The authors thank Dr. F. Cordeiro of the JRC for the useful discussions about statistical treatment of data. Expert laboratories in the analysis of iAs in biological matrices that provided results to be used as assigned value in PTs and laboratories that took part in the studied PTs are acknowledged.

NOTE: All the material used needs to be decontaminated with 10% (m/v) HNO3 and rinsed at least twice with deionized water.
1. Hydrolysis
<ol>
	<li>Weigh accurately ca. 0.5 to 1 g of lyophilized sample (or the equivalent quantity of freshly homogenized sample e.g. 1 to 4 g) in a 50 mL polypropylene centrifuge tube with screw cap.</li>
	<li>Add 4.1 mL of deionized water.</li>
	<li>Agitate with a mechanical shaker for about 5 min until the sample is completely wet.</li>
	<li>Add 18.4 mL of concentrated hydrochloric acid (HCl), not less than 37% m/v.</li>
	<li>Agitate with a mechanical shaker for 15 min.</li>
	<li>Let rest for 12-15 h (for instance overnight).</li>
</ol>
2. Extraction
<ol>
	<li>Add 2 mL of hydrogen bromide (HBr) not less than 48% m/v and 1 mL of hydrazine sulfate (N2H6SO4) solution (15 mg/mL) to the hydrolyzed sample.</li>
	<li>Shake for 30 s with a mechanical shaker.</li>
	<li>Add 10 mL of chloroform (CHCl3).</li>
	<li>Shake for 5 min with a mechanical shaker.</li>
	<li>Centrifuge for 5 min at 800 x g.</li>
	<li>Pipette the chloroform phase (lower phase) into another 50 mL polypropylene centrifuge tube.</li>
	<li>Add again 10 mL chloroform to the remaining acid phase and repeat the extraction. At the end around 20 mL of chloroform should have been collected. Take care to avoid cross-contamination from the acid phase.</li>
</ol>
3. Clean-up of the Chloroform Phase
<ol>
	<li>Centrifuge the pooled chloroform phases for 5 min at 800 x g. The centrifugation time or speed may be increased if needed to achieve a clear separation of the two phases.</li>
	<li>Remove all acid phase residues remaining on the chloroform with a 1 mL pipette. This step is crucial. Any acid phase residues remaining in the chloroform phase will lead to overestimated iAs results because all other arsenic species in the sample are present in the acid phase.</li>
	<li>Filter through a hydrophobic PTFE membrane (25 mm diameter) to remove the remaining solid or acid phase residues present in the chloroform phase and collect the chloroform phase in a 50 mL polypropylene centrifuge tube.</li>
</ol>
4. Back-extraction
<ol>
	<li>Add 10 mL of 1 M HCl to back extract iAs from the chloroform phase collected after the filtration step.</li>
	<li>Shake for 5 min with a mechanical shaker.</li>
	<li>Centrifuge for 5 min at 800 x g.</li>
	<li>Pipette the acid phase (upper phase) and pour it into a 250 mL glass beaker (e.g. Pyrex) for mineralization.</li>
	<li>Repeat the back-extraction and combine the collected HCl phases.</li>
</ol>
5. Sample Mineralization
Note: This step allows elimination of interferences and pre-concentration in samples in which the iAs mass fraction is close to or below the quantification limit, and it is frequently omitted by laboratories which use this protocol with ICP-MS for final determination instead of HG-AAS .
<ol>
	<li>Suspend 20 g of magnesium nitrate hexahydrate [Mg(NO3)26H2O] and 2 g of magnesium oxide (MgO) in 100 mL of deionized water. Add 2.5 mL of this suspension to the glass beaker. Shake the suspension while adding it to avoid precipitation.</li>
	<li>Add 10 mL of concentrated HNO3 of at least 65% m/v and evaporate to dryness in a sand bath (or a thermal plate), avoiding any projections. To verify that the samples are totally dry, place a watch glass on top of the glass beaker and check that no condensation is formed.</li>
	<li>Cover the beakers with watch glasses and place them in a muffle furnace at an initial temperature not exceeding 150 &#186;C and progressively increase the temperature to 425 &#177; 25 &#176;C at a rate of 50 &#176;C/h. Maintain at 425 &#176;C for 12 h. This step is critical. To avoid any projections the rate of increase in temperature must be strictly implemented.</li>
	<li>Allow the ashes to cool down to room temperature.</li>
	<li>Add 0.5 mL of deionized water to wet the ash and add then 5 mL of 6 M HCL. Take care to recover all the ash from the walls of the glass beaker. Dissolve the ash completely, shaking if necessary.</li>
	<li>Add 5 mL of pre-reducing agent, prepared by dissolving 5 g of potassium iodide (KI), and 5 g of ascorbic acid in 100 mL of deionized water, and wait 30 min to achieve a quantitative reduction of iAs to As(III).</li>
	<li>Filter the solution through a Whatman number 1 paper or equivalent and collect it in a 50 mL polypropylene centrifuge tube. Rinse the glass beaker twice with 6 M HCl. Collect the rinsing liquids in a 25 mL tube and make it up to a final volume with 6 M HCl 
	NOTE: When the iAs concentration in a sample is expected to be close or below the quantification limit of the method (0.010 mg/kg), or on the contrary, high, the mineralization steps 5.5-5.7 should be modified using the volumes given in Table 1, which would provide a lower quantification limit. Re-dissolved and pre-reduced samples are stable for 24 h at 4 &#176;C. At least two reagent blanks should be used for the whole analytical process.</li>
</ol>
6. Calibration
NOTE: For quantification purposes use an external calibration curve of As(III) in the range 0.5 - 10 &#181;g/L. Use a 1000 mg/L As(V) commercially available certified standard solution to construct the calibration curve applying subsequent dilutions.
<ol>
	<li>Prepare a 10 mg/L As(V) standard solution by pipetting 1 mL of the 1,000 mg/L standard solution in a 100 mL volumetric flask and filling to the mark with 6 M HCl.</li>
	<li>Prepare a 0.1 mg/L As(V) standard solution by pipetting 1 mL of the 10 mg/L As(V) standard solution in a 100 mL volumetric flask and filling to the mark with 6 M HCl.</li>
	<li>Prepare a 25 &#181;g/L As(V) standard solution pipetting 25 mL of the 0.1 mg/L As(V) standard solution in a 100 mL volumetric flask and filling to the mark with 6 M HCl.</li>
	<li>Prepare the calibration curve of As(III) as follows: pipette from the 25 &#181;g/L As(V) standard solution the volumes given in Table 2 into 50 mL volumetric flask, add 10 mL of the pre-reducing solution in each volumetric flask, wait 30 min, then fill to the mark with 6 M HCl. Other volumes are suitable provided that they maintain the proportions described above.</li>
	<li>Prepare a calibration blank as follows: pipette 10 mL 6 M HCl and 10 mL pre-reducing solution in a 50 mL volumetric flask. Wait 30 min and fill then to the mark with 6 M.</li>
	<li>Use the standards marked as QC1 and QC2 in Table 2 as quality control: QC1 ensures that the quantification at low concentration level is correct and QC2, ensures that the response is stable at high concentrations, with no significant drift in time.</li>
</ol>
7. Determination
<ol>
	<li>Use an atomic absorption spectrometer equipped with an auto-sampler, a flow injection-hydride generation system, and an electro-thermally heated quartz cell for detection and quantification purposes, following the instrumental conditions for quantification of iAs by FI-HG-AAS as listed in Table 3.</li>
</ol>
8. Quantification
<ol>
	<li>Calculate the iAs mass fraction in the samples analyzed (expressed in mg/kg), using the following equation: 
	<img alt="Equation 1" src="/files/ftp_upload/55953/55953eq1.jpg" /> 
	Where: 
	Cx: Concentration in the extract (&#181;g/L), calculated from the calibration curve 
	CBI: Concentration in the reagent blank sample (&#181;g/L), extrapolated from the calibration curve 
	V: Final volume of the sample mineralization step (5.7), usually V= 25 mL 
	w: Weight of sample (in grams)</li>
</ol>

