Journal of AOAC INTERNATIONAL, 104(2), 2021, 300–311

doi: 10.1093/jaoacint/qsaa086
Advance Access Publication Date: 26 November 2020
Article

ANIMAL FOOD, PET FOOD, AND PLANT NUTRIENT METHODS

Validation of the Free Fatty Acid Test Kit for the

Measurement of the Free Fatty Acid Content of

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Vegetable Oils, Fish Oils, Animal Fats (Tallows),
Meat and Fish Meals, and Potato Chips and
Grain-Based Snack Products: AOAC Performance
Tested MethodSM 052004
Virginia C. Gordon I, Christopher C. Rainey*, and Willainia C. Studmire

MP Biomedicals, 29525 Fountain Parkway, Solon, OH 44139, USA

* Corresponding author’s e-mail: Christopher.Rainey@MpBiomedicals.com

Abstract
Background: The Official American Oil Chemists’ Society (AOCS) Ca 5a-40 method for the determination of free fatty acids is
based on titration of an ethanolic solution and requires a large volume of organic solvents, large sample aliquots, and up to
18 hours extraction time for some samples. The SafTest Free Fatty Acid Test Kit is a rapid method designed to measure the
free fatty acid content of vegetable oils; fish oil; animal fats (tallows); meat meal and fish meal products; and crackers,
chips, and other processed grain-based snack products using micro-analytical and membrane separation principles.
Objective: The study objective was to validate the SafTest Free Fatty Acid Test in one internal study, two contracted studies,
and an independent laboratory study studies.
Method: Recovery, limit of quantitation, selectivity, robustness, stability, and consistency of the SafTest Free Fatty Acid Test
were evaluated.
Results: Recoveries from control solutions ranged from 97 to 106%. Repeatability (RSDr) from method developer matrix
studies ranged from 1.1 to 8.1%. Biases, expressed as a percentage recovery from AOCS Ca 5a-40, averaged 96.5% for olive
oils, 94.0% for animal fats, and 103.9% for meat meals. Results observed in the independent laboratory study were similar.
Conclusions: The SafTest Free Fatty Acid Test can measure free fatty acid levels in oils, fats, meal, and snack matrices with
accuracy and precision comparable to AOCS Ca 5a-40.
Highlights: The SafTest Free Fatty Acid Test Kit has the advantage of using reagent volumes, instrumental analysis, and
easy-to-use, standardized procedures with rapid detection times for the determination of free fatty acids.

Received: 18 June 2020; Accepted: 18 June 2020

C AOAC INTERNATIONAL 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com
V

300
 Gordon et al.: Journal of AOAC INTERNATIONAL Vol. 104, No. 2, 2021
Scope of Method
(a) Analyte.—Free fatty acid content as percent oleic acid
equivalent.
(b) Matrices.—Vegetable oils; fish oil; animal fats (tallows);
meat meal and fish meal products; and potato chips, and
crackers and other processed grain-based snack products.
(c) Summary of validated performance claims.—The practical
dynamic range of the calibration is 0.04 to 2.06% free fatty
acids. For materials analyzed neat (e.g., vegetable oils, re-
fined oils), the LOQ is 0.05% free fatty acids. For materials
that are diluted 1:4 prior to testing (e.g., meat meals,
snacks), the LOQ is 0.16% free fatty acids. For materials that
are diluted 1–10 prior to testing (e.g., fats, tallows), the LOQ
is 0.40% free fatty acids. Materials with free fatty acid levels
above the determination range can be analyzed by includ-
ing a dilution step prior to analysis. The relative standard
deviations of repeatability (RSDr) ranged from 1.1 to 8.1%
across all method developer matrix studies. Similar results
were observed in the independent laboratory study where
RSDr was found to be in the range 2.6 to 13%. Free fatty acid
results by this method are in good agreement with results
by AOCS Ca 5a-40 Method.
Intended Users
The SafTest Free Fatty Acid Test can be used by food and pet
food manufacturers to test incoming raw materials, materials
in production, and finished products. The SafTest System
includes a desktop workstation and disposable kits containing
membrane units and reagents (Figure 1). The SafTest Free Fatty
Acid Test is designed for use at lab stations at production sites
or on receiving floors, as well as in fixed food-testing laborato-
ries. The workstation shown in Figure 1 accommodates up to
six samples.
Principle of the Method
The SafTest System, a series of innovative test kits used in de-
termining lipid quality and food freshness, was developed to
address these challenges. The SafTest System is a series of
micro-analytical techniques based on membrane separation
technology as an alternative to sensory and traditional analyti-
cal methods. One test of the series measures free fatty acids
which are a key indicator of hydrolytic degradation associated
with off flavor and textural changes. Results are standardized,
objective, and rapid, with strong correlations to published, offi-
cial test methods. Standardized, well-documented, and user-
Figure 1. The SafTest workstation.
| 301
friendly procedures improve productivity, performance, and
quality control.
One of the methods in the SafTest System is the Free Fatty
Acid Test. Food materials are solubilized in the preparation re-
agent, separated through the membrane separation pack, and
then analyzed by an optical reader.
Liquid test portions are solubilized in the proprietary prepa-
ration reagent and then tested. For dry materials, test portions
are solubilized and then separated through the membrane sep-
aration pack, tested, and then analyzed by an optical reader.
The test is based on a pH indicator reaction that takes place in a
stabilized reagent. Free fatty acid calibrant concentrations are
based on a % oleic equivalent. The decrease in indicator absor-
bance is proportional to the concentration of free fatty acids
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available to interact with the indicator. The indicator visible
spectrum shifts with decreasing pH to absorption maximum in
the 400 to 500 nm range. The resulting absorbance values are
logarithmically related to free fatty acid concentration and de-
crease with increasing free fatty acid concentration.
General Information
Determination of free fatty acids in raw materials and finished
foods is important in the food and pet food industries. Free fatty
acid concentration in a raw material or finished food matrix is a
key indicator of hydrolytic degradation associated with off fla-
vor and textural changes. The free fatty acid concentration is of-
ten used as accept/reject criteria for incoming raw materials to
be used in finished products. Free fatty acid concentration is
also measured in finished products as part of stability criteria.
Elevated free fatty acid concentrations in the incoming raw
materials and finished products can produce unwanted loss of
stability and structural changes in the finished product.
Traditionally, free fatty acid testing has used a visual endpoint
and involved large volumes of solvents, large test portion mass/
volume and up to 18 h of extraction times to obtain the needed
mass of fat. A rapid and objective test is needed to measure free
fatty acid prior to unloading raw materials, during industrial
processes such as frying and predicting stability of finished
products.
Materials and Methods
Test Kit Information
(a) Kit name.—Free Fatty Acid Test Kit.
(b) Cat. No.—07KTFA2000.
(c) Ordering information.—Worldwide: MP Biomedicals, 29525
Fountain Parkway, Solon, OH 44139. Phone: 800-854-0530.
www.mpbio.com/07ktfa2000-free-fatty-acid-test.
Test Kit Components
(a) Preparation reagent.—Part no. PREP2000. Solvent for solubi-
lizing the analyte.
(b) Reagent A.—Part no. AC1010A. Carrier solution for the test.
(c) Reagent B.—Part no. AC1010B. Indicator solution.
(d) Calibrator 1.—Part no. AC1010CAL1. 0.04% free fatty acid
calibrant (oleic acid equivalent).
(e) Calibrator 2.—Part no. AC1010CAL2. 0.26% free fatty acid
(oleic acid equivalent) in Preparation Reagent.
(f) Calibrator 3.—Part no. AC1010CAL3. 0.62% free fatty acid
(oleic acid equivalent) in Preparation Reagent.
 302 | Gordon et al.: Journal of AOAC INTERNATIONAL Vol. 104, No. 2, 2021

