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Analytical methods for the determination of nutrients, inorganic and organic compounds and contaminants, and biological contaminants in livestock feeds

Purpose

This document summarizes the analytical methods used by the Canadian Food Inspection Agency (CFIA) Laboratories to test for various analytes in livestock feeds and feed ingredients in order to verify compliance with the Feeds Act and Feeds Regulations, 1983. Feed analyses are conducted at the CFIA's Ottawa Laboratory (Carling) and Calgary Laboratory.

The scopes of accreditation for these laboratories are available from the Standards Council of Canada. This list is not exhaustive and other methods may be validated and limits of quantification (LOQ) or reporting limits may be used by the CFIA as needed.

Feed sample preparation

The CFIA follows specific procedures to ensure that laboratory sample integrity is maintained from sample reception to final analysis. The production of reliable laboratory sample results is achieved through various steps that include drying, grinding, mixing, and analysing the sample using validated methods. The type of grinder, the sieve size and mixing instructions are provided in analyte specific procedures.

When large samples are received at the lab, a stationary riffle splitter is used to reduce the quantity, processing time and technical resources needed. The equipment is cleaned according to established procedures in order to avoid contamination. The laboratory conducting the analyses ensures homogeneity of the sample prior to taking the test portion.

Nutrients and proximate analysis

Feed samples submitted for nutrients and proximate analysis (except insoluble solids) are ground, mixed and filled in a labelled 500 mL amber sample bottle to ⅔ full with a sufficient representative proportion.

Analytical methods for nutrients and proximate analysis
Analyte Method code LOQ or reporting limit Table Note 1 Brief method description
Ash FD-MIN-ASHF 0.1% A known weight of ground feed is heated at a specified temperature (600°C) for a specified time (2 hours) reducing the feed to ash in a porcelain crucible. The weight of the residue in the crucible is calculated as ash.
Fat FD-MIN-FAT-ANKOM 1% Applicable to solid processed animal feed in a range of 1% to 21% fat, based on the American Oil Chemists' Society (AOCS) Official Procedure Am 5-04 Rapid Determination of Oil/Fat Utilizing High Temperature Solvent Extraction. This method determines crude fat by extracting with petroleum ether utilizing the ANKOM Extraction System. The compounds extracted are predominantly triglycerides. Small amounts of other lipids along with minor components having some solubility in petroleum ether are also extracted.
Fibre, Crude (ANKOM) FD-MIN-FIBRE-FSI 0.25% The crude fibre analytical range is 0.25% to 70%. The method is based on the AOCS Approved Procedure Ba 6a-05. Crude fibre is the organic residue remaining after digestion with sulfuric acid and sodium hydroxide at specific concentrations. The ANKOM Fiber Analyzer analyzes samples individually enclosed in filter bags. Extraction and rinse operations are all accomplished within the same instrument. The Analyzer maintains a solution temperature of 100°C, while providing for proper agitation to ensure a uniform flow of chemical solutions across each sample. After digestion, which removes protein, sugar, starch, lipids and portions of both the structural carbohydrates and lignin, the sample is ashed to determine the crude fibre content.
Insoluble Solids FFIC-INSOL-FAT 0.05% This method is derived from the AOCS Official Method, Ca 3a-46. It determines foreign substances in fat that are insoluble in kerosene and petroleum ether. A known weight of tallow, fat or oil is dissolved in kerosene, filtered and the collected insoluble solids are weighed.
Moisture FFIC-Moisture-105C 0.1% Feeds except those containing urea, high sugar contents, ensiled materials and milk products with sugar content >4% can also be analyzed. The analytical range is 0.1% to 75%. A known amount of feed sample is dried in an air oven under a specific temperature (105°C) for a specified time (16 hours).
Protein (combustion) FD-MIN-PROT-FEED 0.2% Applicable to the analysis of protein in all feed products having a protein content of 0.2% to 100%. Nitrogen is released from feed through combustion at high temperature under high purity oxygen. Reduced nitrogen gas is quantitatively measured by thermal conductivity detection and reported as w/w percent nitrogen in sample. The % nitrogen is converted to protein using the feed protein factor of 6.25.

Table Notes

Table Note 1

The LOQ can on occasion be the reporting limit, which is higher than the LOQ.

Return to table note 1  referrer

Enzyme and microbial supplements

Feed samples submitted for enzyme activity determination or microbial supplements enumeration and identification of microbial viability of fermentation extracts, do not require sample preparation and consequently are not to be ground.

Analytical methods for enzyme and microbial supplements
Analyte Method code LOQ Brief method description
Enzymatic activity Variable: Methods submitted by the registrant at the time of product registration or renewal Variable

Enzyme activity determination methods measure the "work" of the enzyme in a manner that supports the "unit of activity definition". The unit of activity is usually in terms of the metabolite/end product liberated or amount of substrate metabolized per unit time "under the conditions of the assay" (temperature, pH, nature of substrate). The major types of methods are:

End point determination of metabolite produced:

The sample is extracted and diluted to the "analytical range" based on the guaranteed activity. An aliquot of the diluted sample is incubated in the presence of a suitable substrate. After a predefined time, during which the enzyme metabolizes the substrate, the reaction is stopped and the end product is determined against a dose-response curve constructed using solutions of known concentrations of the metabolite/end product. Determination of the end product is often through spectrophotometry or titration.

End point determination using azo-dyed substrates or equivalent:

The sample is extracted and diluted to the "analytical range" based on the guaranteed activity. An aliquot of the diluted sample is incubated in the presence of a suitable substrate to which a dye has been conjugated. After a predefined time, during which the enzyme metabolizes the substrate, the reaction is stopped by the addition of alcohol which will cause the unprocessed substrate to precipitate. The optical density of the solution is compared against a validated dose.

Real time monitoring of substrate breakdown:

The sample is extracted and diluted to the "analytical range" based on the guaranteed activity. An aliquot of the diluted sample is incubated in the presence of a suitable substrate. The reaction is monitored and the time to reach a predefined status is determined. Monitoring can be through spectrophotometry/optical density or viscosity.

Microbial (bacterial or yeast and mould) supplement in CFU/g Variable: Plate Methods submitted by the registrant at the time of product registration or renewal 2500 CFU/g An aliquot of the sample is serially diluted, based on guarantee, to obtain a solution that will contain 250 to 2500 colony forming units (CFU) per milliliter. A 0.1 mL of the solution is spread on, or distributed in, a suitable growth medium in a Petri dish. The dishes are incubated under the appropriate conditions (i.e. temperature and oxygen) for the guaranteed microorganisms until the colonies are large enough to be counted. Single colonies are picked and subjected to an identification step.
Yeast supplement cell count Variable: Counting Chamber Methods submitted by the registrant at the time of product registration or renewal Limit of Detection (LOD): 1000 cells/g
LOQ: 100000 cells/g
This method is used only for yeast cells. An aliquot of the sample is serially diluted, based on guarantee, and mixed with a solution of methylene blue dye to obtain a solution that will contain approximately 100 to 200 cells in the counting area. The counting chamber is loaded with approximately 0.25 mL of the mixture and the cells are counted. Live cells are able to pump out the dye and will appear clear whereas dead cells will remain blue. As heat damaged cells will also appear clear, the solution is additionally counted by a Plate method which also allows for isolation towards identification.
Fermentation extract viability Variable depending on suspected organism LOD: 100 CFU/g
LOQ: 2500 CFU/g
An aliquot of the sample is suspended and diluted in a solution, spread on a medium, and incubated under the conditions suitable for the suspected organism. Samples with a count superior to 10 000 CFU/g are regarded as viable and are isolated for identification.
Microbial identification Variable depending on suspected organism N/A Once the microbe has been isolated, the identity can be confirmed or determined by phenotyping (metabolic profile using systems such as analytical profile index [API] strips or Biolog) or genotyping (DNA sequencing).

