AOAC Research Institute Performance Tested Methods SM Program. Measurement of Free Fatty Acids Using FASafe Standard Test Kit. Certificate Report

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1 Performance Tested Methods SM Program Measurement of Free Fatty Acids Using FASafe Standard Test Kit Certificate Number June 23, 2003 SāfTest, Inc., West 12 th Street, Suite 4 Tempe, Arizona 82581

2 AOAC Performance Tested Method SM : FASafe Standard Test Kit Abstract The FASafe standard kit developed by Safety Associates, Inc. (SAI) is a rapid test kit designed to measure free fatty acid content of oils, tallows, greases, feed, and protein meals using micro-analytical and membrane separation principles. The proprietary FASafe assay kit can be used for testing solubilized dry and wet food matrices by releasing lipid from the sample matrix using a stabilized reagent (i.e. stabilized isopropanol) and employing mechanical mixing and warming techniques. The solubilized food matrix is then filtered through a membrane and the filtrate is analyzed for free fatty acids. The significantly shorter sample preparation of 15 to 20 minutes permits data to be obtained in 15 to 25 minutes. The SAI s FASafe standard test kit can accommodate up to 135 test tubes and it contains all reagents, calibrators, and control standards required for testing. The test parameters for this procedure are presented below: Sample Preparation: Time Requirement: Limit of Quantitation: Interferences: Liquid oils require no heating; semi-solid and solid oils and meals require heating Sample Preparation 5 to 10 minutes Analysis Time approximately 5 to 10 minutes 0.3% Highly colored samples, samples containing acids, bases, or chelating metal ions Method Author Virginia C. Gordon, Ph.D., 2414 West 12 th Street, Suite 4, Tempe, Arizona 82581; Tel: (480) Submitting Company SāfTest, Inc., 2414 West 12 th Street, Suite 4, Tempe, Arizona 82581; Tel: (480) Independent Laboratory Canadian Grain Commission, Main St., Winnepeg, MB Canada R3C 3G8; Tel: (204) Reference Method American Oil Chemists Society (AOCS) Ca 5a-40 official method Reviewers Mr. Luke Hillyard, Deparment of Food Sciences, UC Davis Dr. David Firestone, FDA Ms. Susan Cupett, University of Nebraska-Lincoln Validated Claims FASafe standard test kit is designed to measure free fatty acid (FFA) content of oils and fats in fresh and used oils and in raw and finished products. The FFA is quantitated as percent oleic acid using an indicator that responds to the acids in the sample matrices. The practical dynamic range of the determination has been established as 0.3 to 1.8 percent oleic acid in these matrices. 2

3 This method has been designed to determine FFA (as percent oleic acid) in oils and fats in vegetable oils, seeds oils, olive and other pressed oils, marine oils, animal fats, oils and greases as well as meals. No interferences were measured by fatty amides, amines, alcohols, aldehydes, esters, anhydrides, and acetates and other salts except for n-decylamine acid. Matrices Selected for Validation The FASafe test kit is capable of measuring FFA in the following matrices: A. Vegetable and seed oils B. Marine oils C. Olive oils and pressed oils D. Meals containing fats E. Animal fats and oils Figure 1: The SafTest Workstation Test Kit Information Kit Name: FASafe Standard Test Kit Catalog Number: 1020 Ordering Information: SāfTest, Inc.,, 2414 West 12 th Street, Suite 4, Tempe, Arizona 82581; Tel: (480) ; Fax: (480) ; general customer support: and website: Test Kit Reagents: Preparation Reagent and Reagent A % 2-propanol with antioxidants and stabilizers. Reagent A is tested for presence of low degradants and low levels of antioxidants are added for stabilization. Reagent B - <0.1% ph indicator soluble in water and alcohol mixture. Reagent B is prepared by dissolving the ph indicator at desired concentration in distilled water. Additional Supplies and Reagents Membranes High Non-Specific Adsorption membranes are used for prefiltration and consist of polypropylene or polyethersulfame or polythtrathoro ethylene structures. 3

4 0.05-N NaOH VWR (Lot ); 0.05-, and 0.1-N NaOH; 0.25-N NaOH or 1.0-N NaOH ± Normal Cat. No. VW Whatman Filter paper General all purpose filter paper Apparatus Contents of Bench Top SafTest Platform include: SafTest Filtration Unit Vortex Heating Block with tube Inserts Tube Rocker SafTest Analyzer Tube Racks Positive Displacement Pipette Bottle Top Dispensers Contents of Essential Disposable Labware: 15-mL Conical Vials Disposable test tubes and caps Disposable transfer pipettes Glass beads Positive Displacement Pipette Tips Standard Reference Materials Oleic acid - Sigma % Caproic Acid (hexanoic acid) - Sigma C % Standard Solutions Calibrators One to five fixed concentrations of FFAs prepared in stabilized isopropanol. Free fatty acids include oleic and caproic acids. Controls Low, Medium, and High - Mixture of FFAs, lipid peroxides and malonaldehyde prepared in stabilized isopropanol. See the Validation Study Report for Safety Precautions and Material Safety Data Sheets (MSDSs) and General Method guidelines. Analysis Procedure The Safety Associates, Inc. FASafe Standard Test Kit measures FFA in samples using an alcohol soluble indicator that responds to FFAs in the range of 0.3% to 1.8% expressed as percent oleic. Due to high bias expected below 0.3% oleic, samples having lower FFA concentrations must be analyzed using FASafe High Sensitivity (HSY) Kit. Samples with higher FFA concentrations require a dilution before they can be analyzed by this method. Appendix 4 provides instructions for SafTest Analyzer, Filtration Unit, and Pipetters. 4

