AOAC Research Institute

Performance Tested MethodsSM Program

Measurement of Free Fatty Acids Using

FASafe Standard Test Kit

Certificate Report

Certificate Number 030405

June 23, 2003

SfTest, Inc.,.
2414 West 12 th Street, Suite 4
Tempe, Arizona 82581
AOAC Research Institute Certificate Report
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FASafe Standard Test Kit Date: 06/23/03

AOAC Performance Tested MethodSM: 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 SAIs 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:

Liquid oils require no heating; semi-solid and solid oils and meals require
Sample Preparation:
heating
Sample Preparation 5 to 10 minutes
Time Requirement:
Analysis Time approximately 5 to 10 minutes

Limit of
0.3%
Quantitation:
Highly colored samples, samples containing acids, bases, or chelating
Interferences:
metal ions

Method Author

Virginia C. Gordon, Ph.D., 2414 West 12 th Street, Suite 4, Tempe, Arizona 82581;
Tel: (480) 736-8700.

Submitting Company

SfTest, Inc., 2414 West 12 th Street, Suite 4, Tempe, Arizona 82581;

Tel: (480) 736-8700.

Independent Laboratory

Canadian Grain Commission, 1404-303 Main St., Winnepeg, MB Canada R3C 3G8; Tel: (204) 983-3350

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.
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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: SfTest, Inc., , 2414 West 12th Street, Suite 4, Tempe, Arizona 82581; Tel: (480) 736-
8700; Fax: (480) 736-8717; general e-mail: info@saftest.com; customer support: support@saftest.com; and
website: www.saftest.com

Test Kit Reagents:

Preparation Reagent and Reagent A - 99.9% 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.

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0.05-N NaOH VWR (Lot 0003352); 0.05-, and 0.1-N NaOH; 0.25-N NaOH or 1.0-N NaOH 0.0500 0.0002 Normal Cat.
No. VW3218-1

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 01008 - 99%
Caproic Acid (hexanoic acid) - Sigma C2250 - 99%

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.

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Internal Method Validation Studies

Selectivity and Specificity

For 17 out of 22 substances, FASafe read 0.0% and AOCS read 0.004 to 0.045%. Caproic Acid Methyl Ester read 0.07
on FASafe which was higher than 0.005 in AOCS and Lauryl Sulfate Lithium which read 0.13 on FASafe compared to
0.003 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, r2, 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 Laboratorys 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 FASafeTM and AOCS methods; clearly validating the useful range of FASafe for 0.3 to 1.67%.

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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 Grubbs 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.

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References

Performance Tested Methods Program, AOAC Research Institute.

Official Methods and Recommended Practices of the AOCS, The American Oil Chemists Society, Fifth
Edition, 1993-1997.
Outliers in Statistical Data (3rd edition), V. Barnett and T. Lewis, Wiley, 1994.
SAI Quality Assurance Manual, Revision 1, July 1, 2001.