Measurement and Analysis in the Feed Industry

21 September 202013 min reading

Prof. Raymond D.Coker Emeritus Professor of Food Safety University of Greenwich Raymond Coker Consulting Limited

There will always be a need for accurate, robust and cost-effective procedures for the detection and measurement of toxicants in animal feed, in order to ensure the health and productivity of animals and the safety of consumers.


The maintenance of the quality and safety of feed is of paramount importance. It ensures the health and productivity of animals and, consequently, secures the supply of safe and nutritious food to the consumers of animal products. Fig. 1: Feed value chain
HACCP Flow-diagram: Maize-based
feed in Southeast Asia The production and delivery of high-quality feed requires the careful monitoring of the whole value chain, from the field (where the original crop is grown) to the animal feed producer and then, finally, to the livestock farmer. A typical feed value chain, from the feed mill to the livestock farmer, is illustrated in Fig. 1. It is clear from Fig. 1 that the production of animal feed involves a variety of steps, each of which should be carefully controlled to maintain the safety of the product. The control measures include Good Management, Storage and Agricultural Practices (GMP, GSP and GAP), in combination with the design and implementation of an effective Hazard Analysis and Critical Control Point (HACCP) Plan.  HACCP focuses upon the pro-active control of the process, as opposed to relying entirely on end-product testing. Critical Control Points (CCPs) are those points (steps) in the supply chain where it is essential that the safety of the feed product is strictly controlled. Effective sampling, sample preparation and analysis methods are required at CCPs throughout the supply chain.  It is essential that any sample collected from a batch of raw material or finished product is truly representative of that batch. If not, all future activities will be largely invalidated. HACCP Plans, together with sampling procedures, have been discussed in detail in previous issues of Feed Planet (Nov. 2018, Feb. & Apr. 2019).

The detection and measurement of chemical hazards occurring in feed value chains

The following methods for the determination of pesticides, herbicides and mycotoxins in feed and feed ingredients, serve as examples of analysis procedures which have been developed and reported during the period 2018-2020.


A wide variety of differing types of pesticides are currently employed, including organochlorine, organophosphate, carbamate, synthetic pyrethroids and inorganic pesticides. Methods employed for the detection and measurement of pesticides in feed and feed ingredients include high performance liquid chromatography (HPLC), gas liquid chromatography (GLC) and mass spectrometry (MS). In chromatographic procedures (HPLC and GLC), mixtures of chemical hazards are separated into their individual components by partitioning the mixtures between a liquid and solid, or liquid and liquid phase (HPLC) and by partitioning between a gas and liquid phase (GLC). Mass spectrometry (MS) is frequently employed to detect and measure the individual chemical hazards. Briefly, MS involves the bombardment of a sample with, for example, a beam of electrons which produces multiple charged fragments (a so-called ionisation process), which can then be automatically separated and identified.  The structure of individual molecules within the sample can then be deduced from the nature of their fragmentation pattern.


A 2-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS) method has been developed for the simultaneous determination of 350 pesticides, 16 mycotoxins, tropane alkaloids, and the growth regulators Chlormequat and Mepiquat, in cereals and other products. (Kresse, M. et al, 2019) Separation of the multiple analytes was achieved by employing two liquid chromatography columns in series. The automatic online clean-up of the sample occurred during the HILIC (hydrophilic interaction liquid chromatography) separation on the first column (the first dimension). Components of the cleaned-up sample were then, automatically, either subjected to further separation on a second reversed phase chromatography column (the second dimension) or passed directly to the tandem mass spectrometry system. (Fig. 2)
Fig. 2: A liquid chromatography tandem mass spectrometer
Tandem mass spectrometry is a technique where two or more mass analysers are coupled together to increase their abilities to analyse complex chemical samples. The first mass analyser ionises the molecules within the sample into fragments and separates these ions according to their mass-to-charge ratio (m/z). Ions of specific m/z-ratio are then selected and further ionised into smaller fragment ions, which are then separated and identified within the second mass analyser. Reportedly, the method achieved the performance criteria for the analysis of pesticides in cereals specified in EU Regulation 396/2005.

