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Laboratory Procedure and Methodology for Trans Fat Monitoring Program

The method used by the Trans Fat Monitoring Program for analyzing the trans and saturated fat content of samples collected is the Association of Official Analytical Chemists (AOAC) Method 996.06 (1). This is the recommended method for the determination of total fat and fatty acids in various foods under the current nutrition labelling regulations both in Canada and the United States. This method has been validated through an international collaborative study.

Methodology Comments

Extraction of Fat:

Fat from the samples was performed according to the AOAC (Association of Official Analytical Chemists) Official Method 996.06 (1). The method involves addition of a suitable gas chromatography (GC) standard with the triglyceride (TG) form (such as 13:0 TG, 17:0 TG, or 21:0 TG), release of fat from the food matrix by acid or base hydrolysis, extraction of the released fat using diethyl-ether/hexane, and transesterification of the fat with BF3-MeOH to fatty acid methyl esters (FAME).

FAME analysis by GC:

Analysis of the FAME was performed by capillary GC according to the AOCS (American Oil Chemists' Society) Official Method Ce 1h-05 (2). GC analysis is performed using a 100 metre SP-2560 capillary column. The column oven temperature was operated isothermally at 180oC. Ultra high purity hydrogen or helium was used as the carrier gas at a constant flow rate of 1.0 mL/min. The injector and detector temperatures were 250oC. These GC conditions provided excellent separation of all the FAME with minimum separation of the 18:1 cis and trans isomers.

The FAME peaks on the chromatogram were identified by comparison with published traces (1-5) and also using FAME reference standard mixtures such as Supelco 37, Alltech K110, Matreya No.1131 positional cis/trans isomer, Nu-Chek GLC-463 or AOCS Partially hydrogenated Soybean Oil Sample #5. Identification of some of the trans isomers were further established by comparison with FAME standards of 6t-18:1, 7t-18:1, 9t-18:1, 11t-18:1, 12t-18:1, 13t-18:1, 15t-18:1, 9t,12t-18:2, 9c,12t-18:2 and 9t,12c-18:2 (Sigma, St. Louis, MO).


Calculation of the fatty acid profile was performed according to the procedure described in AOCS Ce 1h-05. The procedure involves correction of the FAME peak areas for the FID response and conversion of the corrected peak areas to mg amounts with respect to the internal standard. Then the weights are converted to TG equivalents and free fatty acid equivalents. The summation of the TG equivalents gives the total fat content. The trans and saturated fatty acids are calculated as percent of total fatty acids.

Quality Assurance:

Staff Competence: The analyses of samples were performed at the Regional Laboratories of Health Products and Food Branch in Toronto, Ontario and Winnipeg, Manitoba under the guidance of Dr. Nimal Ratnayake. Before proceeding with sample analysis, the analysts from the Regional Laboratories of Health Products and Food Branch in Toronto, Ontario and Winnipeg, Manitoba underwent a 2-day laboratory course on trans fatty acid analysis conducted by Dr. Ratnayake in January 2005 in Ottawa.

Analyst Proficiency: Since 2005, the Toronto Laboratory has participated on a regular basis on the AOCS Laboratory Check Sample Program on Trans Fatty Acids. The Winnipeg laboratory participated in the 2005 and 2006 AOCS Check Sample Program on Trans Fatty Acids. Both laboratories performed exceptionally well with acceptable z-scores between -2 and 2 for all trans fatty acids.

Quality Assurance:

Winnipeg laboratory: A well-characterised margarine sample was extracted and analysed with each sample set. The fat extraction from the characterized margarine sample was reproducible throughout the survey (mean extractable fat=75.6%, RSD=6.6%, n=24). The variability in the total trans fatty acids (measured as % total fatty acids), was also very low (mean total trans fatty acids=17.2%, RSD=2.3%, n=24).

Toronto Laboratory: AOCS check sample #5 (partially hydrogenated soybean oil) was analyzed with each set of five samples. In addition, duplicate analyses were performed with one sample in each set of five samples.


  1. AOAC Official Method 996.06. Fat (Total, Saturated, and Unsaturated) in foods, hydrolytic extraction gas chromatographic method, Revised 2001. In: Official Methods of Analysis of AOAC International 18th Edition (Horwitz, W, ed.).
  2. Determination of cis-, trans-, Saturated, Monounsaturated and Polyunsaturated Fatty Acids in Vegetable or Non-Ruminant Animal Oils and Fats by Capillary GLC Method, Official and Recommended Practices of the AOCS, 5th edn., Revisions and Corrections, AOCS Press, Champaign, Official Method Ce 1h-05, approved 2005, revised 2005.
  3. Ratnayake WMN, Pelletier G (1992) Positional and Geometrical Isomers of Linoleic Acid in Partially Hydrogenated Oils. J Am Oil Chem Soc 69:95-105 (1992).
  4. Ratnayake WMN. Overview of Methods for the Determination of trans Fatty Acids by Gas Chromatography, Silver-Ion Thin-Layer Chromatography, Silver-Ion Liquid Chromatography, and Gas Chromatography/Mass Spectrometry.
  5. Ratnayake WMN, Hansen SL, Kennedy MP (2006) Evaluation of the CP-Sil 88 and SP-2560 Columns Used in the Recently Approved AOCS Official Method Ce 1h-05: Determination of cis-, trans-m Saturated, Monounsaturated, and Polyunsaturated Fatty Acids in Vegetable or Non-ruminant Animal Oils and Fats by Capillary GLC Method. J Am Oil Chem Soc 83:475-488.

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