5 Internal Method Validation Studies Selectivity and Specificity For 17 out of 22 substances, FASafe read 0.0% and AOCS read to 0.045%. Caproic Acid Methyl Ester read 0.07 on FASafe which was higher than in AOCS and Lauryl Sulfate Lithium which read 0.13 on FASafe compared to using the AOCS method. Hexanal and 1-Decanol produced a response using the AOCS method but not on the FASafe and would not be expected to produce a response. The two analytes producing a response above the lower cutoff for FASafe are: 1,6-Hexanediamine and n-decylamine. 1,6-Hexanediamine showed a response of 0.27 for 1% test samples. The level of cross reaction (interference) is estimated to be 25%. Cross Reactions for FFA can be summarized as follows: Free Fatty Acids 100% 1,6-Hexanediamine 27% Lauryl Sulfate Lithium 14% n-decylamine 10% Inclusivity Selected FFA constituents responded well to this assay. In addition, a strong correlation (i.e., coefficient of determination, r 2, of 0.98) was observed between the results generated using both FASafe and AOCS methods. This study clearly demonstrates that FASafe Standard Kit is capable of analyzing a wide-range of fatty acids with a high degree of accuracy and precision. Sensitivity: Determination of Limit of Detection and Limit of Quantitation LOQ has been established as 0.3% percent oleic acid and represents the lowest concentration that can be reported with 95% confidence. Repeatability and Accuracy Studies Excellent correlation (i.e., correlation coefficient, r, of 0.99 and 0.98) was observed between the results generated using FASafe and the traditional AOCS method for 40 meal samples and 276 varied oil samples, respectively. Lot-to-Lot Reagent Stability (Ruggedness) Study The lot-to-lot comparability of reagent stability based on studies using n-caproic and Oleic acids. Variations observed between three lots of chemicals were insignificant. Summary of Independent Laboratory s Validation Results Matrices A and B Vegetable and Seed Oils & Marine Oils t-test analyses omitting the low oils (fish 04 and vegetable 06 and 09) demonstrated that, in each case, at the 0.05 level of significance, the two means are not significantly different. On the basis of these results, we have concluded that the means of the two populations are similar. Vegetable oil FFA ranged from 0.3 to 1.4%. Fish oil FFA ranged from 0.3 to 1.67%. Excellent correlation (r of 0.98, 0.98, and 0.95) was observed between the two laboratories (SAI and CGC) comparing the FASafe and AOCS methods; clearly validating the useful range of FASafe for 0.3 to 1.67%. 5

6 Summary of Matrix-Specific Internal Method Validation Studies at SAI Matrices A and B Vegetable and Seed Oils & Marine Oils Close agreement (i.e., correlation coefficient, r, 0.98 and 0.93) was observed between the data generated using the FASafe and the traditional AOCS method for vegetable oils, seed oils, and marine oils. Marine oils presented more difficulties in AOCS official method due to dark oil color in extracts. We believe that the fish oil color did not effect SafTest since the dilutions were high enough to reduce interference. However, intensely colored extracts interfered during the AOCS analysis requiring 28 g of sample in 50 ml of alcohol. Matrix C Olive Oils and Pressed Oils Excellent agreement (i.e., correlation coefficient, r, of 0.97) was observed between the results of FASafe and the traditional AOCS method for olive oils and pressed oils. The t-statistical analysis was performed and in all cases, at the 0.05 level of significance, the two means are not significantly different, that is, the means of the two populations are similar. In addition, the mean of 24 individual RSDs (CV) yielded an estimate of the within-laboratory precision (repeatability) of 3.9 percent for olive oils, after excluding one data point of a high CV (28 percent). This outlier is attributable to random error during the sample preparation and measurement process. The outlier was clearly identified by Grubb s Outlier Test. Matrix D Meals and Protein Powders Close agreement (i.e., correlation coefficient, r, of 0.99) was observed between the data generated using the FASafe and the traditional AOCS method for meals and protein powders. The t-statistical analysis was performed and slight variation and statistical significance were observed for poultry meal. The oil extracted from the meal was very dark and difficult to titrate. With the exception of this matrix, at the 0.05 level of significance, the two means are not significantly different, that is, the means of the two populations are similar. In addition, these data validate the useful range of FASafe from 0 to 23%. In addition, the mean of 20 individual RSD (CV) values yielded an estimate of the within-laboratory precision (repeatability) of 2.2 percent for meals. Matrix E Animal Fats and Oils Excellent agreement (i.e., correlation coefficient, r, of 0.99) was observed between the data generated using the FASafe and the traditional AOCS method for 100 samples of animal fats and oils. Free fatty acid levels ranged from 1.0% to 13.0%. The t-statistical analysis was performed and the results are presented in Table 12. In this evaluation, in each case, at the 0.05 level of significance, the two means are not significantly different. On the basis of these results, we have concluded that the means of the two populations are similar. In addition, the mean of 20 individual RSDs (CV) yielded an estimate of the within-laboratory precision (repeatability) of 2.5 percent for animal fats and oils. Quality Assurance All aspects of internal validation and independent laboratory validation studies were performed in compliance with Good Laboratory Practices (GLP). All data have been reviewed and evaluated by an independent party. 6

7 References Performance Tested Methods Program, AOAC Research Institute. Official Methods and Recommended Practices of the AOCS, The American Oil Chemists Society, Fifth Edition, Outliers in Statistical Data (3 rd edition), V. Barnett and T. Lewis, Wiley, SAI Quality Assurance Manual, Revision 1, July 1,

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