<ol>
	<li>European Commission. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Commission Regulation (EC) 1881/2006 setting maximum levels for certain contaminants in foodstuffs. , (2006).</li><li>EFSA Panel on Contaminants in the Food Chain (CONTAM). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scientific+Opinion+on+Arsenic+in+Food.">Scientific Opinion on Arsenic in Food.</a> EFSA J. 7 (10), 1351 (2009).</li><li>European Food Safety Authority. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dietary+exposure+to+inorganic+arsenic+in+the+European+population.">Dietary exposure to inorganic arsenic in the European population.</a> EFSA J. 12 (3), 3597 (2014).</li><li>de la Calle, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+the+determination+of+inorganic+arsenic+in+rice+depend+on+the+method?.">Does the determination of inorganic arsenic in rice depend on the method?.</a> TrAC. 30 (4), 641-651 (2011).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GB/T5009.11-2003.">GB/T5009.11-2003.</a> Determination of total arsenic and abio-arsenic in foods. , (2003).</li><li>European Committee for Standardisation. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> EN 15517:2008 "Determination of trace elements-Determination of inorganic As in seaweed by hydride generation atomic absorption spectrometry (HG-AAS) after digestion". , (2008).</li><li>de la Calle, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+it+possible+to+agree+on+a+value+for+inorganic+arsenic+in+food?+The+outcome+of+IMEP-112.">Is it possible to agree on a value for inorganic arsenic in food? The outcome of IMEP-112.</a> Anal Bioanal Chem. 404 (8), 2475-2488 (2012).</li><li>Schmeisser, E., Goessler, W., Kienzl, N., Francesconi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volatile+analytes+formed+from+arsenosugars:+determination+by+HPLC-HG-ICPMS+and+implications+for+arsenic+speciation+analyses.">Volatile analytes formed from arsenosugars: determination by HPLC-HG-ICPMS and implications for arsenic speciation analyses.</a> Anal. Chem. 76 (2), 418-423 (2004).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> EN 16802:2016. Foodstuffs. Determination elements and their chemical species. Determination of inorganic arsenic in foodstuffs of marine and plant origin by anion-exchange HPLC-ICP-MS. , (2016).</li><li>European Committee for Standardisation. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Animal feeding stuffs &#8211; Determination of inorganic arsenic by hydride generation atomic absorption spectrometry (HG-AAS) after microwave extraction and separation by solid phase extraction (SPE). , (2012).</li><li>Rasmussen, R. R., Qian, Y., Sloth, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SPE+HG-AAS+method+for+the+determination+of+inorganic+arsenic+in+rice.+Results+from+method+validation+studies+an+a+survey+on+rice+products.">SPE HG-AAS method for the determination of inorganic arsenic in rice. Results from method validation studies an a survey on rice products.</a> Anal Bioanal Chem. 405 (24), 7851-7857 (2013).</li><li>Rasmussen, R. R., Hedegaard, R. V., Larsen, E. H., Sloth, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+validation+of+a+method+for+the+determination+of+inorganic+arsenic+in+rice.+Results+from+method+validation+studies+and+a+survey+on+rice+products.">Development and validation of a method for the determination of inorganic arsenic in rice. Results from method validation studies and a survey on rice products.</a> Anal Bioanal Chem. 403, 2825-2834 (2012).</li><li>Fiamegkos, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+of+a+method+based+on+atomic+absorption+spectrometry+to+determine+inorganic+arsenic+in+food:+Outcome+of+the+collaborative+trial+IMEP-41.">Accuracy of a method based on atomic absorption spectrometry to determine inorganic arsenic in food: Outcome of the collaborative trial IMEP-41.</a> Food Chem. 213, 169-179 (2016).</li><li>Llorente-Mirandes, T., Barbero, M., Rubio, R., L&#243;pez-S&#225;nchez, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Occurrence+of+inorganic+arsenic+in+edible+Shiitake+(Lentinula+edodes)+Products.">Occurrence of inorganic arsenic in edible Shiitake (Lentinula edodes) Products.</a> Food Chem. 158, 207-215 (2014).</li><li>de la Calle, M. B., Linsinger, T., Emteborg, H., Charoud-Got, J., Verbist, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Report+of+the+seventh+interlaboratory+comparison+organised+by+the+European+Union-Reference+Laboratory+for+Heavy+Metals+in+Feed+and+Food.">Report of the seventh interlaboratory comparison organised by the European Union-Reference Laboratory for Heavy Metals in Feed and Food.</a> IMEP-107: Total and inorganic As in rice. , (2010).</li><li>Cordeiro, F., Cizek-Stroh, A., de la Calle, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+total+and+inorganic+arsenic+in+rice.">Determination of total and inorganic arsenic in rice.</a> IRMM-PT-43 Proficiency Test Report. , (2016).</li><li>de la Calle, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> IMEP-112: Total and inorganic arsenic in wheat, vegetable food and algae. , (2011).</li><li>Fiamegkos, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+total+As,+Cd,+Pb,+Hg+and+inorganic+arsenic+in+chocolate.">Determination of total As, Cd, Pb, Hg and inorganic arsenic in chocolate.</a> EURL-HM-20 Proficiency test Report. , (2015).</li><li>ISO-Geneva (CH), International Organization for Standardization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ISO 17043: Conformity assessment - General requirements for proficiency testing. , (2010).</li></ol>

The authors have nothing to disclose.

