of 1-g rice aliquots were also carried out using the same procedure. All digestions were quantified with a 5-point matrix-matched external calibration curve and a blank prepared daily, with internal standardization through the on-line addition of a 5 µg L-1 In solution. Effect of Washing Rice. Room temperature DDW was pumped through a raw rice mini-column with on-line monitoring by ICPMS before sequentially pumping the three artificial bodily fluids through it. Speciation Analysis of As, Cr and Se. 2-mL aliquots of saliva leachates at 37 °C were collected, followed by 4 mL of gastric juice leachates (37 °C), from a packed mini-column off-line at a flow rate of 0.8 mL min-1. Saliva leachates were diluted 5-fold with DDW prior to analysis, while gastric juice leachates were left undiluted as previously recommended.28 A gradient elution was carried out with three HNO3 mobile phases (pH 3.3, 1.1 and 0.1 with mobile phases A and B also containing 1% methanol) (see Table 1). This enabled separation of four As species, two Cr species and two Se species of interest. Blanks and standard solutions that were matrix-matched were injected through the IEC column to create a 5-point external calibration curve (using peak areas), which was used for the quantitative analysis of samples.
RESULTS AND DISCUSSION Separation conditions. Figure 1 shows that the baseline separation of 8 species was achieved within 12 min in 5-fold diluted artificial saliva. However, overlapping of some species of As and Se was observed in gastric juice matrix, as shown in Figure 2. Nonetheless, the separation was still sufficient, as all species of each element were separated from each other and the elements were separated by mass spectrometry. Past work,30 which validated the first two elution steps for the separation of As species with gastro-intestinal extracts of a rice flour CRM, mentioned an interference at around 450 s during the speciation analysis of a gastric juice matrix. This interference was also observed in this study (Figure 2) at m/z 40000
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Analytical Chemistry 70000 Signal (counts/s)
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Figure 2. Chromatogram obtained using the conditions in Table 1 for 100 µg L-1 each of five species in artificial gastric juice 75 and 77, but not at m/z 78. It was thus identified as ArCl+. Although it was baseline-separated from As and Se species, CRI was nonetheless used during speciation analysis of both saliva and gastric juice leachates because of possible interference from polyatomic ions (such as ArC+ and ArO+) on Cr. To help confirm the absence (or presence) of interference, two isotopes each were monitored for Cr and Se. The addition of a third mobile phase of higher nitric acid concentration to the two mobile phases previously used for speciation analysis of As29,30 allows for the elution of not only Se(VI), but also Cr(III) and Cr(VI). Unlike the first two mobile phases, the third mobile phase does not include methanol. The addition of methanol allows for the increased degree of ionization of elements with high ionization potential (i.e. As and Se) through charge transfer with carbon ions.33 It was avoided in the third mobile phase so as not to exacerbate ArC+ interferences on the most abundant Cr isotopes (i.e. at m/z 52 and 53). Because rice is a carbohydrate and thus a significant source of C, tests were conducted with and without CRI gas, which revealed that the CRI gas was required to reduce interferences. The resulting detection limits (measured as three times the standard deviation of the blank signal divided by sensitivity, i.e. the slope of the calibration curve) in saliva and gastric juice leachates are summarized in Table 2, where they are compared to those in DDW. The small degradation observed in saliva stems from the 5-fold dilution that was required to prevent a significant degradation of the separation.28 In the case of gastric juice, detection limits were similar to those in water for As and Se species, because 1% (v/v) MeOH in the mobile phase enhanced sensitivity for As and Se. However, they were degraded for Cr, because of the higher background, despite the use of CRI. Calibrations in each matrix were linear up to at least 100 µg L-1 of each species. Detector saturation resulted above 250 µg L-1. Table 2. Detection Limits in Different Matrices Species
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Water Saliva Gastric Juice Concen- Absolute Concen- Absolute Concen- Absotration (ng) tration (ng) tration lute (µg L-1) (µg L-1) (µg L-1) (ng) 0.2 0.005 0.5 0.01 0.1 0.003 0.06 0.002 0.5 0.01 0.1 0.003 0.1 0.003 0.5 0.01 0.1 0.003 0.3 0.008 5 0.1 0.4 0.01 0.8 0.02 2 0.05 1 0.03 1 0.03 2 0.05 1 0.03 1 0.03 30 1 4 0.1 1 0.03 20 0.5 3 0.08
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Analytical Chemistry Table 3. Percentage (± standard deviation, n=6) of the Total Element Concentration that is Released from Raw Rice by Saliva and Gastric Juice as well as by all Three GastroIntestinal Reagents
Brown Basmati
As 95±18 95±18 72±18 83±20 74±13 78±13
Cr 65±12 79±13 59±16 70±18 70±18 80±20
Se 92±26 92±24 58±13 63±13 83±23 88±24
Speciation analysis of the bio-accessible fraction from raw rice. Given that saliva and gastric juice released most of the bio-accessible As, Se and Cr (Table 3), very little being released by intestinal juice, only speciation analysis of each saliva and gastric juice leachate was carried out. Confirmation of the elution times of species in gastro-intestinal leachates from rice samples was made using standard solution spikes. Figure 3 summarizes the results for the raw rice samples. In each case, mass balance was verified: the sum of species concentrations of each element in each leachate was in agreement with the total concentration of that element measured by direct on-line monitoring of the leachate by ICPMS (labeled “total leached”) according to a Student’s t test at the 95% confidence level (which was performed after an F test to establish if there As concentration (µg/kg)