Gas liquid chromatography

A method for the analysis of pesticides and polychlorinated biphenyls in feed ingredients has been developed, employing a “quick, easy, cheap, effective, rugged, and safe” (QuEChERS) extraction method. This was followed by gas liquid chromatography (GC) involving a programmed temperature vaporizer large-volume injection of the cleaned-up sample, in combination with electron ionization (EI) quadrupole Orbitrap full-scan high-resolution mass spectrometry (GC-EI-Orbitrap HRMS in FS mode). (Tienstra, M., & Mol, H.G.J., 2018). (Fig.3). “Quadrupole” MS employs four parallel metal rods in the mass analyser. Each opposing rod pair is connected electrically, and a radio frequency (RF) voltage is applied between each pair of rods. Fragment ions travel down the quadrupole between the rods, and only ions of a specific mass-to-charge ratio will reach the detector for a given ratio of voltages.
Fig. 3: Gas Chromatography Orbitrap™ GC-MS/MS
In addition to full-scan acquisition, simultaneous full-scan and selected-ion monitoring acquisition was used to improve detectability in those cases where analytes coeluted with intense signals from co-extractants. The method was successfully validated down to 10 μg kg-1 for multiple-feed matrixes using standard addition. Identification according to European Union requirements was achieved in >90% of the analyte/matrix combinations, and suggestions for further increasing identification rates were made. Performance characteristics compared very favourably to an existing method for residue analysis, based on GC with EI tandem MS triple quadrupole mass spectrometry.

Flow injection mass spectrometry

A flow injection mass spectrometry (FI-MS/MS) approach, without any prior chromatography, has been applied to the rapid screening of selected pesticides and mycotoxins in grain and animal feed samples. (Sapozhnikova, Y et al, 2020). The ten selected pesticides represented five pairs of structural isomers. Samples of cereals/grains and animal feed were prepared using QuEChERS extraction, and then diluted and fortified with the selected contaminants to yield their representative maximum levels (MLs) or maximum residue levels (MRLs). The sensitivity of the developed approach was assessed for qualitative screening of the selected contaminants using high resolution triple-quadrupole-time-of-flight MS (HR-QTOFMS) and ion mobility high resolution time-of-flight MS ((IM) HR-TOFMS). In “time of flight” MS an ion's mass-to-charge ratio is determined via a time of flight measurement. The velocity of an ion depends on its mass-to-charge ratio, and the time that it takes to reach the detector over a known distance is measured. This time will depend on the velocity of the ion and is, therefore, a measure of its mass-to-charge ratio. Ion mobility TOFMS did not result in the successful separation of pesticide structural isomers. However, for three out of five pairs of pesticide structural isomers: cyproconazole and uniconazole, methiocarb and ethiofencarb, and vernolate and pebulate, unique fragment ions were found and confirmed with HR-QTOFMS. The developed approach is, reportedly, sufficiently sensitive for the rapid multi-residue screening of pesticides and mycotoxins in complex samples of food and feed, providing a high throughput and effective use of high-end instrumentation.


Enzyme linked immunosorbent assay (ELISA) In ELISA methods, the individual components of a sample are not separated prior to analysis.  The accurate detection and measurement of individual chemical hazards depends upon the ability of antibodies to specifically target individual compounds. ELISA methods are relatively simple to use and enable the simultaneous determination of multiple sample extracts. (Fig.4). A variety of methods exist including direct, indirect, competitive and sandwich ELISAs. A large proportion of commercial ELISAs for small molecules employ a competitive format where, for example, the toxin from a sample competes with a labelled toxin (such as a toxin–enzyme conjugate) for a limited number of immobilised antibodies, within a well in a microtitre plate. After washing to remove any unbound toxin or labelled toxin, and the addition of substrate, the latter reacts with the enzyme associated with the labelled toxin to produce a coloured product. Given the competitive nature of the procedure, the colour intensities within the sample wells are inversely proportional to the concentration of the toxin in the samples, and can be accurately and simultaneously measured, colorimetrically, using a plate reader. Multiple samples are typically measured simultaneously using a 96-well microtitre plate. The determination of the widely used herbicide, glyphosate, in companion animal feeds has been successfully achieved using an enzyme linked immunosorbent assay. (Zhao J, Pacenka S, et al, 2018). Eighteen commercial feeds from eight manufacturers were analysed. Fig 4: Enzyme linked immunosorbent assay (ELISA) Every product contained detectable glyphosate residues in the range of 7.83 × 101-2.14 × 103 μg kg-1 dry weight, with average and median values of 3.57 × 102 and 1.98 × 102 μg kg-1 respectively. Glyphosate concentration was significantly correlated with crude fibre content. However, the relevance of such an exposure to companion animals is currently unknown.