A critical step in the described protocol is the clean-up of the chloroform phase (Step 3.2) because any acid phase residues remaining in the chloroform phase will lead to overestimated iAs results since all other arsenic species in the sample are present in the acid phase. This is of particular relevance when analyzing marine samples due to the presence of a plethora of organic species, which could account for most of the arsenic mass fraction present in the sample. The use of a hydrophobic PTFE (3.3) membrane is of paramount importance. If an emulsion is formed during the extraction of iAs into chloroform, the speed of centrifugation (3.1) can be increased. Other traditional approaches to eliminate emulsions can also be applied. Another critical step is the mineralization (Step 5.3). The rate of increase in temperature must be strictly implemented to avoid any projections which would reduce the iAs recovery leading to an uncontrolled negative bias and could be dangerous for the analyst.
As mentioned above some laboratories have used the evaluated method using ICP-MS instead of FI-HG-AAS. In such a case the dry ashing step (Step 5 in the protocol) is not needed and the 1 M HCl phase can be introduced into the ICP-MS. In the case of HG-AAS, due to its higher detection limit, a pre-concentration step which also eliminates possible interferences, is needed.
The percentage of satisfactory results obtained with the method described in this paper, both with and without the results reported for algae, is comparable to that of HPLC-ICP-MS and higher than that of HG-AAS. The latter technique (HG-AAS) is widely available but prone to interferences from organic arsenic species, especially in food commodities with a complex arsenic species distribution pattern. The lowest percentage of satisfactory results characterizes those obtained with &#34;Other methods&#34; but it must be kept in mind that it covers several analytical approaches, each one of them represented by a small amount of results, Figure 1. The method presented in this paper is an alternative to the more sophisticated/expensive HPLC-ICP-MS, still being characterized by a similar performance even in complex matrices. Frequently the use of hyphenated techniques, such as HPLC-ICP-MS, requires highly qualified operators and expensive infra-structures. The method presented in this paper can be implemented by any analyst trained in basic analytical chemistry.
There are some main drawbacks associated to the method. It is time-consuming since several steps must be followed to separate iAs from other arsenic species and to pre-concentrate iAs down to even sub-ppm levels. It implies the use of chloroform. There is a tendency to avoid the use of chlorinated compounds in the laboratories, due to the negative health effects that they could have. Nevertheless, if good laboratory practices are kept and samples are handled in fume hoods, those negative effects could be avoided. MMA will interfere in the determination of iAs. This must be kept in mind when analyzing samples in which MMA could be present, such as algae, fish and other seafood. However, MMA is normally present in small amounts which would be covered by the uncertainty associated to the results obtained for iAs.

Since January 2016 maximum levels for inorganic arsenic (iAs) in several rice commodities have been included in Commission Regulation (EC) 1881/2006 setting maximum levels for certain contaminants in foodstuffs1 with 0.10 &#181;g/L for rice destined for the production of food for infants and young children, 0.20 &#181;g/L for non-parboiled milled rice (polished or white rice), 0.25 &#181;g/L for parboiled rice and husked rice and 0.30 &#181;g/L for rice waffles, rice wafers, rice crackers and rice cakes. This update of the European legislation for contaminants in food followed the Scientific Opinion on Arsenic in Food of the European Food Safety Authority (EFSA)2 in which it is estimated that the exposure through diet to iAs for average and high consumers in Europe is such that can pose a risk to some consumers, keeping in mind that chronic exposure to iAs causes cancer of the lung, skin and bladder, and skin lesions. In the scientific report of EFSA on Dietary exposure to inorganic arsenic in the European population3, published in 2014, it is concluded that the main contributors to iAs in the diet for consumers of all ages are processed products made of cereals other than rice and that also rice, milk, dairy products and drinking water contribute significantly to iAs intake, with milk and dairy products being the main contributors for toddlers and infants.
In 2010 the European Union Reference Laboratory for Heavy Metals in Feed and food, EURL-HM, ran a proficiency test, IMEP-107, for the determination of iAs in rice, demonstrating that it was possible to determine iAs in rice with sufficient accuracy, regardless of the analytical method used4.
Several analytical methods have been validated for the determination of iAs in foodstuffs. China was the first country to introduce in its legislation a maximum level for iAs in rice. To make the implementation of legislation possible, a standard method was published in 2003 for the determination of what in the standard is called &#34;abio-arsenic&#34;5. The European Committee for Standardisation (CEN), published in 2008 a standardized method, EN 15517:2008, for the determination of iAs in seaweed6. The two methods are based on the use of optimized conditions to generate arsine only from iAs. In that way separation of iAs from other arsenic species that can also generate arsenic hydride is not needed. The final determination is done by atomic fluorescence5 or by hydride generation atomic absorption spectrometry, HG-AAS6. However, it is difficult to set the exact conditions to generate arsenic hydride without suffering from interference of other arsenic compounds and all the iAs mass fractions in algae reported in IMEP-112 (PT organized by the EURL-HM) obtained with those two methods, were scored as unsatisfactory7. Organoarsenic species, such as monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), and arsenosugars present in algae samples, can generate volatile hydrides too and could interfere in the determination of iAs leading to a positive bias in the results8.
Recently, CEN published a new standard method, EN 16802:2016, for the determination of iAs in foodstuffs of marine and plant origin using HPLC-ICP-MS9. Not all laboratories are equipped with that type of instrumentation and non-expensive, straight-forward methods are needed, in particular in countries with less developed laboratory infrastructures.
In 2012 CEN standardized a method for the determination of iAs in animal feeding stuffs by HG-AAS after microwave extraction and off-line separation of iAs by solid phase extraction (SPE), EN 16278:201210. This method which proved to be fit for analyzing iAs in feed could lack the sensitivity required to determine iAs in food of non-marine origin, which according to EFSA seems to be the main dietary contributors in Europe3. However, the same group that developed and validated EN 16278:2012 tested and successfully applied and validated the method to determine iAs in seafood and rice in a collaborative trial11,12.
An alternative method for the determination of iAs in food matrices after selective extraction of iAs into chloroform and further quantification by HG-AAS, was recently validated by the Joint Research Centre (JRC) in a collaborative trial13. The selectivity of the method is better than that of direct HG-AAS and is easy to implement not requiring the use of sophisticated instrumentation such as HPLC-ICP-MS. In this manuscript, the feasibility of using this method to determine iAs in a wide range of food matrices: vegetables, grains, mushrooms and food of marine origin, has been evaluated. Furthermore, the performance of laboratories that used the method in proficiency tests organized by the EURL-HM and the JRC covering several matrices is described.