(g) Calibrator 4.—Part no. AC1010CAL4. 1.02% free fatty acid
(oleic acid equivalent) in Preparation Reagent.
(h) Calibrator 5.—Part no. AC1010CAL5. 2.06% free fatty acid
(oleic acid equivalent) in Preparation Reagent.
(i) Control.—Part no. CTFA2000. 0.10% free fatty acid (oleic acid
equivalent) in Preparation Reagent.
solvents. Protective equipment that becomes contaminated
must be removed and replaced as necessary. Pipetting by mouth
is forbidden. Additional health and safety information are pro-
vided in the Material Safety Data Sheet (https://www.mpbio.
com/media/productattachment/MSDS/MP_MSDS_KTFA2000_
TW_ZH-TW.pdf).

Additional Supplies and Materials Required General Preparation

Preheat the heat block to 37–44 C for all materials except fats
(a) Membranes.—Part no. 07DF1000. High non-specific adsorp-
and tallows. Heat to 46–50 C for solid and semi-solid fats and
tion membranes used for prefiltration consisting of poly-
tallows. Remove the Preparation Reagent from the refrigerator
propylene, polyethenesulfame, or other polyethylene
and allow it to stand at room temperature (18–25 C) for 1–2 h.
structures.
Attach the Preparation Reagent dispenser to the bottle. Set the
(b) Free fatty acid control.—Part no. KTFA2001, 0.10% free fatty
volume on the dispenser as needed (See Table 1). To eliminate

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acid (oleic acid equivalent) in Preparation Reagent. air bubbles and oxidized reagent, dispense 4–5 aliquots of the
reagent into a waste container immediately before use. Turn on
Apparatus SafTest analyzer.

(a) SafTest workstation.—Part no. 07LI2000. The workstation

(Figure 1) includes an analyzer, heat block, membrane sep-
aration unit, pipettors, and one adjustable dispenser for
Preparation Reagent and fixed volume dispensers for each
Reagent A and Reagent B.
Safety Precautions
All testing materials should be handled in accordance with
good laboratory practices. Eye protection that satisfies ANSI
Z87.1, laboratory coat, and appropriate chemical resistant
gloves must be worn while handling standards, reagents, and
Sample Preparation
Critical to proper sample collection and storage is understand-
ing that this test measures degradation. Therefore, samples
must be stored cold and sealed in airtight containers.
Analytical samples must be uniform for reliable results as
some portions of a laboratory sample may be more exposed to
air and environmental conditions leading to heterogeneity and
variability in test results. The analyte concentration may be
changing over time and comparisons of test results should take
into account the date of testing. See Table 1 for specific instruc-
tions for solubilizing and filtering specific matrices.