Microscopic examination

Feed samples submitted for microscopic examination are not to be ground.

Feed samples submitted for microscopic examination
Analyte Method code LOQ Brief method description
Variable
(as per inspector request and sampling program specifications)
FD-BIO-MCR Variable Samples are examined using various magnification and processing techniques:
  • to identify ingredients
  • verify compliance to ingredient definition
  • assess quality of ingredients
  • detect contaminants, adulterants, weed seeds

Biological contaminants

Feed samples submitted for microbiological testing do not require sample preparation and consequently are not to be ground.

Analytical methods for biological contaminants
Analyte Method code LOQ Brief method description
Salmonella spp.

MFLP-29 (preliminary detection)

MFHPB-20 (confirmation)

N/A – qualitative method

Samples are reported as:
Not Detected or Detected

MFLP-29 method: uses a BAX® System which is a screening tool for detecting Salmonella using Polymerase Chain Reaction (PCR) technology for rapid amplification and fluorescent detection. After enrichment, sample preparation involves about 1 hour of user time. The automated procedure then delivers results about 4 hours later. The BAX® System uses the PCR to amplify three specific fragments of bacterial DNA unique to Salmonella and detects the presence of the target amplicons through melting curve analysis.

The size of feed sample is 1 kg and a 100 g analytical portion is tested. This analytical portion is diluted with a total of 900 mL of buffered peptone water which is subjected to a proper blending by mixing, vortexing or stomaching. The sample is enriched by incubation at 35°C for 22-26 h. Transfer 0.1 mL and 1.0 mL of the enriched sample into 9.0 mL of Rappaport Vassiliadis soya peptone (RVS) and Tetrathionate Brilliant Green Bile Enrichment (TBG) broths, respectively. Incubate 24 ± 2 h at 42.5°C. The enriched sample is subjected to the PCR preparation and amplification as specified by the manufacturer (BAX® System).

MFHPB-20 method: Sample for this method is prepared in the same way that it is prepared for the MFLP-29 method. 100 g of a 1 kg feed sample is diluted in 900 mL of buffered peptone water (non-selective enrichment) and it is properly mixed. The sample then undergoes selective enrichment, selective plating, purification, biochemical screening and serological identification.

Elements, metals and minerals (inorganic compounds and contaminants)

Feed samples submitted for heavy metal analysis are ground using a metal free sieve, mixed and filled in a labelled 250 mL amber bottle to ⅔ full with a sufficient representative proportion.