<table><tbody><tr><td>Deionised water</td><td>Any available</td><td></td><td>18.2 M&amp;Omega; cm</td></tr><tr><td>Concentrated hydrochloric acid (HCl).</td><td>Any available</td><td></td><td>Not less than 37 % m/v,&lt;br/&gt; c(HCl) = 12 mol/L, with a&lt;br/&gt; density of&lt;br/&gt; approx. &amp;rho; (HCl) 1.15 g/L</td></tr><tr><td>Concentrated nitric acid (HNO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Any available</td><td></td><td>Not less that 65 % m/v,&lt;br/&gt; c(HNO3) = 14 mol/L, with a&lt;br/&gt; densitiy of approx.&lt;br/&gt; &amp;rho; 1.38 g/L</td></tr><tr><td>Chloroform</td><td>Any available</td><td></td><td>Harmful by inhalation and if swallowed. Irritating to skin.&lt;br/&gt; Wear suitable protective clothing and gloves.</td></tr><tr><td>Hydrogen bromide (HBr)</td><td>Any available</td><td></td><td>Not less than 48 % m/v</td></tr><tr><td>Hydrazine sulphate (N&lt;sub&gt;2&lt;/sub&gt;H&lt;sub&gt;6&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Any available</td><td></td><td>Harmful if swallowed.&lt;br/&gt; Causes burns.&lt;br/&gt; May cause cancer.</td></tr><tr><td>Magnesium nitrate hexahydrate [Mg(NO&lt;sub&gt;3&lt;/sub&gt;)6H&lt;sub&gt;2&lt;/sub&gt;O]</td><td>Any available</td><td></td><td></td></tr><tr><td>Magnesium oxide (MgO)</td><td>Any available</td><td></td><td></td></tr><tr><td>Potassium iodide (KI)</td><td>Any available</td><td></td><td></td></tr><tr><td>Ascorbic acid (C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;8&lt;/sub&gt;O&lt;sub&gt;6&lt;/sub&gt;)</td><td>Any available</td><td></td><td></td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>Any available</td><td></td><td></td></tr><tr><td>Sodium borohydride (NaBH&lt;sub&gt;4&lt;/sub&gt;)</td><td>Any available</td><td></td><td></td></tr><tr><td>Arsenic (V) standard solution</td><td>Any available</td><td></td><td>1,000 mg/L&lt;br/&gt; Use certified standard&lt;br/&gt; solutions commercially available</td></tr><tr><td>Centrifuge</td><td>Any available</td><td></td><td></td></tr><tr><td>Mechanical shaker</td><td>Any available</td><td></td><td></td></tr><tr><td>Sand bath</td><td>Any available</td><td></td><td></td></tr><tr><td>Muffle furnace</td><td>Any available</td><td></td><td></td></tr><tr><td>Polypropylene centrifuge (PC) tubes</td><td>Any available</td><td></td><td>50 mL with screw cap</td></tr><tr><td>Syringe filters with hydrophobic PTFE membrane</td><td>Any available</td><td></td><td>25 mm diameter</td></tr><tr><td>Pyrex glass beaker</td><td>Any available</td><td></td><td>Tall form 250 mL,&lt;br/&gt; capable of withstanding 500 &amp;deg;C</td></tr><tr><td>Watch glasses</td><td>Any available</td><td></td><td></td></tr><tr><td>Volumetric flasks</td><td>Any available</td><td></td><td>10, 25, 100 or 200,&lt;br/&gt; Class A.</td></tr><tr><td>Plastic funnels</td><td>Any available</td><td></td><td></td></tr><tr><td>Whatman n&amp;deg; 1 paper or equivalent</td><td>Any available</td><td></td><td></td></tr><tr><td>Atomic absorption spectrometer equipped with a flow injection system (FI-AAS)</td><td>Any available</td><td></td><td></td></tr></tbody></table>

determination of inorganic arsenic in a wide range of food matrices using hydride generation - atomic absorption spectrometry.

The European Food Safety Authority (EFSA) underlined in its Scientific Opinion on Arsenic in Food that in order to support a sound exposure assessment to inorganic arsenic through diet, information about distribution of arsenic species in various food types must be generated. A method, previously validated in a collaborative trial, has been applied to determine inorganic arsenic in a wide variety of food matrices, covering grains, mushrooms and food of marine origin (31 samples in total). The method is based on detection by flow injection-hydride generation-atomic absorption spectrometry of the iAs selectively extracted into chloroform after digestion of the proteins with concentrated HCl. The method is characterized by a limit of quantification of 10 &#181;g/kg dry weight, which allowed quantification of inorganic arsenic in a large amount of food matrices. Information is provided about performance scores given to results obtained with this method and which were reported by different laboratories in several proficiency tests. The percentage of satisfactory results obtained with the discussed method is higher than that of the results obtained with other analytical approaches.

