Control and characterisation of trace mineral chelates
Chelation plays a vital role in trace mineral nutrition, but inconsistent terminology and regulatory differences have led to confusion in the animal feed industry. This article explores the chemistry of chelation, the importance of verifying complexation and the role of ATR-FTIR spectroscopy in ensuring product integrity.
The process of complexing or chelating elements such as zinc or manganese, to generate a trace mineral chelate, typically involves reacting inorganic mineral salts with a suitable bonding group such as a peptide or amino acid, after which the mineral becomes part of a biologically stable structure.
The chemistry behind chelation has created a great deal of confusion in the animal feed industry. Terms such as ‘metal amino acid complexes’, ‘metal amino acid chelates’, ‘metal polysaccharide complexes’ and ‘metal proteinates’ are routinely used in a misleading and interchangeable fashion. Jurisdictional differences between control agencies also add to the confusion, with different nomenclature for the same class of chelate being used. For example, proteinates (AAFCO) and chelates of protein hydrolysate (EU) refer to the same product type.
Metal proteinates/ chelates of protein hydrolysate, are normally produced by first partially hydrolysing a protein source using either acid or enzymatic procedures. This results in the formation of a hydrolysate containing a mixture of amino acids and peptides of varying chain length. The average chain length will depend on several factors including the degree of hydrolysis. Under appropriate conditions, reaction of a metal salt with such a hydrolysate at an appropriate pH, results in the formation of a spectrum of complexes which vary in size and contain complexed metal ions.
Establishing the degree of complexation is important for both regulatory and practical purposes. Just as regulatory agencies require complete characterisation of medicinal products, similar data is necessary for trace mineral chelates to ensure their safety and efficacy. If the metal ion is not complexed, its use will confer no additional benefit over that of a metal salt. Additionally, the importance of control methods allowing for the detection of products which are merely blends of carrier and inorganic salts are important to prevent the sale of fraudulent materials.
Determining the degree of metal complexation
In 2017, researchers at Alltech’s European Bioscience Centre assessed a number of different methodologies to determine the degree of metal complexation in commercially available chelates of protein hydrolysate (proteinates), thus enabling more complete characterisation and control of these products.
Potential methodologies which were identified included both attenuated total reflectance Fourier transform infrared spectroscopy (ATR–FTIR) and powder X–ray diffraction (PXRD). The ATR–FTIR and PXRD methods have a number of advantages. Both techniques are well established, widely available and data can be acquired rapidly. They are also non–destructive, so the same sample can be analysed using both methods in different laboratories. While both methodologies were assessed and verified as being reliable analytical methods to quantify trace mineral chelation, the ATR–FTIR method was independently verified by the Central Reference Laboratory – Walloon prior to being recommended as an official control method in the EU and as an example further included in the annex of the Reg (EU) 2016/1095 for the determination of chelated zinc content in the feed additive.
Subsequent to this, the method was published under peer review and made available to the industry as a whole, enabling other manufacturers to independently assess their own products. Additional research extended on the earlier work and validated the assay for use with higher concentration trace mineral chelates of protein hydrolysate.
Key advantages
A key advantage of ATR-FTIR spectroscopy as an applicable method for determining the extent of chelation in mineral proteinates is its ability to assess unadulterated samples in the solid state. Methods requiring solubilisation of samples prior to analysis change the chemical characteristics of the products and introduce elements of error not found with ATR-FTIR. The method requires very little sample preparation and performance criteria such as limits of detection, limits of quantification, repeatability and specificity, indicate that this method is suitable for the routine analysis of proteinate products.
The validated ATR-FTIR spectroscopy method is product-specific and, while its use is applicable to the general class of chelates referred to by EFSA as ‘chelates of protein hydrolysate’ (proteinates), the published assay has only been verified for the proteinate produced by Alltech – Bioplex. As the characteristics of any proteinate will depend on the protein source used to produce the hydrolysate, the hydrolysis process and the percentage of metal present, the assay will need to be validated and verified in a product-specific fashion.
Use of validated assays
Product specific assay validation is critical in this instance as the hydrolysis procedure plays a critical role in the production of proteinate products and varies greatly between manufacturers. As such, to obtain accurate data on the extent of chelation for specific chelate products, appropriate standards and calibration curves, along with method verification and validation procedures, must be generated for each specific product type.
With new chelate products continuously being developed, in addition to products with increased mineral concentration, the use of validated assays will ensure that such chelates meet EU regulatory requirements. This further adds to their safety and efficacy characteristics.
Apart from enabling producers meet regulatory requirements, assessment of such chelates via this assay will enable the EU to combat feed additive fraud. In doing so the EU can further protect its agricultural sector by upholding standards through robust monitoring and rigorous enforcement of regulations.
References available upon request.
