EN 16278:2012

Animal feeding stuffs - Determination of inorganic arsenic by hydride generation atomic absorption spectrometry (HG-AAS) after microwave extraction and separation by solid phase extraction (SPE)

This European Standard describes a procedure for the determination of inorganic arsenic in animal feeding stuffs of marine origin by Solid Phase Extraction (SPE) and Hydride Generation Atomic Absorption Spectrometry (HG-AAS). The method has been successfully tested in a collaborative trial with a working range from 0,19 mg/kg to 2,7 mg/kg (HORRATvalues < 2). The LOQ of the method is usually approximately 0,1 mg/kg or lower.

Futtermittel - Bestimmung von anorganischem Arsen mit Atomabsorptionsspektrometrie-Hydridtechnik (HD-AAS) nach Mikrowellen-Extraktion und Trennung durch Festphasenextraktion (SPE)

Diese Europäische Norm beschreibt ein Verfahren für die Bestimmung von anorganischem Arsen in Futtermitteln marinen Ursprungs mittels Festphasenextraktion (SPE) (en: Solid Phase Extraction) und der Atomabsorptionsspektrometrie-Hydridtechnik (HD AAS). Das Verfahren wurde in einem Ringversuch erfolgreich mit einem Messbereich von 0,19 mg/kg bis 2,7 mg/kg (HORRAT-Werte < 2; der HORRAT-Wert ist der Horwitz-Ratio-Wert) geprüft. Die Bestimmungsgrenze LOQ (en: Limit Of Quantification) des Verfahrens beträgt im Allgemeinen etwa 0,1 mg/kg oder weniger.

Aliments des animaux - Dosage de l'arsenic inorganique par spectrométrie d'absorption atomique par génération d'hydrures (SAA-GH) après extraction par micro-ondes

La présente Norme européenne décrit un mode opératoire de dosage de l’arsenic inorganique dans les aliments des animaux d’origine marine par extraction en phase solide (SPE) et par spectrométrie d’absorption atomique par génération d'hydrures (SAA-GH). La méthode a passé avec succès un essai interlaboratoires avec une plage de travail comprise entre 0,19 mg/kg et 2,7 mg/kg (valeurs HORRAT < 2 ; la valeur HORRAT est la valeur du rapport de Horwitz). La LOQ de la méthode est habituellement d’environ 0,1 mg/kg ou moins.

Živalska krma - Določevanje anorganskega arzena s hidridno atomsko absorpcijsko spektrometrijo (HG-AAS) po mikrovalovni ekstrakciji in ločevanju z ekstrakcijo na trdni fazi (SPE)

Ta evropski standard opisuje postopek za določevanje anorganskega arzena v živalski krmi morskega izvora z ekstrakcijo na trdni fazi (SPE) in hibridno atomsko absorpcijsko spektrometrijo (HG-AAS). Metoda je bila uspešno preskušena v medlaboratorijskem poskusu z delovnim razponom od 0,19 mg/kg do 2,7 mg/kg (vrednost HORRAT < 2; vrednost HORRAT je vrednost razmerja, izračunanega iz Horwitzeve enačbe). Meja kvantifikacije je običajno približno 0,1 mg/kg ali manj.

General Information

• Status: Published
• Publication Date: 03-Jul-2012
• ICS: 65.120 - Animal feeding stuffs
• Technical Committee: CEN/TC 327 - Animal feeding stuffs - Methods of sampling and analysis
• Drafting Committee: CEN/TC 327/WG 4 - Heavy metals, trace elements and minerals
• Current Stage: 9093 - Decision to confirm - Review Enquiry
• Start Date: 03-Sep-2023
• Completion Date: 14-Apr-2025
• Directive: 70/373/EC - Council Directive 70/373/EEC of 20 July 1970 on the introduction of Community methods of sampling and analysis for the official control of feeding-stuffs
• Mandate: M/382 - Mandate for standardisation addressed to CEN in the field of Methods of analysis for animal Feedingstuffs
• Ref Project: SIST EN 16278:2012 - Animal feeding stuffs - Determination of inorganic arsenic by hydride generation atomic absorption spectrometry (HG-AAS) after microwave extraction and separation by solid phase extraction (SPE)

Overview

EN 16278:2012 (CEN) defines a validated laboratory procedure for the determination of inorganic arsenic in animal feeding stuffs of marine origin. The method combines microwave-assisted extraction, solid phase extraction (SPE) clean-up and hydride generation atomic absorption spectrometry (HG‑AAS) for quantification of the sum of As(III) and As(V). The procedure was validated in a collaborative trial (working range 0.19–2.7 mg/kg, HORRAT < 2) and typically achieves a limit of quantification (LOQ) ≈ 0.1 mg/kg or lower.