The method was applied to determine the iAs mass fraction in several food commodities purchased from various Spanish markets. The results obtained with this method for a series of different matrices are classified in Table 4 following the categories used by EFSA3 in a report in which dietary exposure to inorganic arsenic in the European population is evaluated on the basis of data reported by Official Control Laboratories (OCL). The results in Table 4 represent the mean of three replicates &#177; the reproducibility standard deviation (SR) for the different food categories, calculated during the collaborative trial in which the present method was validated13. The results shown in Table 4 are in good agreement with other previously published in similar matrices11,12,14.
Of particular relevance are the results obtained for iAs in different types of rice because maximum limits are included for them in the European legislation for contaminants in food1. The highest values being obtained for brown rice and the lowest for white rice, in agreement with the findings of the OCLs3. The highest levels were found for the sea weed Hizikia fusiforme, whose consumption has been discouraged by several authorities as indicated in the report by EFSA.
The performance of laboratories that participated in PTs organized by the EURL-HM and the JRC and that used this method for the determination of iAs, has been compared to the performance of laboratories using other methods. Most of the other methods are based on HPLC-ICP-MS (around 50% of the evaluated results) and on HG-AAS without previous separation of iAs from other arsenic species (25% of the total), Figure 1. Other approaches used (around 15% of the evaluated results), were based on electrothermal atomization (ETAAS), fluorescence detection and ICP coupled to atomic emission spectroscopy (ICP-AES), with and without hydride generation, and are evaluated together under the name &#34;Other methods&#34; because the individual numbers would be too few to be of any statistical significance.
Some of the laboratories that have used the evaluated method introduced some variations to the original protocol and used ICP-MS instead of FI-HG-AAS. Frequently those laboratories did not apply the dry ashing step (Step 5 in the protocol) and just introduced the 1 M HCl phase into the ICP-MS. The PTs evaluated covered various matrices: rice15,16, wheat, spinach, algae17 and chocolate18.
The performance of laboratories was expressed as z-score: 
<img alt="Equation 2" src="/files/ftp_upload/55953/55953eq2.jpg" /> 
Where: 
xlab &#160;&#160;&#160;&#160;is the measurement result reported by a participant in a PT 
Xref &#160;&#160;&#160;&#160;is the assigned value (used to benchmark laboratories). In all PTs dealt within this paper the assigned value was established by a group of expert laboratories in the field of iAs analysis using different analytical methods. 
&#963; &#160;&#160;&#160;&#160;&#160;is the standard deviation for proficiency assessment, fixed by the PT provider taking into consideration the state of the art in a certain area of analysis. In the PTs considered in this paper &#963; was 15% of the assigned value for rice and wheat, 22% in algae and 25% for spinach and chocolate.
The interpretation of the z score is done according to ISO 17043:201019: 
|score| &#8804; 2&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;satisfactory (S) performance 
2 &#60; |score| &#60; 3&#160;&#160;&#160;&#160;questionable (Q) performance 
|score| &#8805; 3&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;unsatisfactory (U) performance
Seventy-five per cent of the results obtained with the method described above, got a satisfactory z-score. The determination of the iAs mass fraction in algae turned out to be challenging as expected, taking into consideration the complex distribution of arsenic species in matrices of marine origin. Two out of the three values reported in IMEP-112 for iAs in algae, using this method, got an unsatisfactory z-score. The same difficulty was observed among the results obtained with other methods. Excluding the results reported for iAs in algae, 85% of the results obtained with the evaluated method were satisfactory.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55953/55953fig1.jpg" /> 
Figure 1: Comparison of Performances (expressed as z-scores) of Laboratories Taking Part in PTs (IMEP-107, IMEP-112, EURL-HM-20 and IRMM-PT-43) with the Method Described in this Paper and with Other Commonly Applied Methods. S: satisfactory, Q: questionable and U: unsatisfactory. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55953/55953fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Expected iAs mass fraction 
			lower than 0.010 mg/kg</td>
			<td>Expected iAs mass fraction 
			higher than what is covered 
			by the calibration curve</td>
		</tr>
		<tr>
			<td>6 mol L-1 HCL volume used to re-dissolve the ashes (mL)</td>
			<td>2</td>
			<td>10</td>
		</tr>
		<tr>
			<td>Pre-reducing agent volume (mL)</td>
			<td>2</td>
			<td>10</td>
		</tr>
		<tr>
			<td>Final volume (mL)</td>
			<td>10</td>
			<td>50</td>
		</tr>
	</tbody>
</table>
Table 1: Modifications of the Protocol when Analyzing Samples in which Very Low or Very High iAs Concentrations are Expected.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Concentration in the 
			calibration curve (&#181;g/L)</td>
			<td>Aliquot (mL)</td>
		</tr>
		<tr>
			<td>0.5</td>
			<td>1</td>
		</tr>
		<tr>
			<td>1</td>
			<td>2 (QC1)</td>
		</tr>
		<tr>
			<td>2.5</td>
			<td>5</td>
		</tr>
		<tr>
			<td>5</td>
			<td>10 (QC2)</td>
		</tr>
		<tr>
			<td>7.5</td>
			<td>15</td>
		</tr>
		<tr>
			<td>10</td>
			<td>20</td>
		</tr>
		<tr>
			<td colspan="2">All As(III) calibration standard solutions shall be prepared freshly before each calibration.</td>
		</tr>
	</tbody>
</table>
Table 2: Aliquots to be taken from the 25 &#181;g/L As(V) standard solution to construct the As(III) calibration curve in a 50 mL final volume.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="4">Flow injection 
			Hydride generation</td>
			<td>&#183;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Loop sample: 0.5 mL (To be adapted when the reconstitution volume of the final pre-reducing solution is different from 25 mL).</td>
		</tr>
		<tr>
			<td>&#183;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Reducing agent: 0.2 % (w/v) NaBH4 in 0.05 % (w/v) NaOH; 5 mL/min flow rate.</td>
		</tr>
		<tr>
			<td>&#183;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; HCl solution 10 % (v/v), 10 mL/min flow rate.</td>
		</tr>
		<tr>
			<td>&#183;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Carrier gas: Argon, 100 mL/min flow rate.</td>
		</tr>
		<tr>
			<td rowspan="5">Atomic absorption 
			spectrometer</td>
			<td>&#183;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Wavelength: 193.7 nm</td>
		</tr>
		<tr>
			<td>&#183;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Spectral band-pass: 0.7 nm</td>
		</tr>
		<tr>
			<td>&#183;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Electrodeless discharge lamp system 2</td>
		</tr>
		<tr>
			<td>&#183;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Lamp current setting: 400 mA</td>
		</tr>
		<tr>
			<td>&#183;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Cell temperature: 900 &#176;C</td>
		</tr>
	</tbody>
</table>
Table 3: Instrumental Conditions used for iAs Quantification by HG-AAS.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Food</td>
			<td>i-As (&#181;g/kg fresh weight)</td>
		</tr>
		<tr>
			<td>Grain and grain-based products&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rice</td>
			<td>White</td>
			<td>113 &#177; 18</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>73 &#177; 12</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>56 &#177; 9</td>
		</tr>
		<tr>
			<td></td>
			<td>Brown</td>
			<td>197 &#177; 32</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>125 &#177; 20</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>275 &#177; 44</td>
		</tr>
		<tr>
			<td></td>
			<td>Parboiled</td>
			<td>134 &#177; 21</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>159 &#177; 25</td>
		</tr>
		<tr>
			<td></td>
			<td>Wafers</td>
			<td>162 &#177; 26</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>127 &#177; 20</td>
		</tr>
		<tr>
			<td>Vegetable and vegetable products</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dehydrated mushroom</td>
			<td>Boletus edulis</td>
			<td>174 &#177; 10</td>
		</tr>
		<tr>
			<td></td>
			<td>Galocybe gambosa</td>
			<td>74 &#177; 4</td>
		</tr>
		<tr>
			<td></td>
			<td>Marasmius oreades</td>
			<td>104 &#177; 6</td>
		</tr>
		<tr>
			<td></td>
			<td>Cantharellus lutescens</td>
			<td>16 &#177; 1</td>
		</tr>
		<tr>
			<td></td>
			<td>Lentinula edodes</td>
			<td>96 &#177; 6</td>
		</tr>
		<tr>
			<td>Sea weed</td>
			<td>Hizikia fusiforme</td>
			<td>97000 &#177; 14550</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>44943 &#177; 6742</td>
		</tr>
		<tr>
			<td></td>
			<td>Fucus vesiculosus</td>
			<td>288 &#177; 43</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>433 &#177; 65</td>
		</tr>
		<tr>
			<td>Fish and other seafood</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fish meat</td>
			<td>Flathead grey mullet</td>
			<td>53 &#177; 12</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>21 &#177; 5</td>
		</tr>
		<tr>
			<td></td>
			<td>European eel</td>
			<td>72 &#177; 16</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>42 &#177; 9</td>
		</tr>
		<tr>
			<td></td>
			<td>Crayfish</td>
			<td>33 &#177; 7</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>20 &#177; 4</td>
		</tr>
		<tr>
			<td></td>
			<td>Tuna</td>
			<td>11 &#177; 2</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>5 &#177; 1</td>
		</tr>
		<tr>
			<td>Molluscs</td>
			<td>Clam</td>
			<td>243 &#177; 54</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>133 &#177; 29</td>
		</tr>
		<tr>
			<td></td>
			<td>Mussel</td>
			<td>32 &#177; 32</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>139 &#177; 31</td>
		</tr>
	</tbody>
</table>
Table 4: Results Obtained for a Range of Different Matrices Applying the Described Method.