Table 1. Preparation instructions

Matrix Preparation instructions Dilution factor Block conditions

Meat meal and fish meal Reduce particle size of >100 g analytical sample with appropriate mill. 1:4 37–44 C for 10 min
Weigh 1.0 g of test portion (60.05 g) into the bottom of each conical tube.
Pack loosely.
Add 10 glass beads to each conical tube.
Set the Preparation Reagent dispenser to 3.0 mL and dispense 1 aliquot
into each conical tube.
Vortex at the highest dial setting for 1 6 0.2 min.
Warm in heat block. See Block Conditions.
Grain-based snack Using a coffee grinder, grind >100 g analytical sample for one minute 1:4 37–44 C for 10 min
products unless fat content is > 20%. If > 20%, then grind for 30 s.
Weigh 1.0 g of test portion (60.05 g) into the bottom of each conical tube.
Pack loosely.
Add 10 glass beads to each conical tube.
Set the Preparation Reagent dispenser to 3.0 mL and dispense 1 aliquot
into each conical tube.
Vortex at the highest dial setting for 1 6 0.2 min.
Warm in heat block. See Block Conditions.
Fats (tallow) Warm >100 g analytical sample to 46–50 C to melt the fat to allow for 1:10 46–50 C for 10 min
thorough mixing.
Weigh 0.20 g of warmed/melted test portion (60.05 g) or aliquot
200 mL test portion using a positive displacement pipette into the bottom
of each conical tube.
Set the Preparation Reagent dispenser to 1.8 mL and dispense 1 aliquot
into each conical tube.
Vortex at the highest dial setting for 1 6 0.2 min
Warm in heat block. See Block Conditions.
Vegetable oils Swirl. Visually check for uniformity.
No preparation needed. (Treated like a control.)
 Gordon et al.: Journal of AOAC INTERNATIONAL Vol. 104, No. 2, 2021
While test solutions are being heated, label a new glass test
tube for each unknown and place the labeled tubes in the
acrylic base of the SafTest filtration unit. Position the mem-
brane holder (with membranes attached) onto the acrylic base.
Remove tubes from the heat block. Pour the test solutions into
the membrane cup, filling to the top. Cap membrane cup to
seal. Turn on the filtration unit and slowly increase the vacuum
pressure to 5–10 in. Hg. Be sure the filtrate is a single-phase
solution. Materials with high fat content may appear slightly
cloudy, which is not a problem unless two layers form.
Improper filtration or too high of a fat content is indicated by
the formation of two layers. Should this occur, discard and pre-
pare using a higher dilution. When filtering is complete, reduce
the pressure to 0 and turn off the filtration unit. Discard the
membrane holder when all six membranes have been used. Do
not reuse membranes. Remove the test tubes containing the fil-
trates. Cap the tubes and place them in the heat block to keep
warm (to fat solubilized) until you perform the test. Complete
test within 2 hours.
Calibration (Weekly)
Power on the SafTest analyzer, allowing 5 min for warm-up.
Preheat the heat block with the 12.75 mm tube insert. Set the
temperature to LOW and dial no. 4. Check with the thermome-
ter and adjust the dial slightly (0–10) to obtain the appropriate
temperature. To set up the calibration curve, label five new
12 mm glass test tubes to correspond to Calibrators 1, 2, 3, 4, and
5; and one new 12 mm tube for the Control. Using a positive dis-
placement pipette, transfer 100 lL of each calibrator and control
into the corresponding labeled test tube. Before dispensing
reagents, gently swirl the contents of each reagent bottle and
eliminate air bubbles and oxidized reagent by dispensing 4–5
aliquots of each reagent into a waste container immediately be-
fore use. Dispense 1 aliquot (fixed volume of 2.35 mL) of Free
Fatty Acid Reagent A into each calibrator and control test tube.
Dispense 1 aliquot (fixed volume of 0.15 mL) of Free Fatty Acid
Reagent B into each calibrator and control test tube. Start the
timer for 10 6 1 min. Cap the test tubes and vortex them at the
fastest dial setting for 30 6 5 s. Place the test tubes in the heat
block at 37–44 C for the remaining time. IMMEDIATELY begin
reading tubes when time is up. If samples are not read immedi-
ately, the tubes must be discarded.
To run the Calibration, press [RUN] on the SafTest analyzer
screen. Press [ACIMSA] icon; when highlighted yellow, press
[NEXT] icon. At the “Blank Tube” prompt, insert a test tube filled
halfway with distilled water in the sample compartment. Do
not remove tube until prompted or after beep. At the “Cal #_ of
5” prompts, insert Calibrators 1–5 in the sample compartment.
At the “DO YOU WANT TO STORE THE CALIBRATORS” prompt,
select YES. Verify that the control falls within the Free Fatty
Acid Control range which can be found on the Free Fatty Acid
Control Package Insert. If it does not, rerun the calibration.
Analysis of Test Solutions on the SafTest Analyzer
Label one new 12 mm glass test tube per prepared test solution.
Using a positive displacement pipette, transfer 100 lL of each
prepared test solution into the corresponding labeled test tube.
Dispense 1 aliquot (fixed volume of 2.35 mL) of Free Fatty Acid
Reagent A into each test tube. Dispense 1 aliquot (fixed volume
of 0.15 mL) of Free Fatty Acid Reagent B into each test tube. Start
the timer for 10 6 1 min. Cap the test tubes and vortex them at
the fastest dial setting for 30 6 5 s. Place the test tubes in the
| 303
heat block at 37–44 C for the remaining time. Analyze samples
by pressing the [STAT] icon and then press the [ACIMSA] icon.
When highlighted yellow, press [NEXT] icon. At the “Blank
Tube” prompt, insert a test tube of distilled water in the sample
compartment. Use the “þ” or “” arrows to select the number of
replicate tubes (1–5) for each unknown (replicate unknowns are
recommended to ensure results are consistent). The SafTest an-
alyzer arbitrarily numbers results 1–99, so be sure to correctly
match results to the proper tube. Press [NEXT] icon. At the
“Smpl #_ Rep #_, Insert Tube” prompt, insert tubes in the sample
compartment. The SafTest analyzer will continue asking for
tubes until the user quits the program by pressing [STOP] and
then [DONE].
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Calculations
The SafTest analyzer will use the Calibrators to calculate the
acid content as percent (%) free fatty acid in the test solution. To
convert the result from the SafTest analyzer to the free fatty
acid content in the test portion, multiply by the appropriate di-
lution factor, as shown in Table 2.
The final result can be expressed on a % fat basis rather than
an as-is basis, if the fat content (% fat) is known. Convert to %
fat basis using the equation
Free Fatty Acids; % As Is
Free Fatty Acids; % of Fat ¼ Fat; %
ð1Þ
100
Validation Study
This validation study was conducted under the AOAC Research
Institute (RI) Performance Tested MethodsSM (PTM) program. The