Analytical methods for elements, metals and minerals
Analyte Method code LOQ or reporting limit Table Note 2 Brief method description
Aluminum FFIC-23-ICP-OES 30 mg/kg The ground sample is digested using a microwave with nitric and hydrochloric acids (3:1). The digestate is transferred quantitatively to a volumetric flask, made to volume with deionized (DI) water, and filtered. Final analysis is done with the addition of internal standard by inductively coupled plasma optical emission spectrometry (ICP-OES).
Arsenic FFIC-23-ICP-OES 5 mg/kg The ground sample is digested using a microwave with nitric and hydrochloric acids (3:1). The digestate is transferred quantitatively to a volumetric flask, made to volume with DI water, and filtered. Final analysis is done with the addition of internal standard by ICP-OES.
Arsenic FFIC-MULTI-ICP-MS 0.050 mg/kg (typical matrix) The ground sample is digested using a microwave with nitric and hydrochloric acids (3:1). The digestate is transferred quantitatively to a volumetric flask, made to volume with DI water, and filtered. Final analysis is done with the addition of internal standard by inductively coupled plasma mass spectrometry (ICP-MS). (5.0 mg/kg using higher dilution) Table Note 3
Boron FFIC-23-ICP-OES 25 mg/kg The ground sample is digested using a microwave with nitric and hydrochloric acids (3:1). The digestate is transferred quantitatively to a volumetric flask, made to volume with DI water, and filtered. Final analysis is done with the addition of internal standard by ICP-OES.
Cadmium FFIC-23-ICP-OES 2 mg/kg The ground sample is digested using a microwave with nitric and hydrochloric acids (3:1). The digestate is transferred quantitatively to a volumetric flask, made to volume with DI water, and filtered. Final analysis is done with the addition of internal standard by ICP-OES.
Cadmium FFIC-MULTI-ICP-MS 0.010 mg/kg (typical matrix) The ground sample is digested using a microwave with nitric and hydrochloric acids (3:1). The digestate is transferred quantitatively to a volumetric flask, made to volume with DI water, and filtered. Final analysis is done with the addition of internal standard by ICP-MS. (1.0 mg/kg using higher dilution) Table Note 3
Calcium FFIC-23-ICP-OES 300 mg/kg The ground sample is digested using a microwave with nitric and hydrochloric acids (3:1). The digestate is transferred quantitatively to a volumetric flask, made to volume with DI water, and filtered. Final analysis is done with the addition of internal standard by ICP-OES.
Cobalt FFIC-23-ICP-OES 2 mg/kg The ground sample is digested using a microwave with nitric and hydrochloric acids (3:1). The digestate is transferred quantitatively to a volumetric flask, made to volume with DI water, and filtered. Final analysis is done with the addition of internal standard by ICP-OES.
Cobalt FFIC-MULTI-ICP-MS 0.050 mg/kg (typical matrix) The ground sample is digested using a microwave with nitric and hydrochloric acids (3:1). The digestate is transferred quantitatively to a volumetric flask, made to volume with DI water, and filtered. Final analysis is done with the addition of internal standard by ICP-MS. (5.0 mg/kg using higher dilution) Table Note 3
Copper FFIC-23-ICP-OES 25 mg/kg The ground sample is digested using a microwave with nitric and hydrochloric acids (3:1). The digestate is transferred quantitatively to a volumetric flask, made to volume with DI water, and filtered. Final analysis is done with the addition of internal standard by ICP-OES.
Copper FFIC-MULTI-ICP-MS 1.6 mg/kg (typical matrix) The ground sample is digested using a microwave with nitric and hydrochloric acids (3:1). The digestate is transferred quantitatively to a volumetric flask, made to volume with DI water, and filtered. Final analysis is done with the addition of internal standard by ICP-MS. (160 mg/kg using higher dilution) Table Note 3
Chromium FFIC-23-ICP-OES 22 mg/kg The ground sample is digested using a microwave with nitric and hydrochloric acids (3:1). The digestate is transferred quantitatively to a volumetric flask, made to volume with DI water, and filtered. Final analysis is done with the addition of internal standard by ICP-OES.
Chromium FFIC-MULTI-ICP-MS 0.5 mg/kg (typical matrix) The ground sample is digested using a microwave with nitric and hydrochloric acids (3:1). The digestate is transferred quantitatively to a volumetric flask, made to volume with DI water, and filtered. Final analysis is done with the addition of internal standard by ICP-MS. (50 mg/kg using higher dilution) Table Note 3
Iodine FLS-2015-002 0.43 ppb Samples are prepared prior to weighing into digitubes. Tetramethylammonium hydroxide is added to the samples along with water before placing the tubes into a heated oven. Extraction of iodine is carried out in the oven for 3 hours. After cooling, the samples are diluted with water. Samples are filtered before analysing on the ICP-MS instrument. The internal standard used in this method is Tellurium.
Iron FFIC-23-ICP-OES 10 mg/kg The ground sample is digested using a microwave with nitric and hydrochloric acids (3:1). The digestate is transferred quantitatively to a volumetric flask, made to volume with DI water, and filtered. Final analysis is done with the addition of internal standard by ICP-OES.
Lead FFIC-23-ICP-OES 5 mg/kg The ground sample is digested using a microwave with nitric and hydrochloric acids (3:1). The digestate is transferred quantitatively to a volumetric flask, made to volume with DI water, and filtered. Final analysis is done with the addition of internal standard by ICP-OES.
Lead FFIC-MULTI-ICP-MS 0.2 mg/kg (typical matrix) The ground sample is digested using a microwave with nitric and hydrochloric acids (3:1). The digestate is transferred quantitatively to a volumetric flask, made to volume with DI water, and filtered. Final analysis is done with the addition of internal standard by ICP-MS. (20 mg/kg using higher dilution) Table Note 3
Magnesium FFIC-23-ICP-OES 300 mg/kg The ground sample is digested using a microwave with nitric and hydrochloric acids (3:1). The digestate is transferred quantitatively to a volumetric flask, made to volume with DI water, and filtered. Final analysis is done with the addition of internal standard by ICP-OES.
Manganese FFIC-23-ICP-OES 2 mg/kg The ground sample is digested using a microwave with nitric and hydrochloric acids (3:1). The digestate is transferred quantitatively to a volumetric flask, made to volume with DI water, and filtered.  Final analysis is done with the addition of internal standard by ICP-OES.
Mercury FFIC-Hg-DMA 0.044 mg/kg The concept of the analysis of total Hg by direct analysis uses sequential thermal decomposition of the sample in an oxygen stream, catalytic conversion to elemental Hg, amalgamation to trap the Hg and a final heating step to release the Hg from the amalgam for atomic absorption spectrophotometry detection at 253.65 nm. Spectral interferences are removed using a 254 nm filter and 9 mm bandwidth.
