Novel method offers way to extract, analyze organoarsenic in feed

By Aerin Einstein-Curtis contact

- Last updated on GMT

© iStock
© iStock
A new method of extracting arsenic compounds from animal feed was found to support results and be simpler, say researchers.

A team of researchers from the University of Nuevo Leon and the Laboratory of Forensic Chemistry in Mexico examined the potential of a novel way to detect the presence of organoarsenic additives in livestock feed. The group published its work in the journal of Food Chemistry​.

“The goal of this work was to develop a method for quantifying three phenyl As [arsenic] compounds in livestock feed additives by high performance liquid chromatography coupled to ultraviolet oxidation hydride generation atomic fluorescence spectrometry (HPLC-UV/HG-AFS),”​ said the researchers. Additionally, work was done to assess the use of the microwave assisted extraction (MAE) method for removing organoarsenic from feed samples.

The MAE method is easier to set up compared to other procedures, they said. The parameters considered for optimizing the method were temperature, extraction time and extractant concentration.

The method was used to check feed samples and the results were validated using mass balance, they said.

The group found that the method was able to find total arsenic and organoarsenic compounds including p-arsanilic acid (ASA), roxarsone (ROX) and nitarsone (NIT) in feed samples.

“In this work, a new method for the determination of phenyl As compounds in livestock feed samples based on a microwave assisted extraction procedure followed by HPLC-UV/HG-AFS detection was developed,” ​said the researchers. “The HPLC-UV/HG-AFS analytical method offered several advantages, including low cost, relative simplicity, high sensitivity, and good reproducibility.”

Why look at organoarsenic additives and analytical methods?

Phenylated arsenic compounds may be used as feed additives in animal production as the organoarsenic derivatives have been linked with disease prevention, improved growth and feed efficiency and better meat pigmentation, the researchers said. ROX and ASA have been used to control hemorrhagic enteritis in swine and to boost productivity in poultry and NIT can be used to address histomoniasis in turkeys.

The organoarsenic compounds have little bioaccumulation potential and are predominately excreted unchanged, they said. Poultry litter contaminated with arsenic however may be sold as fertilizer and a source of As contamination in crops, soil and water when phenylated As species undergo biogeochemical degradation.

Those compounds can transform into stable, but more toxic, inorganic arsenic compounds like arsenite (AsIII) and arsenate (AsV), they said. In that state, they may pose potential human and environmental health risks.

Maximum allowable doses of the feed additives ROX an ASA in some locations vary from 50 to 100 mg kg-1 in China to 187.5 mg kg-1 by the European Food Safety Authority, said the researchers. “Therefore, it is very important to develop rapid and simple analytical methods for determination of organoarsenic compounds in feed samples,”​ they added.

Current analytical techniques are hyphenated techniques based on use of a chromatographic separation technique and an element-selective/specific detector, they said. “Different separation techniques, including high performance liquid chromatography (HPLC), gas chromatography (GC) and capillary electrophoresis (CE) coupled with inductively coupled plasma mass spectrometry (ICP-MS) and atomic absorption or fluorescence spectrometry in combination with hydride generation (HG-AAS and HG-AFS), have been evaluated for the determination of these organoarsenic compounds in different environmental samples,”​ they added.

The use of HPLC-ICP-MS has been found to be the most effective process for finding phenylarsenic compounds in complex sample matrices as it does not need derivatization and uses a simple interface, they said. But the determination of arsenic species using HPLC with HG-AFS presents a potential alternative.

HG-AFS offers some advantages for the analysis of species because it is simpler and less expensive, they said. Other HPLC separation modes like ion exchange (IE), reversed phase (RP) and reserved phase ion pairing (RP-IP) also have been used to check for arsenic speciation in feed additives.

The project seeks to find a way to quantify three phenyl arsenic compounds in livestock feed additives using HPLC with other processes so the conditions for the extraction, separation and determination of ASA, ROX and NIT were documented, said the researchers.

Preparation of samples is an important part of analysis as it isolates compounds of interest from the sample matric and potential interferences, they said.

Little work has been done examining the use of microwave assisted extraction (MAE) protocol using phosphoric acid as the extracting agent for organoarsenic compounds in feed samples, the researchers said. Previously, organoarsenic species are extracted from environmental samples using different solutions including methanol and water, acetic acid and methanol, phosphoric acid or sodium hydroxide and different processing techniques.

Methods and materials

In the trials, seven commercial animal feed samples for species including pigs, chickens and roosters were collected from factories that produce and sell feed, said the researchers. Samples were then crushed and analyzed.

To determine total arsenic about 200mg of a feed sample was processed with a microwave digestion system using 3mL of concentrated nitric acid (HNO3), 1mL of hydrochloric acid (HCI) and 1mL of hydrogen peroxide (H2O2), they said. Temperatures were then increased to 210 degrees centigrade and centrifuged, after cooling.

After additional filtering and dilution the sample was mixed with a reductant solution, they said.

To check for organoarsenics, phosphoric acid was used to extract the arsenic compounds from feed samples, said the researchers. About 500mg of a sample was weighed, 10mL of 1.5 mole (M) H3PO4 and then the sample was extracted for 45 minutes at 120 degrees centigrade using the MAE system and the extracts were centrifuged for 10 minutes.

The supernatant was mixed with 2mL of 2% CuSO4, mixed and diluted with water, they said.

To check for stability of the arsenic species, a control and a poultry feed sample spiked with ROX, ASA and NIT was preformed, they said. Samples were filtered and injected into the chromatographic column used for As-species separation.

For arsenic speciation analysis by HG-AFS, phenylated arsenic molecules were destroyed and quantification was done by external calibration using a standard As species as reference.  


Using the microwave assisted extraction on phenylated arsenic compounds with 1.5 M phosphoric acid (H3PO4) for 45 minutes at 120 decree centigrade was found to have the most recoveries of arsenic and organoarsenic types, said the researchers. It also did not degrade the compounds.

“The extraction of total As was approximately 97%, and the As species recoveries were between 95.2 and 97.0%,”​ they said. This suggests that the method was suitable for determining phenylarsenic additives in feed, they added.

Levels of total arsenic found in the feed with the trial method and the certified values showed a lack of significant difference, they said.

Mass balance comparisons of the amount of extracted As and the total concentration of As in the samples were used to test the results of the analysis, they said.

In the chicken feed samples, ASA was the only As species found, the researchers said. It had a concentration ranging from 0.72 and 12.91 mg kg-1.

Source: Food Chemistry

Title: Speciation analysis of organoarsenic compounds in livestock feed by microwave-assisted extraction and high performance liquid chromatography coupled to atomic fluorescence spectrometry

DOI: 10.1016/j.foodchem.2017.04.012

Authors: A. Saucedo-Velez, L. Hinojosa-Reyes, M. Villanueva-Rodríguez, A. Caballero-Quintero, A. Hernández-Ramírez, J. Guzmán-Mar

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