Free Fatty Acid Method was validated for: fresh and used vege-
table oils; fish oil; animal fats (tallows); meat meal and fish
meal products; and crackers, chips, and other processed grain-
based snack products. The method developer studies included
calibration, LOQ, matrix studies, selectivity, stability, robust-
ness, and bias.
Reference Method
The method chosen as a reference method, AOCS Official
Method Ca 5a-40 (1), is the traditional global approach for deter-
mining free fatty acids. This method estimates free fatty acid
content by titration against KOH in the presence of phenol-
phthalein indicator. The acid number is defined as the KOH re-
quired to neutralize the free fatty acids present per gram;
however, the free fatty acid content is expressed as oleic acid
equivalents. For oils and fats, this is a direct titration of the oil
or fat. For food matrices, a fat extraction step is required to ex-
tract sufficient fat to accommodate a titration, which is per-
formed on the extracted fat. For low-fat materials, it may
Table 2. Applying the dilution factor, %
Dilution SafTest Result x dilution Final result,
Dilution factor result factor %a
1:4 4 0.19 0.19% x 4 0.76
1:10 10 0.19 0.19% x 10 1.9
Neat (1:1) 1 0.19 0.19% x1 0.19
a
Final result, in test portion, on an as-is basis.
 304 | Gordon et al.: Journal of AOAC INTERNATIONAL Vol. 104, No. 2, 2021
require large masses of material to obtain sufficient fat to per-
form the titration.
Method Developer Studies
Matrix studies conducted prior to 2005 through the AOAC
Research Institute (unpublished) comparing results on 12 vege-
table and 11 marine oils by the SafTest Free Fatty Acid Test to
AOCS Ca 5a-40 (1) indicated good agreement. The free fatty acid
mean of 10 replicates of each of the 23 materials by the SafTest
Free Fatty Acid Test was 0.769% 6 0.124, while the mean by the
AOCS method was 0.767% 6 0.188. The correlation coefficient
between the two methods was 0.98 for vegetable oils and 0.93
for marine oils.
Calibration and Control Study
(a) Methodology.—The five calibrators (0.04, 0.26, 0.62, 1.02, and
2.06%) were used to create the calibration curve and pro-
vide the range of the test. Test values for the Control 0.71%
must be between 0.64 and 0.78% or the test must be rerun.
(b) Results.—Method of calibration is based on a logarithmic re-
lationship (see Figure 2). The lowest and highest calibrators
determine the analytical range between which analyte
concentrations can be calculated.
Selectivity
(a) Methodology.—To demonstrate selectivity of the SafTest
Free Fatty Acid Test, two experiments were performed. In
the first, a wide variety of 20 fatty acids with long and short
chains as well as saturated even- and odd-carbon fatty
acids were tested for response in the method at a concen-
tration of 1%. In a second experiment, a selection of 22
molecules similar in structure to fatty acids was tested by
the SafTest Free Fatty Acid Test to determine positive in-
terference in the method.
(b) Results.— Results for the 20 fatty acids responses in the
SafTest Free Fatty Acid Test are provided in Table 3. All 20
fatty acids were recovered within acceptable criteria to
AOCS Aa 5-40. Results for each of the 22 potential interfer-
ents are provided in Table 4. All but five of the compounds
produced responses below the LOQ of the SafTest Free
Fatty Acid Test. The five compounds producing a response
above the LOQ for the SafTest Free Fatty Acid Test are: n-
decylamine, capric anhydride, caproic acid methyl ester,
1,6-hexanediamine, and lauryl sulfate lithium indicating
1.6
1.4
1.2
Opcal Density
1 (b)
0.8
0.6
0.4
0.2
0
0 0.5 1 1.5 2 2.5
Free Fay Acid %
Figure 2. Typical polygonal calibration curve for the SafTest Free Fatty Acid Test.
cross reactivity with the test. Two compounds producing a
notable response were 1,6-hexanediamine and lauryl sul-
fate lithium. Materials containing these compounds might
not be suitable for the SafTest Free Fatty Acid Test.
LOD and LOQ
(a) Methodology.—Ten replicates of a 0.10% free fatty acid con-
trol were tested and mean and standard deviations were
calculated. Following the empirical determination of LOD
and LOQ using a control solution, the test method LOQ de-
termined for five matrices (vegetable oil, poultry fat, poul-
try meal, cracker, and potato chip) by testing 10 replicates
of a product matrix with free fatty concentrations near the
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LOQ. All LODs were calculated as 3  sr, and LOQs were cal-
culated as 10  sr.
(b) Results.—For the SafTest Free Fatty Acid Test, the LOQ was
empirically determined as 0.05% oleic acid equivalent,
therefore, the LOQ accounting for a 1:4 dilution from a food
matrix is 0.20% oleic acid equivalent. In this experiment,
the LOQs determined using materials representing five ma-
trices, calculated as 10 time sr and incorporating the dilu-
tion factor are as follows: 0.06% for vegetable oil, 0.09% for
poultry fat, 0.20% for poultry meal, 0.33% for snack crack-
ers, and 0.48% for potato chips.
Stability Study
(a) Methodology.—The stability of the kit components was
tested at 2, 7, and 11 months by measuring low (0.30%), me-
dium (0.52%), and high (1.00%) free fatty acid test solutions
using one SafTest Free Fatty Acid Test kit stored at 4 C over
the 11-month shelf life period. Control test solutions were
prepared by diluting fatty acids into preparation reagent.
(b) Results.—Results are presented in Table 5. RSDs for the low
(0.30%), medium (0.52%), and high (1.00%) free fatty test sol-
utions ranged from 2 to 7%, well within generally accept-
able precision for the method. Results are displayed
graphically in Figure 3 and no trends were observed. This
data supports a shelf life of 11 months.
Lot-to-Lot Studies
(a) Methodology.—The purpose of the study was to determine
the consistency of SafTest Free Fatty Acid Test Kit reagents
from lot-to-lot. Three lots of SafTest Free Fatty Acid Test
kits manufactured in March (Lot A), July (Lot B), and in
December 2016 (Lot C) were evaluated by testing n-caproic
acid at low (0.30%), medium (0.52%), and high (1.00%) con-
centration test solutions and oleic acid at low (0.30%), me-
dium (0.63%), and high (1.25%) concentration test
solutions.
Results.—Table 6 provides the results of the lot-to-lot tests.
Variations observed between three lots of each free fatty
acid test kit components were insignificant with sr ranging
from 0.010 to .088.
Assessment of Controls
(a) Methodology.—To determine within-laboratory accuracy
and precision, replicate (8 to 10) measurements of low
(0.30% oleic acid), medium (0.63% oleic acid), and high
(1.25% oleic acid) controls representing the dynamic range
 Gordon et al.: Journal of AOAC INTERNATIONAL Vol. 104, No. 2, 2021 | 305