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was a difference in variance and then use the appropriate t test). In each case, the sum of the concentrations of bioaccessible species and the total concentration left in the residue (not shown) was also in agreement with the total concentration found after digestion of an aliquot of rice, according to a Student’s t test at the 95% confidence level. Except for As and Se in white rice, the bio-accessibility of all three elements is significantly less than 100%. Taking bioaccessibility into account is thus important for realistic risk assessment. Only Se(VI) was detected in all leachates. All leachates also contained As(III), As(V) and Cr(III). In basmati and organic brown rice, some of the As(V) and As(III) was replaced by the less toxic DMA. The fact that all saliva leachates included both Cr(III) and Cr(VI) but gastric juice only contained Cr(III) suggests that HCl reduced Cr(VI). Speciation analysis of the bio-accessible fraction from cooked rice. Comparison of Figure 4 to Figure 3 reveals that cooking rice converted any and all Cr(VI) into the safer Cr(III). This is very encouraging as rice is typically consumed after being cooked. This trend is however not observed for As and Se where the more toxic forms (As(III), As(V) and Se(VI)) are still present after cooking. However, in the case of white rice, cooking converted some inorganic As into the less toxic DMA. On the other hand, the reverse was observed for basmati rice: cooking converted DMA into the more toxic
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Figure 4. Total and bio-accessible concentrations of As, Se and Cr and their species in cooked organic white, organic brown and basmati rice (n=6, error bars=standard deviation).
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Analytical Chemistry
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Figure 5. Percentage of As, Se and Cr that is removed from raw rice by washing rice with DDW (n=6, error bars= standard deviation). inorganic As forms. As for raw rice, no MMA or Se(IV) was detected. In all cases, the sum of species concentrations was in agreement with the total concentration measured separately by on-line leaching. As well, the sum of bio-accessible concentrations with the concentration left in the residue was in agreement with the total concentration found after digestion of an aliquot of rice, according to a Student’s t test at the 95% confidence level. Effect of washing rice prior to cooking. Figure 5 shows that at least half of the Cr and As was removed by simply washing rice with high purity water. On the other hand, DDW removed at the most 15% of the Se. Nonetheless, in all cases, a significant amount of toxic elements was removed by a quick washing step. All rice should thus be washed with Asfree water before cooking. This should also be done before processing (such as grinding to make flour, puffing to make rice cereals, etc.) or using rice for other rice products (such as rice milk). CONCLUSIONS The simultaneous speciation analysis method that was developed in this work provides valuable information within 12 min on the chemical forms of As, Cr and Se in the bioaccessible fraction of food, which is required for realistic risk assessment of food safety. Its application to three different rice samples revealed that 72-95% of As, 59-70% of Cr and 58-92% of Se were bio-accessible from raw rice and that a significant portion of the bio-accessible fraction was in a toxic form for As and Cr, while all bio-accessible Se was in the toxic Se(VI) form. Furthermore, it also revealed that cooking rice had a positive impact: it converted the toxic Cr(VI) into the safer Cr(III) form. On the other hand, it had no effect on Se. Depending on the rice, cooking could have no effect on As speciation, convert inorganic As into less toxic species or convert DMA into the more toxic inorganic As. This new speciation analysis method will enable a detailed study on what governs species inter-conversion during cooking. For example, in the case of rice, does pressure cooking, as opposed to atmospheric pressure cooking done in this work, have a different effect? It can also be used to study the stability of species in leachates to determine how long they can be stored without change in speciation before analysis. This study also showed the significant beneficial effect of adding a washing step before cooking rice, as over half of the As and Cr could be removed. In the cases of the three rice samples considered, the amount of inorganic As left after washing was less than the European Union recommended
maximum of 100 µg kg-1 for young children and 200 µg kg-1 for adults.34 Hence, all three rice samples are safe for consumption by adults and children. Future work will include application of this method to other staple food (such as wheat, corn, etc.) and a close look at rice cereals for babies. Indeed, the inorganic As concentration in rice products (cereals, crackers, etc.) was reported to constitute a health risk for infants because infants and young children have three times the food consumption rate of adults based on body weight, and are at greater risk of experiencing toxicological issues as they are going through rapid development.35,36
AUTHOR INFORMATION Corresponding Author * E-mail:
[email protected]; telephone: 1-613533-2619; fax: 1-613-533-6669.
Present Addresses † McGill University, Department of Food Science and Agricultural Chemistry, 21111 Lakeshore, Ste Anne de Bellevue, QC H9X 3V9. Author Contributions All authors have given approval to the final version of the manuscript.
Funding Sources The Natural Sciences and Engineering Research Council of Canada funded this research.
ACKNOWLEDGMENT The authors gratefully acknowledge the financial support of the Natural Sciences and Engineering Research Council of Canada (grant number 39487-2013). NWS is grateful for a Marie Mottashed scholarship and graduate awards from Queen’s School of Graduate Studies and Research.
ABBREVIATIONS IEC, ion exchange chromatography; ICPMS, inductively coupled plasma mass spectrometry; DDW, doubly deionized water.
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