Key topics & technical requirements

• Scope: Inorganic arsenic in marine-derived animal feeds; test portion and sample prep follow EN ISO 6497/6498 guidance.
• Sample extraction: ~0.2 g homogenised sample, extracted with 10 mL of 3% H2O2 in 0.055 mol/L HCl using microwave program (90 °C, 25 min), then centrifuged (≥ 4 000 rpm).
• SPE clean-up: Strong anion-exchange cartridges (e.g., Strata SAX 500 mg/6 mL recommended); cartridges conditioned with methanol and equilibration solution, sample buffered to pH ~6.5, washed and eluted with 1.25 mL 0.4 mol/L HCl. Low drop rate and vacuum drying between steps are emphasised.
• Pre-reduction: Convert As(V) to As(III) before hydride generation - example procedure: add KI/ascorbic pre-reduction solution and 2.6 mol/L HCl with 60 min incubations (applied equally to samples and calibration standards).
• HG‑AAS measurement: Automated hydride-generation flow system with a heated quartz cell (typical cell temp 900 °C, wavelength 193.7 nm). Calibration recommended across 0.25–8 µg/L (instrument dependent) with matching acid matrix.
• Reagents & safety: Analytical‑grade reagents, high-purity HCl and low‑arsenic water (EN ISO 3696). Sodium borohydride use requires strict ventilation controls because it generates hydrogen with acids - safety warnings are explicit.
• Performance: Working range and LOQ from collaborative trials; guidance on storage times for extracts/eluates and method precision included.

Applications & users

This standard is intended for:

• Accredited testing laboratories performing routine monitoring of inorganic arsenic in marine-derived feeds.
• Feed manufacturers and QC departments seeking compliance and risk assessment for arsenic content.
• Regulatory authorities and enforcement laboratories assessing feed safety and conformity with limits.
• Method developers and instrument vendors implementing SPE + HG‑AAS workflows for low-level arsenic analysis.

Practical benefits include a validated, SPE-based separation that reduces interference from organic arsenic species and a reproducible HG‑AAS measurement route suitable for routine monitoring.

Related standards

• EN ISO 3696 - Water for analytical laboratory use
• EN ISO 6497 - Animal feeding stuffs - Sampling
• EN ISO 6498 - Animal feeding stuffs - Guidelines for sample preparation

Keywords: EN 16278:2012, inorganic arsenic, animal feeding stuffs, marine origin, HG‑AAS, hydride generation, SPE, microwave extraction, LOQ, feed testing.

Standards Content (Sample)

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.RFuttermittel - Bestimmung von anorganischem Arsen mit Atomabsorptionsspektrometrie-Hydridtechnik (HD-AAS) nach Mikrowellen-Extraktion und Trennung durch Festphasenextraktion (SPE)Aliments des animaux - Dosage de l'arsenic inorganique par spectrométrie d'absorption atomique par génération d'hydrures après extraction par micro-ondesAnimal feeding stuffs - Determination of inorganic arsenic by hydride generation atomic absorption spectrometry (HG-AAS) after microwave extraction and separation by solid phase extraction (SPE)65.120KrmilaAnimal feeding stuffsICS:Ta slovenski standard je istoveten z:EN 16278:2012SIST EN 16278:2012en,fr,de01-september-2012SIST EN 16278:2012SLOVENSKI
STANDARD
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 16278
July 2012 ICS 65.120 English Version
Animal feeding stuffs - Determination of inorganic arsenic by hydride generation atomic absorption spectrometry (HG-AAS) after microwave extraction and separation by solid phase extraction (SPE)
Aliments des animaux - Dosage de l'arsenic inorganique par spectrométrie d'absorption atomique par génération d'hydrures après extraction par micro-ondes
Futtermittel - Bestimmung von anorganischem Arsen mit Atomabsorptionsspektrometrie-Hydridtechnik (HD-AAS) nach Mikrowellen-Extraktion und Trennung durch Festphasenextraktion (SPE) This European Standard was approved by CEN on 17 May 2012.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
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Avenue Marnix 17,
B-1000 Brussels © 2012 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 16278:2012: ESIST EN 16278:2012

Results of the interlaboratory tests. 12Annex B (informative)
Flowchart Animal feeding stuffs — Determination of inorganic arsenic by hydride generation atomic absorption spectrometry (HG-AAS) after microwave extraction and separation by solid phase extraction (SPE) . 13Bibliography . 18 SIST EN 16278:2012