Mercury FFIC-HG-ICP-MS 0.025 mg/kg This method is applicable to the determination of total acid extractable Hg in a wide range of feed matrix. The digestate is transferred quantitatively to a volumetric flask, made to volume with DI water, and filtered. Holmium internal standard is added in-line. L-Cysteine is added to all solutions to improve stability and decrease memory effect. Final analysis is done by ICP-MS
Molybdenum FFIC-23-ICP-OES 0.20 mg/kg The ground sample is digested using a microwave with nitric and hydrochloric acids (3:1). The digestate is transferred quantitatively to a volumetric flask, made to volume with DI water, and filtered. Final analysis is done with the addition of internal standard by ICP-OES.
Molybdenum FFIC-MULTI-ICP-MS 0.2 mg/kg (typical matrix) The ground sample is digested using a microwave with nitric and hydrochloric acids (3:1). The digestate is transferred quantitatively to a volumetric flask, made to volume with DI water, and filtered. Final analysis is done with the addition of internal standard by ICP-MS. (20 mg/kg using higher dilution) Table Note 3
Nickel FFIC-23-ICP-OES 3 mg/kg The ground sample is digested using a microwave with nitric and hydrochloric acids (3:1). The digestate is transferred quantitatively to a volumetric flask, made to volume with DI water, and filtered. Final analysis is done with the addition of internal standard by ICP-OES.
Nickel FFIC-MULTI-ICP-MS 0.5 mg/kg (typical matrix) The ground sample is digested using a microwave with nitric and hydrochloric acids (3:1). The digestate is transferred quantitatively to a volumetric flask, made to volume with DI water, and filtered. Final analysis is done with the addition of internal standard by ICP-MS. (50 mg/kg using higher dilution) Table Note 3
Palladium FFIC-23-ICP-OES 17 mg/kg The ground sample is digested using a microwave with nitric and hydrochloric acids (3:1). The digestate is transferred quantitatively to a volumetric flask, made to volume with DI water, and filtered. Final analysis is done with the addition of internal standard by ICP-OES.
Palladium FFIC-MULTI-ICP-MS 0.5 mg/kg (typical matrix) The ground sample is digested using a microwave with nitric and hydrochloric acids (3:1). The digestate is transferred quantitatively to a volumetric flask, made to volume with DI water, and filtered. Final analysis is done with the addition of internal standard by ICP-MS. (50 mg/kg using higher dilution) Table Note 3
Phosphorus FFIC-23-ICP-OES 300 mg/kg The ground sample is digested using a microwave with nitric and hydrochloric acids (3:1). The digestate is transferred quantitatively to a volumetric flask, made to volume with DI water, and filtered. Final analysis is done with the addition of internal standard by ICP-OES.
Potassium FFIC-23-ICP-OES 300 mg/kg The ground sample is digested using a microwave with nitric and hydrochloric acids (3:1). The digestate is transferred quantitatively to a volumetric flask, made to volume with DI water, and filtered. Final analysis is done with the addition of internal standard by ICP-OES.
Salt FFIC-SALT-POTIT 0.1% The method is based on a potentiometric titration. A weighed sample is placed in water, and nitric acid is added to ensure solubility, and the chlorides present are titrated with silver nitrate solution 0.1N. The titration is performed with a potentiometer equipped with a silver-silver chloride reference electrode and a silver indicating electrode. Feed results are reported as percent salt.
Selenium FFIC-23-ICP-OES 22 mg/kg The ground sample is digested using a microwave with nitric and hydrochloric acids (3:1). The digestate is transferred quantitatively to a volumetric flask, made to volume with DI water, and filtered. Final analysis is done with the addition of internal standard by ICP-OES.
Selenium FFIC-MULTI-ICP-MS 0.05 mg/kg (typical matrix) The ground sample is digested using a microwave with nitric and hydrochloric acids (3:1). The digestate is transferred quantitatively to a volumetric flask, made to volume with DI water, and filtered. Final analysis is done with the addition of internal standard by ICP-MS. (5.0 mg/kg using higher dilution) Table Note 3
Sodium FFIC-23-ICP-OES 300 mg/kg The ground sample is digested using a microwave with nitric and hydrochloric acids (3:1). The digestate is transferred quantitatively to a volumetric flask, made to volume with DI water, and filtered. Final analysis is done with the addition of internal standard by ICP-OES.
Sulphur FFIC-23-ICP-OES 300 mg/kg The ground sample is digested using a microwave with nitric and hydrochloric acids (3:1). The digestate is transferred quantitatively to a volumetric flask, made to volume with DI water, and filtered. Final analysis is done with the addition of internal standard by ICP-OES.
Uranium FFIC-23-ICP-OES 20 mg/kg The ground sample is digested using a microwave with nitric and hydrochloric acids (3:1). The digestate is transferred quantitatively to a volumetric flask, made to volume with DI water, and filtered.  Final analysis is done with the addition of internal standard by ICP-OES.
Uranium FFIC-MULTI-ICP-MS 0.02 mg/kg (typical matrix) The ground sample is digested using a microwave with nitric and hydrochloric acids (3:1). The digestate is transferred quantitatively to a volumetric flask, made to volume with DI water, and filtered. Final analysis is done with the addition of internal standard by ICP-MS. (2.0 mg/kg using higher dilution) Table Note 3
Vanadium FFIC-23-ICP-OES 2 mg/kg The ground sample is digested using a microwave with nitric and hydrochloric acids (3:1). The digestate is transferred quantitatively to a volumetric flask, made to volume with DI water, and filtered. Final analysis is done with the addition of internal standard by ICP-OES.
Vanadium FFIC-MULTI-ICP-MS 0.2 mg/kg (typical matrix) The ground sample is digested using a microwave with nitric and hydrochloric acids (3:1). The digestate is transferred quantitatively to a volumetric flask, made to volume with DI water, and filtered. Final analysis is done with the addition of internal standard by ICP-MS. (20 mg/kg using higher dilution) Table Note 3
Zinc FFIC-23-ICP-OES 10 mg/kg The ground sample is digested using a microwave with nitric and hydrochloric acids (3:1). The digestate is transferred quantitatively to a volumetric flask, made to volume with DI water, and filtered. Final analysis is done with the addition of internal standard by ICP-OES.
Zinc FFIC-MULTI-ICP-MS 5.0 mg/kg (typical matrix) The ground sample is digested using a microwave with nitric and hydrochloric acids (3:1). The digestate is transferred quantitatively to a volumetric flask, made to volume with DI water, and filtered. Final analysis is done with the addition of internal standard by ICP-MS. (500 mg/kg using higher dilution) Table Note 3