Watch this Scientific Journal Video about Determination of Inorganic Arsenic in a Wide Range of Food Matrices using Hydride Generation - Atomic Absorption Spectrometry. at JoVE.com

Determination of Inorganic Arsenic in a Wide Range of Food Matrices using Hydride Generation - Atomic Absorption Spectrometry.

The usefulness of an analytical method to determine inorganic arsenic in a wide range of food matrices is demonstrated. The method consists of selective extraction of inorganic arsenic into chloroform with a final determination by hydride generation-atomic absorption spectrometry.

The European Food Safety Authority (EFSA) underlined in its Scientific Opinion on Arsenic in Food that in order to support a sound exposure assessment to ...

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14.6K Views.  Joint Research Centre. The usefulness of an analytical method to determine inorganic arsenic in a wide range of food matrices is demonstrated. The method consists of selective extraction of inorganic arsenic into chloroform with a final determination by hydride generation-atomic absorption spectrometry.

Video: Determination of Inorganic Arsenic in a Wide Range of Food Matrices using Hydride Generation - Atomic Absorption Spectrometry.

Quantification of Heavy Metals and Other Inorganic Contaminants on the Productivity of Microalgae

Increasing demand for renewable fuels has researchers investigating the feasibility of alternative feedstocks, such as microalgae. Inherent advantages include high potential yield, use of non-arable land and integration with waste streams. The nutrient requirements of a large-scale microalgae production system will require the coupling of cultivation systems with industrial waste resources, such as carbon dioxide from flue gas and nutrients from wastewater. Inorganic contaminants present in these wastes can potentially lead to bioaccumulation in microalgal biomass negatively impact productivity and limiting end use. This study focuses on the experimental evaluation of the impact and the fate of 14 inorganic contaminants (As, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb, Sb, Se, Sn, V and Zn) on Nannochloropsis salina growth. Microalgae were cultivated in photobioreactors illuminated at 984 &#181;mol m-2 sec-1 and maintained at pH 7 in a growth media polluted with inorganic contaminants at levels expected based on the composition found in commercial coal flue gas systems. Contaminants present in the biomass and the medium at the end of a 7 day growth period were analytically quantified through cold vapor atomic absorption spectrometry for Hg and through inductively coupled plasma mass spectrometry for As, Cd, Co, Cr, Cu, Mn, Ni, Pb, Sb, Se, Sn, V and Zn. Results show N. salina is a sensitive strain to the multi-metal environment with a statistical decrease in biomass yieldwith the introduction of these contaminants. The techniques presented here are adequate for quantifying algal growth and determining the fate of inorganic contaminants.

Integration of microalgal cultivation with industrial flue gas will ultimately introduce heavy metals and other inorganic compounds into the growth media. This study presents a procedure used to determine the end fate and impact of heavy metals and inorganic contaminants on the growth of Nannochloropsis salina grown in photobioreactors.

Increasing demand for renewable fuels has researchers investigating the feasibility of alternative feedstocks, such as microalgae. Inherent advantages ...

Experimental Column Setup for Studying Anaerobic Biogeochemical Interactions Between Iron (Oxy)Hydroxides, Trace Elements, and Bacteria

Fate and speciation of trace elements (TEs), such as arsenic (As) and mercury (Hg), in aquifers are closely related to physio-chemical conditions, such as redox potential (Eh) and pH, but also to microbial activities that can play a direct or indirect role on speciation and/or mobility. Indeed, some bacteria can directly oxidize As(III) to As(V) or reduce As(V) to As(III). Likewise, bacteria are strongly involved in Hg cycling, either through its methylation, forming the neurotoxin monomethyl mercury, or through its reduction to elemental Hg&#176;. The fates of both As and Hg are also strongly linked to soil or aquifer composition; indeed, As and Hg can bind to organic compounds or (oxy)hydroxides, which will influence their mobility. In turn, bacterial activities such as iron (oxy)hydroxide reduction or organic matter mineralization can indirectly influence As and Hg sequestration. The presence of sulfate/sulfide can also strongly impact these particular elements through the formation of complexes such as thio-arsenates with As or metacinnabar with Hg.
Consequently, many important questions have been raised on the fate and speciation of As and Hg in the environment and how to limit their toxicity. However, due to their reactivity towards aquifer components, it is difficult to clearly dissociate the biogeochemical processes that occur and their different impacts on the fate of these TE.
To do so, we developed an original, experimental, column setup that mimics an aquifer with As- or Hg-iron-oxide rich areas versus iron depleted areas, enabling a better understanding of TE biogeochemistry in anoxic conditions. The following protocol gives step by step instructions for the column set-up either for As or Hg, as well as an example with As under iron and sulfate reducing conditions.

Experimental Column Setup for Studying Anaerobic Biogeochemical Interactions Between Iron OxyHydroxides, Trace Elements, and Bacteria

Fate and speciation of arsenic and mercury in aquifers are closely related to physio-chemical conditions and microbial activity. Here, we present an original experimental column setup that mimics an aquifer and enables a better understanding of trace element biogeochemistry in anoxic conditions. Two examples are presented, combining geochemical and microbiological approaches.

Fate and speciation of trace elements (TEs), such as arsenic (As) and mercury (Hg), in aquifers are closely related to physio-chemical conditions, such as ...

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

Dimethylated thioarsenicals such as dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV), which are produced by the metabolic pathway of dimethylarsinic acid (DMAV) thiolation, have been recently found in the environment as well as human organs. DMMTAV and DMDTAV can be quantified to determine the ecological effects of dimethylated thioarsenicals and their stability in environmental media. The synthesis method for these compounds is unstandardized, making replicating previous studies challenging. Furthermore, there is a lack of information about storage techniques, including storage of compounds without species transformation. Moreover, because only limited information about synthesis methods is available, there may be experimental difficulties in synthesizing standard chemicals and performing quantitative analysis. The protocol presented herein provides a practically modified synthesis method for the dimethylated thioarsenicals, DMMTAV and DMDTAV, and will help in the quantification of species separation analysis using high performance liquid chromatography in conjunction with inductively coupled plasma mass spectrometry (HPLC-ICP-MS). The experimental steps of this procedure were modified by focusing on the preparation of chemical reagents, filtration methods, and storage.