Table 3. Method selectivity for free fatty acid compounds

Free fatty acid, %

a
ID Constituent CAS No. SafTest FFA AOCS Ca 5a-40 Biasb

2858 Linoleic Acid 60-33-3 0.91 1.05 0.14

2859 Oleic Acid 112-80-1 0.88 1.02 0.14
2860 Linolenic Acid 463-40-1 0.99 1.01 0.02
2861 n-Caproic Acid 142-62-1 2.00 2.39 0.39
2862 Capric Acid 334-48-5 1.46 1.71 0.25
2864 Lignoceric Acid 557-59-5 0.71 0.69 0.02
2865 Lauric Acid 143-07-77 1.30 1.41 0.11
2866 Nonadecanoic Acid 646-30-0 0.84 0.97 0.13
2867 Heptanoic Acid 111-14-8 1.98 2.23 0.24
2868 Heptadecanoic Acid 506-12-7 0.90 1.01 0.11

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2869 Erucic Acid 112-86-7 0.72 0.83 0.11
2870 Arachidonic Acid 506-32-1 0.85 0.88 0.03
2871 Myristic Acid 544-63-8 1.15 1.29 0.14
2876 Adipic Acid 124-04-9 4.17 3.99 0.18
2881 Caprylic Acid 124-07-2 1.83 1.99 0.16
2882 Palmitic Acid 57-10-3 0.99 1.05 0.06
2883 Palmitoleic Acid 373-49-9 1.35 1.53 0.18
2884 Myristoleic Acid 544-64-9 1.39 1.45 0.06
2885 cis-11,14,17-Eicosatrienoic Acid 2091-27-2 0.80 0.84 0.04
2886 cis-13-Eicosenoic Acid 17735-94-3 0.81 0.82 0.01

a
SafTest Free Fatty Acid Test.
b
Bias calculated as SafTest Free Fatty Acid Test result minus AOCS Ca 5a-40 result.

Table 4. Method selectivity for potential chemical interferents

Free fatty acid, %

ID Constituent CAS No. SafTest FFAa AOCS Ca 5a-40 Biasb

2844 1-Octanol 111-87-5 0.00 0.01 0.01

2845 n-Decylamine 2016-57-1 0.06 0.02 0.04
2846 n-Decyl Aldehyde (a.k.a, Caprinaldehyde) 112-31-2 0.00 0.02 0.02
2847 n-Decyl Alcohol (a.k.a, 1-Decanol) 112-30-1 0.00 0.05 0.05
2848 Glycerol 56-81-5 0.00 0.01 0.01
2849 Capric Anhydride 2082-76-0 0.11 0.05 0.06
2850 1-Hexanol 111-27-3 0.00 0.00 0.00
2851 Hexanal 66-25-1 0.00 0.04 0.04
2852 Caproic Acid Methyl Ester 106-70-7 0.07 0.01 0.06
2853 Capric Acid Methyl Ester 110-42-9 0.00 0.00 0.00
2854 Oleyl Acetate 693-80-1 0.00 0.00 0.00
2855 Oleyl Alcohol 143-28-2 0.00 0.00 0.00
2856 Oleic Anhydride 24909-72-6 0.00 0.00 0.00
2857 Oleic Ethyl Ester (a.k.a, Oleic Acid Ethyl Ester or Ethyl Oleate) 111-62-6 0.00 0.00 0.00
2863 2-Heptanone 110-43-0 0.00 0.00 0.00
2873 n-Hexadecane 544-76-3 0.00 0.01 0.01
2874 1,6-Hexanediamine 124-09-4 0.27 0.02 0.25
2875 Adipic Acid Diethyl Ester (a.k.a, Diethyl Adipate) 141-28-6 0.00 0.01 0.01
2877 Lauryl Acetate 112-66-3 0.00 0.01 0.01
2878 Lauryl Chloride 112-16-3 0.00 0.01 0.01
2879 Lauric Acid Methyl Ester 111-82-0 0.00 0.01 0.01
2880 Lauryl Sulfate Lithium 2044-56-6 0.13 0.00 0.13

a
SafTest Free Fatty Acid Test.
b
Bias calculated as SafTest Free Fatty Acid Test result minus AOCS Ca 5a-40 result.

(i.e., quantitation range) of the method were made with Robustness

one test kit.
(b) Results.—RSDr of controls were 5, 6, and 6% (Table 7). (a) Methodology.—Three parameters known to be critical were
Recovery ranged from 97 to 106%. evaluated to demonstrate impact on the Free Fatty Acid
 306 | Gordon et al.: Journal of AOAC INTERNATIONAL Vol. 104, No. 2, 2021

Table 5. SafTest Free Fatty Acid Test Kit reagent stability

Low control Medium control High control

a a
Age of reagents A & B SafTest FFA sr RSD, % SafTest FFA sr RSD, % SafTest FFAa sr RSD, %

2 Months 0.322 0.020 6 0.521 0.034 7 1.000 0.076 7

7 Months 0.292 0.011 4 0.531 0.011 2 1.031 0.042 4
11 Months 0.299 0.019 7 0.512 0.020 4 0.995 0.028 3

a
Mean of SafTest Free Fatty Acid Test results.