Table Notes

Table Note 2

The LOQ can on occasion be the reporting limit, which is higher than the LOQ.

Return to table note 2  referrer

Table note 3

Considering the wide range of matrices typically received and unusual sample types, the lab may increase the LOQ or reporting limit as needed based on higher dilution factors needed to mitigate interferences, protect the instrumentation and/or use proper extraction.

Return to table note 3  referrer

Analytical methods for organic compounds and contaminants

Veterinary drugs

Feed samples submitted for veterinary drug analysis are ground in a sequential order to prevent carry over, mixed and filled in a labelled 250 mL amber sample bottle to ⅔ full: non-medicated samples for residue analysis (1st), followed by the medicated complete feeds for residue testing (2nd), then, the medicated complete feeds for guarantee level testing (3rd), followed by the medicated premix samples for guarantee level testing (4th) and the medicated premix for residue level testing (5th).

Analytical methods for veterinary drugs
Analyte Method code LOQ or reporting limit Table Note 4 Brief method description
Amprolium FD-DRUGS-AMP 2 mg/kg Amprolium is extracted from  ground feed samples by shaking with 100 mL methanol/water (2+1) containing 5 mM sodium dioctylsulfosuccinate (DOSS) and 10 mM CaCl2. The extract is filtered through a 0.45 μm nylon filter and late eluting interferences are removed on a chromatography cartridge of acidic alumina. Determination is by reversed phase liquid chromatography (LC) using a mobile phase containing DOSS to increase the retention time of amprolium beyond that of co-extractives.
Arsanilic Acid, Roxarsone, Nitarsone FD-DRUGS-ORGANOARSENIC 0.50 mg/kg The organoarsenical drugs are extracted from feed samples with 2% K2HPO4 in methanol/water (10+90) using mechanical shaking for 30 minutes. A portion of the extract is then centrifuged, filtered and diluted in methanol/water (10+90). The drugs are determined and detected by anion exchange liquid chromatography-tandem mass spectrometry (LC-MS/MS) and quantified by using a product ion for each analyte and confirmed by another product ion of the same analyte.
Bacitracin FD-DRUGS-BACR 0.3 mg/kg (residue) Bacitracin A is extracted using methanol/water (70+30, v/v) with mechanical shaking for 1 hour. A centrifuged portion of the extract is diluted, then filtered, and bacitracin A is detected and determined by reverse phase LC-MS/MS. The result is quantified using a product ion and confirmed by another product ion of the same analyte.
Chlortetracycline hydrochloride FD-BIO-CTC 2 mg/kg Chlortetracycline hydrochloride is extracted from feed samples in an acid-acetone solution, maintained at or below pH 1.2, using mechanical agitation. For sample guarantees ≤ 50 ppm, an aliquot of clarified supernatant is pH adjusted to 4.5 and then diluted to the reference concentration of the standard response line. For sample guarantees > 50 ppm, an aliquot of clarified supernatant is diluted to the reference concentration. A series of standard concentrations and the sample solutions are pipetted into wells cut into agar inoculated with Bacillus cereus. The agar plates are incubated overnight and the resulting zones of inhibition are measured and the potency calculated using the dose-response curve.
Decoquinate FD-DRUGS-DEC 0.5 mg/kg Decoquinate is extracted from ground feed samples with 1% CaCl2/methanol solution. After filtration and dilution an aliquot is diluted with H2O, and analysed by reversed phase LC with fluorescence detection. Positive trace level samples are confirmed by LC analysis using an alternate excitation wavelength.
Erythromycin FD-DRUGS-LCMSMS2 0.10 mg/kg In this multi-residue method erythromycin is extracted using methanol/ammonium acetate 5 mM (90+10) with mechanical shaking for 1 hour. A portion of the extracts are filtered, and then diluted, and the veterinary drugs are determined and detected by reverse phase LC-MS/MS. Results are quantified using a product ion for each analyte and confirmed by another product ion of the same analyte.
Hygromycin B FD-DRUGS-HILIC1 0.40 mg/kg Hygromycin B is extracted using 1% formic acid with mechanical shaking for 1 hour. A portion of the extracts are filtered and the veterinary drugs are determined and detected by hydrophilic interaction chromatography (HILIC) - LC-MS/MS. Results are quantified using a product ion for each analyte and confirmed by another product ion of the same analyte.
Lasalocid sodium FD-DRUGS-LAS_RP 1.0 mg/kg Lasalocid sodium is extracted from feed samples into an acidified methanol solution using an ultrasonic bath at 40°C, or by shaking and overnight extraction (shaking and overnight extraction is mandatory for mineral premixes). Sample extracts are centrifuged, diluted to the target concentration, if necessary, and analyzed by reversed phase LC with fluorescence detection. Lasalocid sodium consists of 5 homologs; lasalocid A predominates (> 90 % of the total), with the remainder being homologs B, C, D and E. All homologs have equal bioactivity, and therefore no biopotency correction factors are required to calculate the total lasalocid content of test samples.
Lasalocid sodium FD-DRUGS-LCMSMS1 0.10 mg/kg
0.01 mg/kg (traceback)
In this multi-residue method lasalocid sodium is extracted using methanol/ammonium acetate 5 mM (90+10) with mechanical shaking for 1 hour. A portion of the extracts are filtered, then diluted, and the veterinary drugs are determined and detected by reverse phase LC-MS/MS. Results are quantified using a product ion for each analyte and confirmed by a second product ion.
Lincomycin FD-BIO-LINC 1 mg/kg Lincomycin is extracted from feed samples in a methanolic buffer solution using mechanical agitation and centrifugation. Clarified supernatants of the low level Lincomycin feeds (<22 ppm) are concentrated, then washed with hexane in a separatory funnel. The aqueous phase is pH adjusted to 8.0 and then diluted to the reference concentration of the standard response line. For feeds at intermediate guarantees (≥22 to 88 ≤ ppm), the extract is transferred to a separatory funnel and washed with hexane. An aliquot of the aqueous phase is pH adjusted to 8.0 and diluted to the reference level. For feeds containing medication at higher levels (≥ 88 ppm), an aliquot of clarified supernatant is simply diluted to the reference concentration. A series of standard concentrations and the sample solutions are pipetted into cylinders set on the surface of agar inoculated with Kocuria rhizophila (ATCC 9341). The agar plates are incubated overnight, the resulting zones of inhibition are measured and the antibiotic potency is calculated using the dose-response curve.
Lincomycin FD-DRUGS-LCMSMS2 0.10 mg/kg In this multi-residue method lincomycin is extracted using methanol/ammonium acetate 5 mM (90+10) with mechanical shaking for 1 hour. A portion of the extracts are filtered, then diluted, and the veterinary drugs are determined and detected by reverse phase LC-MS/MS. Results are quantified using a product ion for each analyte and confirmed by another product ion of the same analyte.
Maduramicin FD-DRUGS-LCMSMS1 0.10 mg/kg
0.01 mg/kg (traceback)
In this multi-residue method maduramicin is extracted using methanol/ammonium acetate 5 mM (90+10) with mechanical shaking for 1 hour. A portion of the extracts are filtered, then diluted, and the veterinary drugs are determined and detected by reverse phase LC-MS/MS. Results are quantified using a product ion for each analyte and confirmed by a second product ion.
Melengestrol acetate (MGA) FD-DRUGS-MGA 0.08 mg/kg Melengestrol acetate (MGA) is extracted from an aqueous slurry of feed sample with hexane, partitioned from hexane into aqueous methanol, and then into dichloromethane. After evaporation of the dichloromethane the dried extract is transferred with chloroform into a volumetric flask. An aliquot is pipetted into a mixed column of silica gel and acid aluminium oxide, and the MGA is eluted with a hexane-acetone solution. The residue is dissolved in mobile phase and the MGA is quantitated by LC.
Melengestrol acetate (MGA) FD-DRUGS-MGAR 4 µg/kg Melengestrol acetate (MGA) is extracted using methanol/water (75:25) with mechanical shaking for 1 hour. A portion of the extract is transferred to a CHEM-ELUT cartridge, eluted with hexane and reconstituted in methanol. The extract is then purified with C18 solid phase extraction column. Concentration of MGA is determined by reverse phase LC-MS/MS. Results are quantified using a product ion and confirmed by another product ion of the same precursor. 4,6-Pregnadien-6-methyl-16-methylene-17-ol-3,20-dione-2,2-d2 Acetate (Melengestrol Acetate – d2, MGA-d2) is used as an internal standard.
Monensin sodium FD-DRUGS-IONO4 1 mg/kg The ionophores monensin sodium, narasin and salinomycin sodium are extracted using methanol/water (90+10) with mechanical shaking for 1 hour, then the extracts are filtered. The ionophores are determined by reverse phase LC using post-column derivatization with vanillin, and detection at 520 nm. Results can be confirmed using a hexane extraction or post-column derivatization with dimethylaminobenzaldehyde (DMAB) and detection at 592 nm.
Monensin sodium FD-DRUGS-LCMSMS1 0.10 mg/kg
0.01 mg/kg (traceback)
In this multi-residue method monensin sodium is extracted using methanol/ammonium acetate 5 mM (90+10) with mechanical shaking for 1 hour. A portion of the extracts are filtered, diluted, and the veterinary drugs are determined and detected by reverse phase LC-MS/MS. Results are quantified using a product ion for each analyte and confirmed by a second product ion.
Narasin FD-DRUGS-IONO4 2 mg/kg The ionophores monensin sodium, narasin and salinomycin sodium are extracted using methanol/water (90+10) with mechanical shaking for 1 hour, then the extracts are filtered. The ionophores are determined by reverse phase LC using post-column derivatization with vanillin, and detection at 520 nm. Results can be confirmed using a hexane extraction, or post-column derivatization with dimethylaminobenzaldehyde (DMAB) and detection at 592 nm.
Narasin FD-DRUGS-LCMSMS1 0.10 mg/kg
0.01 mg/kg (traceback)