Mycotoxins are highly potent fungal toxins which occur in a wide variety of crops and, consequently, also in human foods and animal feeds. The occurrence and control of mycotoxins has been described in previous issues of Feed Planet (Nov. 2018, Feb. & Apr. 2019).


Liquid chromatography in combination with tandem mass spectrometry is widely employed for the determination of mycotoxins in animal feeds and feed ingredients. The identification and quantitation of the emerging fusariotoxins, enniatins and beauvericin, in animal feeds and their ingredients, has recently been performed using a liquid chromatography/tandem mass spectrometry, employing a hybrid triple-quadrupole linear ion trap mass spectrometer (LC-QTRAP/MS/MS) (Tolosa et al. 2019). For example, an analytical procedure for the simultaneous determination of emerging fusariotoxins was developed, involving an acetonitrile-based extraction followed by a QuEChERS and dispersive solid-phase clean-up, prior to LC-MS/MS. The co-occurrence of mycotoxins was observed in 47 % of samples, enniatin B and beauvericin being the most common combination. The analytical method was validated and the key performance characteristics fulfilled the criteria described by European Commission Decision 2002/657/EC. The development and validation of an ultra-performance liquid chromatography tandem mass spectrometry method (UPLC–MS/MS), for the simultaneous determination of citrinin and ochratoxin A in a variety of chicken and swine feed has also been reported (Meerpoel et al. 2018). A QuEChERS extraction method was employed, without any further clean-up step. The developed method was validated according to the criteria described in European Commission Regulation No. 401/2006/EC and Commission Decision No. 2002/657/EC. The limits of concentration of citrinin and ochratoxin A were 3.9 and 5.6 μg kg-1, respectively. The simultaneous occurrence of citrinin and ochratoxin A was reported in more than 50 % of 90 Belgian chicken and pig feed samples.


A variety of immunochemical methods can, potentially, be applied to the determination of mycotoxins, including enzyme-linked immunosorbent assay, flow injection immunoassay, chemiluminescence immunoassay, lateral flow immunoassay, and flow-through immunoassay.  (Rahman, H., et al, 2019). A novel, rapid magnetic beads-based direct competitive ELISA (MB-dcELISA) has been developed for the rapid analysis of aflatoxin B1 in feeds, and other commodities, utilizing an aflatoxin nanobody, Nb28, and its mimotope, ME17. (Zhao et al. 2019). A nanobody is an antibody fragment which can bind selectively to a specific antigen; a mimotope is a macromolecule, often a short-chain peptide, which can take on the structural appearance of the epitope of a harmful biological substance; and, an epitope is the part of an antigen that is recognized by the immune system. A nanobody, with a known amino acid sequence, is easier and less expensive to prepare than a monoclonal antibody. Similarly, mimotopes can be produced by peptide synthesis, thus obviating the production of toxic aflatoxin conjugates. The reported detection limit of the MB-dcELISA was 0.13 ng mL1, with a linear range of 0.24–2.21 ng mL-1. A good recovery (84.2-116.2%) was reported, with a low coefficient of variation (2.2-15.9%) in spiked maize, feeds and other commodities. The development of an immunoassay based on a nanobody and mimotope provides a potential new approach for the monitoring of aflatoxin B1 and other toxic small molecular weight compounds.