Stability Study
1.2

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1 1 1.031 0.995
Free Fay Acid, %

n-caproic acid,
0.8 low

0.6 n-caproic acid,

0.521 0.531 0.512 medium
0.4
0.322 0.292 0.299 n-caproic acid,
0.2 high

0
2 3 4 5 6 7 8 9 10 11
Months
Figure 3. Trend chart for stability study.

Table 6. Lot-to-lot comparison study

Lot A (March) Lot B (July) Lot C (December)

a a
Test material n SafTest FFA, % sr SafTest FFA, % sr SafTest FFA, %a sr

n-Caproic Acid Low 15 0.32 0.020 0.29 0.011 0.30 0.019

Medium 15 0.52 0.034 0.53 0.011 0.51 0.020
High 15 1.00 0.076 1.03 0.042 1.00 0.028
Oleic Acid Low 15 0.29 0.022 0.26 0.010 0.29 0.021
Medium 15 0.68 0.036 0.65 0.024 0.67 0.043
High 15 1.23 0.055 1.36 0.088 1.26 0.069

a
Mean of SafTest Free Fatty Acid Test results.

Table 7. Assessment of controls

Control sample n True value, free fatty acid, % Measured value SafTest FFAa RSD, % Recovery, %

Low 10 0.30 0.29 5 97

Medium 8 0.63 0.67 6 106
High 8 1.25 1.26 6 101

a
Mean of SafTest Free Fatty Acid Test results.

Test: (1) reaction time, (2) temperature of sample prepara- control at 10 6 1 min read 1.05% 6 0.02%. For three repli-
tion, and (3) grinding time of dry matrices. Parameters cates, the control at 20 6 1 min read 0.99% 6 0.05%, which
were evaluated individually by testing three replicates of is not in the acceptable range for the control. Testing time
each condition comparing the standard condition and an must be controlled to 10 6 1 min. (2) Tallow was prepared
exaggerated condition. Matrices known to be sensitive to at 46–50 C as stated in the protocol and at 37–44 C, a tem-
the parameter were chosen for temperature and grinding perature used for materials without saturated fats. At a
time. See Table 8. preparation temperature of 37–44 C the tallow did not
(b) Results.—(1) Reaction times for the SafTest Free Fatty Acid completely dissolve at a 1:10 dilution and was untestable.
Test were compared for 10 6 1 min and 20 6 1 min on the At a 1:25 dilution, particulates were still observed in the fi-
0.05% fatty acid control solution. For three replicates, the nal test solution, so a temperature of 37–44 C was
 Gordon et al.: Journal of AOAC INTERNATIONAL Vol. 104, No. 2, 2021 | 307

Table 8. Robustness parameters

Matrix Parameter n Condition 1 Condition 2

1.05% Fatty acid control solution Reaction time Parameter condition 10 min 20 min
results, SafTest FFA % 6 sr 3 1.05% 6 0.02% 0.99% 6 0.05%
Tallow Temperature Parameter condition 46–50 C 37–44 C
Results, SafTest FFA % 6 sr 3 3.52% 6 0.26% No result
High fat potato chip Grinding time Parameter condition 30 s 60 s
results, SafTest FFA % 6 sr 3 0.34% 6 0.11% (36% crude fat) <LOQ (25% crude fat)

unacceptable. At a correct preparation temperature, the Table 9. Method developer matrix study on olive oils
tallow could be tested at 1:10 dilution with a free fatty acid
Free fatty
result of 3.52% 6 0.26%, in agreement with the AOCS Ca 5a-
Matrix Method n acid, %a sr RSD, % Biasb