1 Scope This European Standard describes a procedure for the determination of inorganic arsenic in animal feeding stuffs of marine origin by Solid Phase Extraction (SPE) and Hydride Generation Atomic Absorption Spectrometry (HG-AAS). The method has been successfully tested in a collaborative trial with a working range from 0,19 mg/kg to 2,7 mg/kg (HORRATvalues < 2; HORRAT value is Horwitz –Ratio value). The LOQ of the method is usually approximately 0,1 mg/kg or lower.
2 Normative references The following documents, in whole or in part, are normatively referenced in this document and are indispensable for its application. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. EN ISO 3696, Water for analytical laboratory use — Specification and test methods (ISO 3696) EN ISO 6497, Animal feeding stuffs — Sampling (ISO 6497) EN ISO 6498, Animal feeding stuffs — Guidelines for sample preparation (ISO 6498) 3 Principle Inorganic arsenic consists of arsenite, As(III) and arsenate, As(V). This standard describes a method for the determination of inorganic arsenic (i.e. the sum of As(III) and As(V)). A representative test portion of the sample is treated with a diluted hydrochloric acid and hydrogen peroxide solution using microwave assisted heating. Hereby inorganic arsenic species are extracted and As(III) is oxidised to As(V). The inorganic arsenic is selectively separated from other arsenic compounds using solid phase extraction (SPE) and the concentration of inorganic arsenic is determined by HG-AAS in the SPE eluate. The gaseous hydride is transferred into a heated measuring cell (cuvette) by means of a carrier gas stream and decomposed.
Since arsenic (III) and arsenic (V) show a different sensitivity to the hydride technique, it is necessary to reduce arsenic (V) to arsenic (III) in order to avoid incorrect measurements.
WARNING — The use of this standard can involve hazardous materials, operations and equipment. This standard does not purport to address all the safety problems associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and
determine the applicability of regulatory limitations prior to its use. 4 Reagents The concentration of arsenic in the reagents and water used shall be low enough not to affect the results of the determination. All reagents shall be of analytical grade or similar unless otherwise specified. Use water conforming to grade 2 of EN ISO 3696. 4.1 Hydrochloric acid (HCl), mass fraction of ≥ 30 % of approximately
(HCl) ≥ 1,15 g/ml. NOTE Only use hydrochloric acid available with high purity or which was cleaned by a sub-boiling distillation.
4.2 Diluted hydrochloric acid. 4.2.1 0,4 mol/l as eluent for the SPE. Dilute e.g. 20 ml of hydrochloric acid (4.1) to 0,5 l with water. 4.2.2 0,055 mol/l for the extractant solution. SIST EN 16278:2012

(CH3COOH) = 1,05 g/ml. 4.6 Diluted acetic acid, 0,5 mol/l for washing of SPE columns. Dilute e.g. 15 ml of acetic acid (4.5) to 0,5 l with water. 4.7 Sodium hydroxide pellets, mass fraction of ≥ 98 %. 4.8 Sodium hydroxide, 0,1 mol/l for adjustment of pH. Dilute 0.4 gram sodium hydroxide pellets (4.7) to 100 ml with water. 4.9 Ammonium carbonate, mass fraction of ≥ 99,999 %. 4.10 Ammonium carbonate buffer solution, 40 mmol/l for sample buffering. Dissolve e.g. 0,384 g ammonium carbonate (4.9) in water and dilute to 100 ml with water. 4.11 Methanol (CH3OH), HPLC grade, for conditioning of SPE sorbent. 4.12 Sodium borohydride, mass fraction of ≥ 96 %. 4.13 Sodium borohydride solution for hydride generation. Dissolve e.g. 2,5 g of sodium hydroxide pellets (4.7) in a small amount of water; add 2,5 g of sodium borohydride (4.12) and dilute to 500 ml with water. Prepare a fresh solution daily prior to the hydride generation step and, when necessary, filter before use.
Commercially available sodium borohydride solutions may also be used. NOTE The required concentrations may vary slightly with the system and the instructions of the relevant manufacturer shall therefore be observed. WARNING — It is essential to observe the safety instructions for working with sodium borohydride. Sodium borohydride forms hydrogen with acids and this can result in an explosive air/hydrogen mixture. A permanent extraction system shall be provided at the point where the measurements are carried out.
4.14 L-Ascorbic acid, mass fraction of ≥ 99,7 %. 4.15 Potassium iodide, mass fraction of ≥ 99,5 %. SIST EN 16278:2012

4.19 Diluted inorganic arsenic solutions. Dilute the arsenic stock solution (4.18) by water in several steps, such as in the following example: 1 000 mg/l
10 mg/l
1 mg/l
0,1 mg/l 4.20 Calibration solutions. Prepare six external standards including a blank calibration solution that covers the linear range of the element to be determined by diluting the diluted inorganic arsenic solutions (4.19). The linear range is instrument dependent. Appropriate matching of the calibration solutions shall be performed (prereduction according to Table 2). It is important to adjust the acid concentration of the standards to the acid concentration of the samples. The following calibration range is recommended: 0,25 µg/l to 8 µg/l. These calibration solutions should be prepared freshly before use. NOTE
An example on the production of calibration standard is given in Annex B, Table B.2. 4.21 Silicone antifoam agent ≥ 30 %. 5 Apparatus and equipment To minimise contamination, all apparatus and equipment that come into direct contact with the sample and the solutions shall be carefully pre-treated. It is recommended to avoid the use of glassware, since this may cause contamination with arsenate [1]. 5.1 Laboratory grinder, capable of grinding to a particle size of less than or equal to 0,5 mm. 5.2 Analytical balance, capable of weighing to an accuracy of 1 mg. 5.3 Microwave oven with closed vessels, capable of programming temperature. 5.4 pH indicator paper/sticks, for pH range 4 to 7. 5.5 Centrifuge with containers, capable of a minimum centrifuge speed of 4 000 rpm (2 100 g). 1/100 1/10 1/10 SIST EN 16278:2012
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