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

This article presents modified experimental protocols for dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV) synthesis, inducing dimethylarsinic acid (DMAV) thiolation through mixing of DMAV, Na2S, and H2SO4. The modified protocol provides an experimental guideline, thereby overcoming limitations of the synthesis steps that could have caused experimental failures in quantitative analysis.

Dimethylated thioarsenicals such as dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV), which are produced by the metabolic ...

Removal of Arsenic Using a Cationic Polymer Gel Impregnated with Iron Hydroxide

In this work, we prepared an adsorbent composed of a cationic polymer gel containing iron hydroxide in its structure designed to adsorb arsenic from groundwater. The gel we selected was the N,N-dimethylamino propylacrylamide methyl chloride quaternary (DMAPAAQ) gel. The objective of our preparation method was to ensure the maximum content of iron hydroxide in the structure of the gel. This design approach enabled simultaneous adsorption by both the polymer structure of the gel and the iron hydroxide component, thus, enhancing the adsorption capacity of the material. To examine the performance of the gel, we measured reaction kinetics, carried out pH sensitivity and selectivity analyses, monitored arsenic adsorption performance, and conducted regeneration experiments. We determined that the gel undergoes a chemisorption process and reaches equilibrium at 10 h. Moreover, the gel adsorbed arsenic effectively at neutral pH levels and selectively in complex ion environments, achieving a maximum adsorption volume of 1.63 mM/g. The gel could be regenerated with 87.6% efficiency and NaCl could be used for desorption instead of harmful NaOH. Taken together, the presented gel-based design method is an effective approach for constructing high-performance arsenic adsorbents.

In this work, we prepared an adsorbent composed of the cationic N,N-dimethylamino propylacrylamide methyl chloride quaternary (DMAPAAQ) polymer gel and iron hydroxide for adsorbing arsenic from groundwater. The gel was prepared via a novel method designed to ensure the maximum content of iron particles in its structure.

In this work, we prepared an adsorbent composed of a cationic polymer gel containing iron hydroxide in its structure designed to adsorb arsenic from ...

Atomic Absorbance Spectroscopy to Measure Intracellular Zinc Pools in Mammalian Cells

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in proteins and generating redox stress. Pathological conditions that lead to metal depletion or accumulation are causal agents of different human diseases. Some examples include anemia, acrodermatitis enteropathica, and Wilson&#8217;s and Menkes&#8217; diseases. It is therefore important to be able to measure the levels and transport of transition metals in biological samples with high sensitivity and accuracy in order to facilitate research exploring how these elements contribute to normal physiological functions and toxicity. Zinc (Zn), for example, is a cofactor in many mammalian proteins, participates in signaling events, and is a secondary messenger in cells. In excess, Zn is toxic and can inhibit absorption of other metals, while in deficit, it can lead to a variety of potentially lethal conditions.

Graphite furnace atomic absorption spectroscopy (GF-AAS) provides a highly sensitive and effective method for determining Zn and other transition metal concentrations in diverse biological samples. Electrothermal atomization via GF-AAS quantifies metals by atomizing small volumes of samples for subsequent selective absorption analysis using wavelength of excitation of the element of interest. Within the limits of linearity of the Beer-Lambert Law, the absorbance of light by the metal is directly proportional to concentration of the analyte. Compared to other methods of determining Zn content, GF-AAS detects both free and complexed Zn in proteins and possibly in small intracellular molecules with high sensitivity in small sample volumes. Moreover, GF-AAS is also more readily accessible than inductively coupled plasma mass spectrometry (ICP-MS) or synchrotron-based X-ray fluorescence. In this method, the systematic sample preparation of different cultured cell lines for analyses in a GF-AAS is described. Variations in this trace element were compared in both whole cell lysates and subcellular fractions of proliferating and differentiated cells as proof of principle.

Cultured primary or established cell lines are commonly used to address fundamental biological and mechanistic questions as an initial approach before using animal models. This protocol describes how to prepare whole cell extracts and subcellular fractions for studies of zinc (Zn) and other trace elements with atomic absorbance spectroscopy.

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in ...

Biochemistry

Quantification of Humic and Fulvic Acids in Humate Ores, DOC, Humified Materials and Humic Substance-Containing Commercial Products

The purpose of this method is to provide an accurate and precise concentration of humic (HA) and/or fulvic acids (FA) in soft coals, humic ores and shales, peats, composts and humic substance-containing commercial products. The method is based on the alkaline extraction of test materials, using 0.1 N NaOH as an extractant, and separation of the alkaline soluble humic substances (HS) from nonsoluble products by centrifugation. The pH of the centrifuged alkaline extract is then adjusted to pH 1 with conc. HCl, which results in precipitation of the HA. The precipitated HA are separated from the fulvic fraction (FF) (the fraction of HS that remains in solution,) by centrifugation. The HA is then oven or freeze dried and the ash content of the dried HA determined. The weight of the pure (i.e., ash-free) HA is then divided by the weight of the sample and the resulting fraction multiplied by 100 to determine the % HA in the sample. To determine the FA content, the FF is loaded onto a hydrophobic DAX-8 resin, which adsorbs the FA fraction also referred to as the hydrophobic fulvic acid (HFA). The remaining non-fulvic acid fraction, also called the hydrophilic fulvic fraction (HyFF) is then removed by washing the resin with deionized H2O until all nonabsorbed material is completely removed. The FA is then desorbed with 0.1 N NaOH. The resulting Na-fulvate is then protonated by passing it over a strong H+-exchange resin. The resulting FA is oven or freeze dried, the ash content determined and the concentration in the sample calculated as described above for HA.

This method provides a gravimetric quantification of humic substances (e.g., humic and fulvic acids) on an ash-free basis, in dry and liquid materials from soft coals (i.e., oxidized and non-oxidized lignite and sub-bituminous coal), humate ores and shales, peats, composts and commercial fertilizers and soil amendments.

The purpose of this method is to provide an accurate and precise concentration of humic (HA) and/or fulvic acids (FA) in soft coals, humic ores and ...

Biological Samples Preparation for Speciation at Cryogenic Temperature using High-Resolution  X-Ray Absorption Spectroscopy

The study of elements with X-ray absorption spectroscopy (XAS) is of particular interest when studying the role of metals in biological systems. Sample preparation is a key and often complex procedure, particularly for biological samples. Although X-ray speciation techniques are widely used, no detailed protocol has been yet disseminated for users of the technique. Further, chemical state modification is of concern, and cryo-based techniques are recommended to analyze the biological samples in their near-native hydrated state to provide the maximum preservation of chemical integrity of the cells or tissues. Here, we propose a cellular preparation protocol based on cryo-preserved samples. It is demonstrated in a high energy resolution fluorescence detected X-ray absorption spectroscopy study of selenium in cancer cells and a study of iron in phytoplankton. This protocol can be used with other biological samples and other X-ray techniques that can be damaged by irradiation.