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40 result of 4.2%. Temperature for tallow preparation must
be 46–50 C. (3) Potato chips were prepared with a grinding Olive oil A AOCS Ca 5a-40 10 0.4298 0.0104 2.4 0.014
time as recommended of 30 s and a grinding time used for SafTest FFA 10 0.4159 0.0151 3.6
lower fat test portions of 60 s. Potato chips ground for 60 s Olive oil B AOCS Ca 5a-40 10 0.2155 0.0028 1.3 0.004
produced a lower fat test solution of 25% and a free fatty SafTest FFA 9c 0.2115 0.0100 4.7
acid result <LOQ. Potato chips ground for 30 s produced a Olive oil C AOCS Ca 5a-40 10 0.3349 0.0058 1.7 0.051
fat test solution with 36% fat and a free fatty acid result of SafTest FFA 10 0.3490 0.0110 3.2
0.34% 6 0.11%. Fat analysis by AOCS extraction established Olive oil D AOCS Ca 5a-40 10 1.2466 0.0204 1.6 0.1222
a fat content of 36% and free fatty acid result of 0.24%. SafTest FFA 10 1.1284 0.0161 1.4
Grinding for high fat materials must not exceed 30 6 5 s in Olive oil E AOCS Ca 5a-40 10 0.6762 0.0144 2.1 0.034
a coffee grinder to avoid separation of fat leading to low SafTest FFA 10 0.6323 0.0281 4.4
test results. Test parameters of reaction time, temperature Olive oil F AOCS Ca 5a-40 10 1.2401 0.0188 1.5 0.339
SafTest FFA 10 0.9213 0.0754 8.1
of preparation and grinding of dry matrices are critical to
Olive oil G AOCS Ca 5a-40 10 0.9550 0.0159 1.7 0.095
determine accurate free fatty results using the SafTest Free
SafTest FFA 10 0.8599 0.0448 5.2
Fatty Acid Test. See Table 8.
Olive oil H AOCS Ca 5a-40 10 1.3073 0.0083 0.6 0.195
SafTest FFA 10 1.1123 0.0330 2.9
Matrix Study: Olive Oils Olive oil I AOCS Ca 5a-40 10 0.2363 0.0034 1.4 0.002
SafTest FFA 10 0.2382 0.0092 3.9
(a) Methodology.—Each of 12 different refined or pressed olive Olive oil J AOCS Ca 5a-40 10 0.3007 0.0051 1.7 0.100
oil products were portioned into 20 blind coded containers SafTest FFA 10 0.3107 0.0101 3.3
(240 portions total). Ten portions of each product were ana- Olive oil K AOCS Ca 5a-40 10 0.2306 0.0029 1.3 0.180
lyzed by the SafTest Free Fatty Acid Test using the proce- SafTest FFA 10 0.2486 0.0087 3.5
dures outlined above and ten portions were analyzed by Olive oil L AOCS Ca 5a-40 10 0.3920 0.1094 28d 0.208
the AOCS Ca 5a-40 method (see Table 9). SafTest FFA 10 0.1885 0.0106 5.6
(b) Results.—The mean, standard deviation(s), RSDr, and bias
a
Mean fatty acid test, %.
were calculated for each product and the results are pre- b
Bias calculated as SafTest Free Fatty Acid Test result minus AOCS Ca 5a-40
sented in Table 9. In this evaluation, the RSD ranged from result.
1.4 to 8.8% for the SafTest Free Fatty Acid Test. The bias, c
Container broke and test was lost.
expressed as a percent recovery from AOCS Ca 5a-40, d
Olive oil was very dark and difficult to titrate.
ranged from 79 to 108%, and averaged 96.5%.
estimate of the within-laboratory precision (repeatability)
Matrix Study: Meat Meal and Fish Meal of 2.2% for meals (Table 10). The bias, expressed as a per-
cent recovery from AOCS Ca 5a-40, ranged from 92 to 109%,
(a) Methodology.—Each of ten different meat meal and fish and averaged 103.9% (this excludes the problematic poultry
meal products were portioned into 20 blind coded contain- meal for which the recovery was 125%).
ers (200 portions total). Ten portions were analyzed using
the SafTest Free Fatty Acid Test as described above and an- Matrix Study: Animal Fat
other 10 portions were analyzed by AOCS Ca 5a-40 after
solvent extraction of the fat using AOCS Aa 4-38 (2). (a) Methodology.—Each of 10 different fat products were por-
(b) Results.—The mean, standard deviation(s), RSDr, and bias tioned into 20 blind coded containers (200 test portions to-
were calculated for each product. The results are presented tal). Ten portions of each product were analyzed by the
in Table 10. For the Free Fatty Acid Test, means for meat SafTest Free Fatty Acid Test using the procedure outline
meal products ranged from 3.9 to 19.05 with RSDr of 0.9 to above and ten portions were tested by AOCS Ca 5a-40.
4.9% and for fish meal a mean of 24.05 with RSDr of 1.1%. (b) Results.—The mean, standard deviation(s), and RSD were
Close agreement was observed between the data generated calculated for each product. The results are presented in
using the SafTest Free Fatty Acid Test and the AOCS Ca 5a- Table 11. Excellent agreement was observed between the
40 method for meals. This data validates the useful range SafTest Free Fatty Acid Test and the AOCS Ca 5-40 for 100
of the SafTest Free Fatty Acid Test from 3.3 to 23%. In addi- test portions of ten different animal fats and oils. Free fatty
tion, the mean of 10 individual RSDr values yielded an acid levels ranged from 1.0 to 12.84%. In addition, the mean
 308 | Gordon et al.: Journal of AOAC INTERNATIONAL Vol. 104, No. 2, 2021

Table 10. Method developer matrix study on meat meal and fish meal

Sample matrix Method n Free fatty acid, %a sr RSD, % Biasb

Lamb meal No. 5 AOCS Ca 5a-40 10 17.649 0.354 2.0 1.355

SafTest FFA 10 19.054 0.274 1.4
Poultry meal AOCS Ca 5a-40 10 10.234 0.105 1.0 2.579
SafTest FFA 10 12.763 0.245 1.9
Fish meal No. 2 AOCS Ca 5a-40 10 19.417 0.290 1.5 0.577
SafTest FFA 10 18.840 0.237 1.23
A poultry meal No. 21 AOCS Ca 5a-40 10 11.622 0.109 0.9 0.598
SafTest FFA 10 12.210 0.139 1.1
Fish meal No. 1 AOCS Ca 5a-40 10 23.115 0.437 1.9 0.939
SafTest FFA 10 24.054 0.262 1.1
Lamb meal No. 12 AOCS Ca 5a-40 10 2.904 0.082 2.8 0.996
SafTest FFA 10 3.900 0.190 4.9

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Meal No. 13 AOCS Ca 5a-40 9c 18.488 0.471 2.5 1.026
SafTest FFA 10 19.514 0.436 2.2
Meat bone meal AOCS Ca 5a-40 10 3.353 0.282 8.4 0.390
SafTest FFA 10 3.740 0.158 4.2
Poultry meal No. 4 AOCS Ca 5a-40 10 11.22 0.036 0.3 0.040
SafTest FFA 10 11.26 0.272 2.4
Poultry meal No. 35 AOCS Ca 5a-40 10 12.84 0.085 0.7 0.40
SafTest FFA 10 13.24 0.281 2.1

a
Mean fatty acid test, %.
b
Bias calculated as SafTest Free Fatty Acid Test result minus AOCS Ca 5a-40 result.
c
Insufficient fat for titration in test portion.