In this multi-residue method narasin is extracted using methanol/ammonium acetate 5 mM (90+10) with mechanical shaking for 1 hour. A portion of the extracts are filtered, diluted, and the veterinary drugs are determined and detected by reverse phase LC-MS/MS. Results are quantified using a product ion for each analyte and confirmed by a second product ion.
Nicarbazin FD-DRUGS-NIC-LC 2.0 mg/kg Nicarbazin is extracted from samples and determined by reversed phase LC with UV detection. Feed, supplements and premixes containing < 4000 mg/kg are extracted with acetonitrile/water (80+20,v/v). Concentrated samples containing over 4000 mg/kg are extracted with dimethylformamide. An aliquot of the extract is filtered, diluted if necessary, and the 4,4-dinitrocarbanilide moiety of nicarbazin is determined by LC using a UV detector at 350 nm.
Novobiocin FD-DRUGS-LCMSMS2 0.30 mg/kg In this multi-residue method novobiocin is extracted using methanol/ammonium acetate 5 mM (90+10) with mechanical shaking for 1 hour. A portion of the extracts are filtered, then diluted, and the veterinary drugs are determined and detected by reverse phase LC-MS/MS. Results are quantified using a product ion for each analyte and confirmed by another product ion of the same analyte.
Oxytetracycline hydrochloride (HCl) FD-DRUGS-OTC-LC 2.0 mg/kg
10.0 mg/kg (in fish feed)
Oxytetracycline HCl is extracted from ground feed samples in an acid-methanol solution using mechanical agitation. After centrifugation for 5 minutes at 2000 rpm, an aliquot of the extract is diluted with water and/or acid-methanol so that the concentration of Oxytetracycline HCl is approximately the same as in the working standard, and the solutions contain at least 50% water. The extracts are filtered and analyzed by reversed phase LC with fluorescence detection.
Penicillin (from Procaine Penicillin G) FD-BIO-PEN 1 mg/kg Procaine Penicillin G is extracted from feed samples in an acetone-buffer solution using mechanical agitation. An aliquot of clarified supernatant is diluted to the reference concentration of the standard response line. A series of standard concentrations and the sample solutions are pipetted into cylinders set on the surface of agar inoculated with Kocuria rhizophila (ATCC 9341). The agar plates are incubated overnight and the resulting zones of inhibition are measured and the antibiotic potency calculated using the dose-response curve.
Ractopamine FD-DRUGS-RAC 2 mg/kg
1 mg/kg (in turkey and swine feeds)
Ractopamine is extracted from ground feed samples with acidic methanol/water solution. An aliquot of the extract is diluted with a 2% acetic acid solution. Determination is by reversed phase LC with Fluorescence detection.
Ractopamine FD-DRUGS-RACMSR 0.05 mg/kg Ractopamine is extracted using methanol/ammonium acetate 5 mM (90+10) with mechanical shaking for 1 hour. A portion of the extract is filtered, then diluted and the veterinary drug is detected and determined by reverse phase LC-MS/MS. Result is quantified using a mass transition and confirmed by another mass transition. Ractopamine-d6 HCl is used as an internal standard (IS).
Salinomycin sodium FD-DRUGS-IONO4 2 mg/kg The ionophores monensin sodium, narasin and salinomycin sodium are extracted using methanol/water (90+10) with mechanical shaking for 1 hour, and then the extracts are filtered. The ionophores are determined by reverse phase LC using post-column derivatization with vanillin, and detection at 520 nm. Results can be confirmed using a hexane extraction, or post-column derivatization with dimethylaminobenzaldehyde (DMAB) and detection at 592 nm.
Salinomycin sodium FD-DRUGS-LCMSMS1 0.10 mg/kg
0.01 mg/kg (traceback)
In this multi-residue method salinomycin is extracted using methanol/ammonium acetate 5 mM (90+10) with mechanical shaking for 1 hour. A portion of the extracts are filtered, then diluted, and the veterinary drugs are determined and detected by reverse phase LC-MS/MS. Results are quantified using a product ion for each analyte and confirmed by a second product ion.
Semduramicin sodium FD-DRUGS-LCMSMS1 0.10 mg/kg
0.01 mg/kg (traceback)
In this multi-residue method semduramicin sodium is extracted using methanol/ammonium acetate 5 mM (90+10) with mechanical shaking for 1 hour. A portion of the extracts are filtered, then diluted, and the veterinary drugs are determined and detected by reverse phase LC-MS/MS. Results are quantified using a product ion for each analyte and confirmed by a second product ion.
Spectinomycin FD-DRUGS-HILIC1 0.20 mg/kg Spectinomycin is extracted using 1% formic acid with mechanical shaking for 1 hour. A portion of the extracts are filtered and the veterinary drugs are determined and detected by HILIC - LC-MS/MS. Results are quantified using a product ion for each analyte and confirmed by another product ion of the same analyte.
Sulfadiazine FD-DRUGS-SDZR 0.10 mg/kg Sulfadiazine is extracted from ground feed with 0.3 N HCl and 1.5% diethylamine in 25% methanol by shaking for 1 hour. The clarified extract is subjected to LC separation and post-column derivatization (LC-PCD) with dimethylaminobenzaldehyde (DMAB). Sulfamerazine (SMR) is used as the internal standard for sulfadiazine (SDZ). Compounds are detected at 450 nm.
Sulfadiazine FD-DRUGS-SDZ 20 mg/kg Sulfadiazine is extracted from ground feed with 0.2 N HCl and 1.5% diethylamine (DEA) in 25% methanol by shaking for 1 hour. Clarified extract is subjected to LC separation and post-column derivatization (LC-PCD) with dimethylaminobenzaldehyde (DMAB). Sulfamerazine (SMR) is used as internal standard for sulfadiazine (SDZ). Compounds are detected at 450 nm.
Sulfamethazine residue FD-DRUGS-SQNR 0.10 mg/kg Sulfamethazine is extracted from ground feed with 0.3 N HCl and 1.5% diethylamine (DEA) in 25% methanol by shaking for 1 hour. The clarified extract is subjected to LC separation and post-column derivatization (LC-PCD) with dimethylaminobenzaldehyde (DMAB). Sulfamerazine (SMR) is used as the internal standard for sulfamethazine (SMT). Compounds are detected at 450 nm.
Sulfamethazine FD-DRUGS-SQN 20 mg/kg Sulfamethazine is extracted from ground feed with 0.2 N HCl and 1.5% diethylamine (DEA) in 25% methanol by shaking for 1 hour. Clarified extract is subjected to LC separation and post-column derivatization (LC-PCD) with dimethylaminobenzaldehyde (DMAB). Sulfamerazine (SMR) is used as internal standard for sulfamethazine (SMT). Compounds are detected at 450 nm.
Tiamulin FD-DRUGS-TIA 4.0 mg/kg The feed sample is treated with a sodium carbonate solution to remove tiamulin from its carrier and to convert tiamulin to the free base. The tiamulin base is extracted into an organic solvent mixture and then concentrated by back extraction into an aqueous tartaric acid solution. Tiamulin is then analyzed by reversed phase liquid chromatography with detection at 254 nm.
Tilmicosin FD-DRUGS-TIL 5.0 mg/kg Tilmicosin is extracted from feed with water/acetonitrile/buffer feed extractant, and from drug premixes using a premix extractant. The extract is filtered, diluted if necessary, and analysed by LC. A gradient mobile phase is used to separate tilmicosin from coextractants. The method elutes the two tilmicosin isomers as a single peak.
Tylosin phosphate FD-BIO-TYL 4 mg/kg Tylosin is extracted from feedsamples in a methanolic buffer solution using mechanical agitation. An aliquot of clarified supernatant is diluted to target the reference concentration of the standard response line. A series of standard concentrations and the sample solutions are pipetted into wells cut into agar inoculated with Kocuria rhizophila (ATCC 9341). The agar plates are incubated overnight and the resulting zones of inhibition are measured and the antibiotic potency calculated using the dose-response curve.
Tylosin phosphate FD-DRUGS-LCMSMS2 0.10 mg/kg In this multi-residue method tylosin phosphate is extracted using methanol/ammonium acetate 5 mM (90+10) with mechanical shaking for 1 hour. A portion of the extracts are filtered, diluted, and the veterinary drugs are determined and detected by reverse phase LC-MS/MS. Results are quantified using a product ion for each analyte and confirmed by another product ion of the same analyte.
Virginiamycin FD-BIO-VMY 5 mg/kg Virginiamycin is extracted from the sample in 50% citric acid-acetone solution using mechanical agitation. An aliquot of clarified supernatant is adjusted to a pH of 6.0 and is then washed with petroleum ether and cyclohexane, concentrated to small volume and finally diluted to the reference concentration of the standard response line. A series of standard concentrations and the sample solutions are pipetted into wells cut into agar inoculated with Kocuria rhizophila (ATCC 9341). The agar plates are incubated overnight and the resulting zones of inhibition are measured and the antibiotic potency calculated using the dose-response curve.
Virginiamycin FD-DRUGS-LCMSMS2 0.10 mg/kg In this multi-residue method virginiamycin is extracted using methanol/ammonium acetate 5 mM (90+10) with mechanical shaking for 1 hour. A portion of the extracts are filtered, then diluted, and the veterinary drugs are determined and detected by reverse phase LC-MS/MS. Results are quantified using a product ion for each analyte and confirmed by another product ion of the same analyte.
Zilpaterol HCl in cattle feed and supplements FD-DRUGS-ZIL 0.2 mg/kg Zilpaterol HCl is extracted from the feed or supplement using phosphate buffer at pH 2.1. The sample is then sonicated and shaken. For solid samples, the mixture is then centrifuged, and this process is repeated, pooling the extracts. The extracts are then filtered before determination by reversed phase LC with fluorescence detection at an excitation of 284 nm and emission of 320 nm.
Zilpaterol HCl FD-DRUGS-ZILR 0.3 mg/kg Zilpaterol HCl is extracted from the various types of feed by sonication and mechanical shaking using a phosphate buffer at pH 2.1. The extract is cleaned up using a combination of NH2 and C18 solid phase extraction. Determination is performed by reversed phase LC with fluorescence detection at an excitation of 284 nm and emission of 320 nm. The presence of residue of Zilpaterol HCl in a sample above LOQ is confirmed by LC-MS/MS.