This protocol presents a detailed procedure to prepare biological cryosamples for synchrotron-based X-ray absorption spectroscopy experiments. We describe all the steps required to optimize sample preparation and cryopreservation with examples of the protocol with cancer and phytoplankton cells. This method provides a universal standard of sample cryo-preparation.

The study of elements with X-ray absorption spectroscopy (XAS) is of particular interest when studying the role of metals in biological systems. Sample ...

Chemistry

Food Sample Homogenization and Digestion for Multi-Element Analysis

Preparation of Food Samples Using Homogenization and Microwave-Assisted Wet Acid Digestion for Multi-Element Determination with ICP-MS

Sample preparation is crucial for elemental determination, and various techniques are available, one of which involves homogenization followed by acid digestion. Special care is required during sample handling in the preparation stage to eliminate or minimize potential contamination and analyte loss. Homogenization is a process that simultaneously reduces particle size and uniformly distributes sample components. Following homogenization, the sample undergoes acid digestion, wherein it is digested with acids and auxiliary chemicals at elevated temperatures, transforming solid samples into a liquid state. In this process, metals in the original sample react with acids to form water-soluble salts. Samples prepared through acid digestion are suitable for elemental analysis using techniques such as inductively coupled plasma mass spectrometry, inductively coupled plasma optical emission spectroscopy, atomic absorption spectroscopy, electrochemical methods, and other analytical techniques. This work details the preparation of food samples for multi-element determination using inductively coupled plasma mass spectrometry. The step-by-step procedure involves the homogenization process using a laboratory-sized mixer with ceramic blades, followed by acid digestion in closed vessels using microwave-assisted wet acid digestion. A mixture of 5.0 mL of 68 wt% HNO3 and 1.0 mL of 30 wt% H2O2 serves as an auxiliary reagent. This guide provides an explanation of the processes involved in both stages.

Author Spotlight: Technologies and Challenges in Elemental Analysis of Food Samples

The presented protocol describes sample homogenization with a laboratory mixer, acid digestion of food samples using a mixture of 68 wt% HNO3 and 30 wt% H2O2&#160;via microwave-assisted wet acid digestion, and multi-element determination performed with inductively coupled plasma mass spectrometry.

Sample preparation is crucial for elemental determination, and various techniques are available, one of which involves homogenization followed by acid ...

Enhanced Isomeric Separation of Ergot Alkaloids Using LC-IMS-MS

Detection of Regulated Ergot Alkaloids in Food Matrices by Liquid Chromatography-Trapped Ion Mobility Spectrometry-Time-of-Flight Mass Spectrometry

Ion mobility mass spectrometry (IMS) acts as an additional separation dimension when integrated into liquid chromatography-mass spectrometry (LC-MS) workflows. LC-IMS-MS methods provide higher peak resolution, enhanced separation of isobaric and isomeric compounds, and improved signal-to-noise ratio (S/N) compared to traditional LC-MS methods. IMS provides another molecular characteristic for the identification of analytes, namely the collision cross section (CCS) parameter, reducing false positive results. Therefore, LC-IMS-MS methods address important analytical challenges in the field of food safety (i.e., detection of compounds at trace levels in complex food matrices and unambiguous identification of isobaric and isomeric molecules).
Ergot alkaloids (EAs) are a family of mycotoxins produced by fungi that attack a wide variety of grass species, including small grains such as rye, triticale, wheat, barley, millet, and oats. Maximum levels (MLs) of these mycotoxins have been established in several foodstuffs, as detailed in the Commission Regulation EC/2023/915. This new legislation includes six main EAs and their corresponding epimers, so an efficient methodology is required to properly distinguish these isomeric molecules considering their co-occurrence.
Therefore, the goal of this protocol is to show how the integration of IMS in LC-MS workflows contributes to the separation of isomeric EAs, enhancing the selectivity of the analytical method. Additionally, it illustrates how the generation of CCS libraries through the characterization of analytical standards provides higher confidence for the identification of mycotoxins. This protocol is designed to clearly explain the benefits of implementing IMS in food safety, taking as an example the determination of EAs in cereals. A QuEChERS-based extraction followed by an LC-trapped ion mobility spectrometry (TIMS)-MS analysis provided limits of quantification ranging from 0.65 to 2.6 ng/g with acceptable accuracy (although low recovery for ergotaminine) at 1.5x, 1x, and 0.5x the ML and exhibited a negligible matrix effect.

This protocol presents a validated liquid chromatography-ion mobility-high resolution mass spectrometry method to determine the presence of ergot alkaloids in food in compliance with the recently released Commission Regulation (EU) 2023/915.

Ion mobility mass spectrometry (IMS) acts as an additional separation dimension when integrated into liquid chromatography-mass spectrometry (LC-MS) ...

Lead Analysis of Soil Using Atomic Absorption Spectroscopy

Source: Laboratories of Margaret Workman and Kimberly Frye - Depaul University
Lead occurs naturally in soil, in levels ranging from 10-50 ppm. However, with the widespread use of lead in paint and gasoline in addition to contamination by industry, urban soils often have concentrations of lead significantly greater than background levels &#8211; up to 10,000 ppm in some places. Ongoing problems arise from the fact that lead does not biodegrade, and instead remains in the soil.
Serious health risks are associated with lead poisoning, where children are particularly at risk. Millions of children in the U.S. are exposed to soil containing lead. This exposure can cause developmental and behavioral problems in children. These problems include learning disabilities, inattention, delayed growth, and brain damage. The Environmental Protection Agency has set a standard for lead in soil at 400 ppm for play areas and 1,200 ppm for non-play areas.
Lead is also of concern in soil, when it&#8217;s used for gardening. Plants take up lead from the soil. Therefore, vegetables or herbs grown in contaminated soil can lead to lead poisoning. In addition, contaminated soil particles can be breathed in while gardening or brought into the house on clothing and footwear. It is recommended that soils with lead levels greater than 400 ppm should not be used for gardening. It is further recommended that soil with lead levels between 100 and 400 ppm not be used for leafy vegetables or herbs, because lead can be stored in the leaves. On a similar note, root vegetables should not be grown in this soil, because lead can also accumulate in plant roots.

Source: Laboratories of Margaret Workman and Kimberly Frye - Depaul University
Lead occurs naturally in soil, in levels ranging from 10-50 ppm. However, ...

Determination of Inorganic Arsenic in a Wide Range of Food Matrices using Hydride Generation - Atomic Absorption Spectrometry.

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