Raman spectroscopy

Raman spectroscopy provides a structural fingerprint by which molecules can be identified. A source of monochromatic light, usually from a laser in the visible, near infrared, or near ultraviolet range is used. The laser light interacts with molecular vibrations within the compound under investigation, resulting in an increase or decrease in the energy of the laser photons. The scattered light passes through a spectrometer which is typically equipped with an array detector (e.g. a charge-coupled device, CCD), to produce a Raman spectrum. The quantitative determination of zearalenone in maize, without sample extraction, has been performed using Raman spectroscopy in combination with chemometrics (multivariate analysis algorithms)  (Guo et al. 2019). Raman spectra were collected using a DXR SmartRaman spectrometer (Thermo Fisher Scientific, Madison, WI, USA) which was equipped with a universal platform sampling accessory, and configured with a near infrared laser at excitation wavelength of 785 nm and a high-resolution grating. (Fig.5). A procedure based on a synergy interval partial least squares-ant colony optimization (siPLS-ACO) algorithm, achieved coefficients of correlation (Rp) of 0.9260 and Root Mean Square Error of Prediction (RMSEP) of 87.9132 μg kg-1, respectively, in the prediction set. This procedure showed promising results for the rapid screening of large numbers of maize samples for zearalenone, without the need for sample-extraction steps. Fig. 5: A SmartRaman Spectrometer


There will always be a need for accurate, robust and cost-effective procedures for the detection and measurement of toxicants in animal feed, in order to ensure the health and productivity of animals and the safety of consumers. Traditional laboratory-based procedures, for example gas and liquid chromatography, are widely used, together with immunochemical and spectroscopic methods. Raman spectroscopy offers a means of analysis which avoids the sample extraction step. However, when using this method, the need to produce a small laboratory sample which is representative of the batch under examination is a challenge. Undoubtedly, there is an urgent need for simpler, accurate and inexpensive methods, which can be used successfully by non-scientists outside of a formal laboratory environment, throughout the feed supply chain.


Guo et al. (2019). Quantitative assessment of zearalenone in maize using multivariate algorithms coupled to Raman spectroscopy. Food Chemistry, 286, 282-288. doi:10.1016/j.foodchem.2019.02.020 Kresse, M., Drinda, H., Romanotto, A., & Speer, K. (2019). Simultaneous determination of pesticides, mycotoxins, and metabolites as well as other contaminants in cereals by LC-LC-MS/MS. Journal of Chromatography B, 1117, 86–102. doi:10.1016/j.jchromb.2019.04.013 Meerpoel et al. (2018). Development and validation of an LC–MS/MS method for the simultaneous determination of citrinin and ochratoxin a in a variety of feed and foodstuffs. Journal of Chromatography A, 1580, 100-109. doi:10.1016/j.chroma.2018.10.039 Rahman, H., Yue, X., Yu, Q., Xie, H., Zhang, W., Zhang, Q., & Li, P. (2019). Specific antigen‐based and emerging detection technologies of mycotoxins. Journal of the Science of Food and Agriculture, 99(11), 4869-4877. REGULATION (EC) NO 396/2005 OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 23 February 2005 on maximum residue levels of pesticides in or on food and feed of plant and animal origin and amending Council Directive 91/414/EEC Sapozhnikova, Y., Zomer, P., Gerssen, A., Nuñez, A., & Mol, H. G. J. (2020). Evaluation of flow injection mass spectrometry approach for rapid screening of selected pesticides and mycotoxins in grain and animal feed samples. Food Control, 116, 107323. doi:10.1016/j.foodcont.2020.107323 Tienstra, M., Mol, H.G.J., 2018. Application of Gas Chromatography Coupled to Quadrupole-Orbitrap Mass Spectrometry for Pesticide Residue Analysis in Cereals and Feed Ingredients. Journal of AOAC International. doi:10.5740/jaoacint.17-0408 Tolosa et al. (2019). Tolosa, J., Rodríguez-Carrasco, Y., Ferrer, E., & Mañes, J. (2019). Identification and Quantification of Enniatins and Beauvericin in Animal Feeds and Their Ingredients by LC-QTRAP/MS/MS. Metabolites, 9(2), 33. doi:10.3390/metabo9020033  Zhao J, Pacenka S, Wu J, et al. (2018). Detection of glyphosate residues in companion animal feeds. Environ Pollut. 2018;243(Pt B):1113-1118. doi:10.1016/j.envpol.2018.08.100 Zhao et al. (2019). A novel nanobody and mimotope based immunoassay for rapid analysis of aflatoxin B1. Talanta, 195, 55-61. doi:10.1016/j.talanta.2018.11.013
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