Table Notes

Table Note 4

Considering the wide range of matrices typically received and unusual sample types, the lab may increase the LOQ or reporting limit as needed based on higher dilution factors needed to mitigate interferences, protect the instrumentation and/or use proper extraction.

Return to table note 4  referrer

Mycotoxins

Feed samples submitted for Mycotoxin analysis are ground, mixed and filled in a labelled 500 mL amber sample bottle to two-thirds full with a sufficient representative proportion.

Analytical methods for mycotoxins
Analyte Method code LOQ or reporting limit Table Note 5 Brief method description
Ergot Alkaloids (6 pairs) FD-TOXINS-ERG Ergometrine + Ergometrinine – 20 μg/kg
Ergosine + Ergosinine - 20 μg/kg
Ergotamine + Ergotaminine - 20 μg/kg
Ergocornine + Ergocorninine - 20 μg/kg
Ergocryptine + Ergocryptinine -20 μg/kg
Ergocristine + Ergocristinine -20 μg/kg
Sum Ergot Alkaloid - 120 μg/kg
Ergot Alkaloids are extracted by shaking with acetonitrile/ammonium carbonate (2.08 mmol/L) solution. The extract is filtered and purified using commercially available mycosep column. The purified extract is then diluted with acetonitrile prior to determination by liquid chromatography-tandem mass spectrometry (LC-MS/MS).
Fumonisins (total of B1 and B2) FD-TOXINS-FUM-LCMS 0.30 mg/kg Fumonisins are extracted using methanol:water (75:25) with mechanical shaking for an hour. A portion of the extract is filtered, then diluted and the fumonisins are determined by reverse phase LC-MS/MS. Results are quantified using a product ion for each analyte. Identification of the analyte is confirmed by comparing the relative ion intensities of two transitions in the standard solutions and the relative ion intensities of the same two transitions in the sample.
Multiple Mycotoxins FD-TOXINS-MULTITOX Zearalenone – 100 μg/kg
Aflatoxin B1 – 1.0 μg/kg
Aflatoxin B2 - 1.0 μg/kg
Aflatoxin G1 – 1.0 μg/kg
Aflatoxin G2 - 1.0 μg/kg
Total Aflatoxins - 4.0 μg/kg
Ochratoxin A - 10 μg/kg
Targeted mycotoxins are extracted from ground feed by shaking with acetonitrile-water. The extract is diluted with methanol-water containing formic acid and ammonium formate, and then filtered for toxin determination by LC-MS/MS with labeled internal standards.
Trichothecenes FD-TOXINS-TRICO-LCMS

Feed:
0.050 mg/kg for Nivalenol, Fusarenon-X, 15-acetyldeoxynivalenol, 3-acetyldeoxynivalenol, Neosolaniol, and Diacetoxyscirpenol
0.12 mg/kg for Deoxynivalenol
0.020 mg/kg for HT-2 0.015 mg/kg for T-2

Dried Distillers Grains (DDGs):
0.10 mg/kg for Nivalenol, Fusarenon-X, 15-acetyldeoxynivalenol, 3-acetyldeoxynivalenol, Neosolaniol, and Diacetoxyscirpenol,
0.30 mg/kg for Deoxynivalenol
0.027 mg/kg for HT-2
0.015 mg/kg for T-2

Trichothecenes are extracted using acetonitrile:water (84:16) with mechanical shaking for two hours. A portion of the extract is passed through a MycoSep column. Trichothecenes are determined by reverse phase LC-MS/MS. Results are quantified using a product ion for each analyte. Identification of the analyte is confirmed by comparing the relative ion intensities of two transitions in the standard solutions and the relative ion intensities of the same two transitions in the sample. Quantitation is performed using labelled isotopes internal standards and both ionization polarities.

Table Notes

Table Note 5

Considering the wide range of matrices typically received and unusual sample types, the lab may increase the LOQ or reporting limit as needed based on higher dilution factors needed to mitigate interferences, protect the instrumentation and/or use proper extraction.

Return to table note 5  referrer

Dioxins, furans and polychlorinated biphenyls (pcbs)

Feed samples submitted for environmental contaminant analysis should arrive in a glass amber jar with an aluminum foil-lined lid, not more than ¾ full, and be a minimum of 1 kg weight.

Analytical methods for dioxins, furans and polychlorinated biphenyls (pcbs)
Analyte Method code Limit of detection (LOD) Brief method description
Seven dioxin congeners, ten furan congeners and twelve dioxin-like PCB congeners (refer to Appendix I) EC-001 refer to Appendix I Feed samples are thoroughly homogenized. A representative sample is taken, mixed with Ottawa sand and is extracted overnight by soxhlet. The extract is concentrated, dried, filtered and passed through acidified silica, silica and alumina columns on the PowerPrep system. Palm Oils are dissolved in hexane and put through additional acidified silica prior to cleanup on the FMS. A carbon column separates samples into two fractions, dioxin/furans/coplanar-PCB's, and World Health Organization (WHO)-PCB's/marker PCB's. The fractions are concentrated and analyzed by gas chromatography with high resolution mass spectrometry (GC-HRMS) detection (resolution of greater than 10,000). Quantitation is calculated on isotope dilution, and congener (analyte) results are multiplied by toxic equivalent factors (TEF). Results are reported as the upper-bound sum of toxic equivalents (TEQs).

Pesticides

Feed samples submitted for pesticide analysis should arrive in a glass amber jar with an aluminum foil-lined lid, not more than ¾ full, and be a minimum of 1 kg weight.

Analytical methods for pesticides
Analyte Method code Reporting limit Brief method description
The list of pesticides can be found in Appendix II FF-001 refer to Appendix II The feed sample is homogenised, then a 50 g sample is shaken with water and acetonitrile, salted out with NaCl, centrifuged, and a portion of the acetonitrile is decanted and evaporated to 5mL. The concentrated extract is dried with anhydrous sodium sulfate, solvent exchanged to cyclohexane, quantitatively transferred to a 10mL volumetric flask and made to volume with 1:3 acetone:cyclohexane. Gel Permeation Chromatography (GPC) is used for initial cleanup, Envicarb and aminopropyl solid phase extraction is the final cleanup. The extract is then split, and half is analyzed by gas chromatography-mass selective detector (GC-MSD), and the other portion is analyzed by liquid chromatography-mass spectrometry (LC-MS/MS).

Appendix I - The World Health Organization (WHO) 2005 Toxic Equivalency Factors (TEFs) and CFIA Calgary Laboratory Limits of Detection (LOD) for dioxins, furans, dioxin-like polychlorinated biphenyls (PCBs) and indicator PCBs

Polychlorinated Dibenzo-para-dioxins (PCDDs)

Congener TEF value LOD for feed (pg/g w/w) Table Note 6 LOD for hydrogenated palm oil (pg/g w/w) Table Note 7
2,3,7,8-TCDD 1 0.075 0.026
1,2,3,7,8-PeCDD 1 0.075 0.032
1,2,3,4,7,8-HxCDD 0.1 0.100 0.043
1,2,3,6,7,8-HxCDD 0.1 0.100 0.047
1,2,3,7,8,9-HxCDD 0.1 0.100 0.046
1,2,3,4,6,7,8-HpCDD 0.01 0.100 0.054
OCDD 0.0003 0.200 0.150

Table Notes

Table Note 6

LOD estimate for feed matrix is based on 5.0 g sample size

Return to table note 6  referrer

Table Note 7

LOD estimate for hydrogenated palm oil matrix is based on 6.0 g sample size

Return to table note 7  referrer

Polychlorinated Dibenzofurans (PCDFs)

Congener TEF value LOD for feed (pg/g w/w) Table Note 8 LOD for hydrogenated palm oil (pg/g w/w) Table Note 9
2,3,7,8-TCDF 0.1 0.075 0.049
1,2,3,7,8-PeCDF 0.03 0.075 0.034
2,3,4,7,8-PeCDF 0.3 0.100 0.030
1,2,3,4,7,8-HxCDF 0.1 0.100 0.035
1,2,3,6,7,8-HxCDF 0.1 0.100 0.025
1,2,3,7,8,9-HxCDF 0.1 0.100 0.034
2,3,4,6,7,8-HxCDF 0.1 0.100 0.025
1,2,3,4,6,7,8-HpCDF 0.01 0.100 0.027
1,2,3,4,7,8,9-HpCDF 0.01 0.100 0.026
OCDF 0.0003 0.200 0.060

Table Notes

Table Note 8

LOD estimate for feed matrix is based on 5.0 g sample size

Return to table note 8  referrer

Table Note 9

LOD estimate for hydrogenated palm oil matrix is based on 6.0 g sample size

Return to table note 9  referrer

Dioxin-Like PCBs (DL-PCBs) - Non-ortho PCBs

Congener TEF value LOD for feed (pg/g w/w) Table Note 10 LOD for hydrogenated palm oil (pg/g w/w) Table Note 11
PCB 77 0.0001 0.200 0.641
PCB 81 0.0003 0.100 0.442
PCB 126 0.1 0.200 0.428
PCB 169 0.03 0.150 0.471

Table Notes

Table Note 10

LOD estimate for feed matrix is based on 5.0 g sample size

Return to table note 10  referrer

Table Note 11

LOD estimate for hydrogenated palm oil matrix is based on 6.0 g sample size

Return to table note 11  referrer

Dioxin-Like PCBs (DL-PCBs) - Mono-ortho PCBs

Congener TEF value LOD for feed (pg/g w/w) Table Note 12 LOD for hydrogenated palm oil (pg/g w/w) Table Note 13
PCB 105 0.00003 6.000 2.010
PCB 114 0.00003 1.000 0.0945
PCB 118 0.00003 20.000 5.000
PCB 123 0.00003 2.00 2.007
PCB 156 0.00003 1.000 0.556
PCB 157 0.00003 1.000 0.496
PCB 167 0.00003 5.000 1.029
PCB 189 0.00003 0.500 0.659

Table Notes

Table Note 12

LOD estimate for feed matrix is based on 5.0 g sample size

Return to table note 12  referrer

Table Note 13

LOD estimate for hydrogenated palm oil matrix is based on 6.0 g sample size

Return to table note 13  referrer

Indicator PCBs (Marker PCBs)

Congener LOD for feed (pg/g w/w) Table Note 14 LOD for hydrogenated palm oil (pg/g w/w) Table Note 15
PCB 28 2.000 15.789
PCB 52 10.000 46.345
PCB 101 20.000 21.869
PCB 138 40.000 9.464
PCB 153 40.000 9.371
PCB 180 10.000 2.309

Table Notes

Table Note 14

LOD estimate for feed matrix is based on 5.0 g sample size

Return to table note 14  referrer

Table Note 15

LOD estimate for hydrogenated palm oil matrix is based on 6.0 g sample size

Return to table note 15  referrer

Appendix II – List of pesticides analyzed in feed samples and reporting limits

Pesticide Reporting limit (ppm)
Aldicarb 0.04 ppm
Aldicarb Sulfone 0.04 ppm
Aldicarb Sulfoxide 0.04 ppm
Aldrin 0.04 ppm
Allethrin, d-trans- 0.08 ppm
Ametryne 0.04 ppm
Atrazine 0.04 ppm
Benalaxyl 0.04 ppm
BHC, α- 0.04 ppm
BHC, β- 0.04 ppm
Bifenthrin 0.04 ppm
Butylate 0.04 ppm
Captan Degradation (THPI) monitoring
Captan monitoring
Carbendazim 0.04 ppm
Carbaryl 0.04 ppm
Carbofuran 0.04 ppm
Carboxin 0.04 ppm
Chlordane, trans- Internal standard, not monitored
Chlorfenvinphos 0.04 ppm
Chloridazon 0.04 ppm
Chlormephos 0.04 ppm
Chloroneb 0.04 ppm
Chlorpropham 0.04 ppm
Chlorpyrifos 0.04 ppm
Chlorpyrifos-methyl 0.04 ppm
Chlorthalonil 0.08 ppm
Clomazone 0.04 ppm
Cyfluthrin 0.04 ppm
Cyanazine 0.04 ppm
Cypermethrin 0.04 ppm
DDD, p,p'- [or p,p'-TDE] 0.04 ppm
DDE, p,p'- [or p,p'-DDE] 0.04 ppm
DDT, o,p'- 0.04 ppm
DDT, p,p'- 0.04 ppm
Deltamethrin 0.04 ppm
Demeton-S-methyl 0.04 ppm
Diazinon 0.04 ppm
Dichlorvos 0.04 ppm
Dicloran 0.04 ppm
Dieldrin 0.04 ppm
Diethofencarb 0.04 ppm
Difenoconazole 0.04 ppm
Dimethoate 0.04 ppm
Dioxacarb 0.04 ppm
Diphenamid 0.04 ppm
Disulfoton 0.04 ppm
Endosulfan, α- 0.04 ppm
Endosulfan, β- 0.04 ppm
Endrin 0.04 ppm
EPTC 0.04 ppm
Esfenvalerate 0.04 ppm
Ethiofencarb 0.04 ppm
Ethiofencarb sulfone 0.04 ppm
Ethiofencarb sulfoxide 0.04 ppm
Etrimfos 0.04 ppm
Fenchlorophos 0.04 ppm
Fenitrothion 0.04 ppm
Fenpropathrin 0.04 ppm
Fensulfothion 0.04 ppm
Fenvalerate 0.08 ppm
Fonofos 0.04 ppm
Furthiocarb 0.04 ppm
Heptachlor 0.04 ppm
Heptachlor Epoxide, exo- 0.04 ppm
Heptachlor Epoxide, endo- 0.04 ppm
Hexachlorobenzene 0.04 ppm
3-Hydroxycarbofuran 0.04 ppm
Iambda-Cyhalothrin 0.04 ppm
Indoxacarb 0.04 ppm
Iprodione 0.08 ppm
Iprovalicarb 0.04 ppm
Isoprocarb 0.04 ppm
Isofenphos 0.04 ppm
Lindane [or gamma-BHC] 0.04 ppm
Linuron 0.04 ppm
Malathion 0.04 ppm
Metalaxyl 0.04 ppm
Methacrifos 0.04 ppm
Methamidophos 0.04 ppm
Methidathion 0.04 ppm
Methiocarb 0.04 ppm
Methiocarb sulfone monitoring
Methiocarb sulfoxide monitoring
Methomyl 0.04 ppm
Methoprotryne 0.04 ppm
Methoxychlor 0.04 ppm
Metobromuron 0.04 ppm
Metolachlor 0.04 ppm
Metolcarb 0.04 ppm
Metribuzin 0.04 ppm
Mexacarbate 0.04 ppm
Monocrotophos 0.04 ppm
Monolinuron 0.08 ppm
Naled 0.04 ppm
Omethoate 0.04 ppm
Oxadixyl 0.04 ppm
Oxamyl 0.04 ppm
Oxamyl-oxime 0.04 ppm
Oxycarboxin 0.04 ppm
Parathion 0.04 ppm
Parathion-methyl 0.04 ppm
Permethrin, cis- 0.04 ppm
Permethrin, trans- 0.04 ppm
Phorate 0.04 ppm
Phosalone 0.04 ppm
Phosphamidon 0.04 ppm
Pirimiphos-methyl 0.04 ppm
Procymidon 0.04 ppm
Propamocarb 0.04 ppm
Propanil 0.04 ppm
Propiconazole 0.04 ppm
Propoxur 0.04 ppm
Pyrazophos 0.04 ppm
Quinalphos 0.04 ppm
Tefluthrin 0.04 ppm
Terbufos 0.04 ppm
Terbutylazine 0.04 ppm
Tetrachlorvinphos 0.04 ppm
Thiodicarb 0.04 ppm
Thiamethoxam 0.04 ppm
Thiofanox 0.04 ppm
Thiofanox sulfoxide 0.04 ppm
Triadimenol 0.04 ppm
Tri-allate 0.04 ppm
Triazophos 0.04 ppm
Triadimefon 0.04 ppm
Trifluralin 0.04 ppm
Trimethacarb 0.04 ppm
Vernolate 0.04 ppm
Vinclozolin 0.04 ppm
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