Table 11. Method developer matrix study on animal fats and oils

Matrix Method n Free fatty acid, %a sr RSD, % Biasb

Animal fat No. 1 AOCS Ca 5a-40 10 1.160 0.023 1.9 0.085

SafTest FFA 10 1.075 0.025 2.3
Poultry fat No. 1 AOCS Ca 5a-40 10 1.031 0.008 0.8 0.046
SafTest FFA 10 0.985 0.025 2.5
Poultry fat No. 3 AOCS Ca 5a-40 10 1.897 0.008 0.4 0.049
SafTest FFA 10 1.848 0.056 3.0
Turkey fat No. 1 AOCS Ca 5a-40 10 1.202 0.104 8.7 0.066
SafTest FFA 10 0.936 0.049 5.2
Turkey fat B AOCS Ca 5a-40 10 1.031 0.008 0.8 0.046
SafTest FFA 10 0.985 0.025 2.5
Poultry fat No. 10 AOCS Ca 5a-40 10 1.840 0.029 1.6 0.060
SafTest FFA 10 1.900 0.078 4.1
Animal fat No. 19 AOCS Ca 5a-40 10 1.765 0.022 1.2 0.035
SafTest FFA 10 1.733 0.066 3.8
Poultry fat from AOCS Ca 5a-40 10 12.844 0.085 0.7 0.036
meal No. 35 SafTest FFA 10 12.483 0.453 3.6
Poultry fat from AOCS Ca 5a-40 10 11.222 0.036 0.3 1.702
meal No. 4 SafTest FFA 10 9.528 0.217 2.3
Turkey fat No. 7 AOCS Ca 5a-40 10 0.940 0.006 0.6 0.019
SafTest FFA 10 0.959 0.034 3.5

a
Mean fatty acid test, %.

of 10 individual RSDr values yielded an estimate of the
within-laboratory precision (repeatability) of 2.5% for ani-
mal fats and oils (Table 11). The bias, expressed as a per-
cent recovery from AOCS Ca 5a-40, ranged from 80 to 102%,
and averaged 94.0%.
(b)
Matrix Study: Potato Chips and Processed Grain-Based
Snack Products
(a) Methodology.—Eight snack products were ground to reduce
particle size and then coded and tested at Medallion Labs.
Each of the eight products was portioned into 8 containers
(64 total test portions) for the SafTest Free Fatty Acid Test
and each of the 8 products were portioned into 3 containers
(24 total test portions) for the AOCS Ca 5a-40 method on
the extracted fat at Medallion Labs.
Results.—Unexpectedly, the results on the snack products,
except for one, were below the LOQ of the Free Fatty Acid
method (data not shown). This was confirmed with results
by the AOCS Ca 5a-40 at Medallion Labs that were all less
than 0.4%. It was not expected to have such low results, so
the only conclusion to be drawn is that both methods were
 Gordon et al.: Journal of AOAC INTERNATIONAL Vol. 104, No. 2, 2021 | 309

Table 12. Independent laboratory matrix study

Free fatty acid, %

SafTest FFA SafTest FFA AOCS Ca 5a-40 AOCS Ca 5a-40 Recovery, Recovery,
Matrix n Parameter (as-is basis) (fat basis)a (as-is basis)b (fat basis)a % (as-is basis)c % (fat basis)d

Olive oil 8 Mean 0.308 0.308 0.313 0.313 98.5 (p ¼ 0.789) 98.5
RSDr, % 13.3 13.3 9.0 9.0
Soybean oil 8 Mean <0.04 <0.04 0.090 0.090 NCe NCe
RSDr, % 20.6 20.6
Clarified butter 8 Mean 0.236d 0.236d 0.258 0.258 91.8f (p ¼ 0.012) 91.8f
RSDr, % 6.7 6.7 5.0 5.0
Beef fat 8 Mean 0.593 0.593 0.519 0.519 114.4f (p < 0.00) 114.4f
RSDr, % 6.1 6.1 6.6 6.6
Crackers 8 Mean <0.16 <0.58 0.069 0.235 NCe NCe

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RSDr, % 16.4 10.9
Baked corn scoops 8 Mean <0.16 <2.00 0.035 0.443 NCe NCe
RSDr, % 15.3 9.5
Potato chips 8 Mean <0.16 <0.43 0.064 0.170 NCe NCe
RSDr, % 16.6 6.3
Chicken meal 8 Mean 1.28d 9.28d 0.401 2.91 320.2f (p < 0.001) 318.7f
RSDr, % 2.6 2.6 5.9 3.9
Meat and bone meal 8 Mean 2.21 22.45 NAg NAg NAg NAg
RSDr, % 5.5 5.5
Fish meal 8 Mean 2.17d 19.36d 1.963d 17.503d 110.5d (p < 0.001) 110.6d
RSDr, % 5.2 5.2 5.8 5.5

a
Fat tested by respective reference method.
b
AOCS ¼ AOCS Method Ca 5a-40 (Reference Method).
c
Recovery calculated as SafTest Free Fatty Acid Test (as is basis) result divided by AOCS Ca 5a-40 (as is basis) result  100.
d
Recovery calculated as SafTest Free Fatty Acid Test (fat basis) result divided by AOCS Ca 5a-40 (fat basis) result  100.
e
NC ¼ Not calculated.
f
Values are significantly different (p < 0.05).
g
NA ¼ Not available (could not extract enough fat).

in agreement and determined that the free fatty acid
results were low in processed grain-based snack products.
Independent Laboratory Study
The Independent Laboratory Validation Study was conducted
by Merieux NutriSciences Silliker Food Science Center (FSC) un-
der the guidance of the AOAC Research Institute.
Calibration
Verification of the LOQ
(a) Methodology.—LOQ of the method was calculated based on
the lowest point of the calibration curve of the method
which is 0.04%. The accuracy of the calibration is validated
in each batch of analysis by analysis of a control sample.
(b) Results.—The lowest calibrant establishes the LOQ at 0.04%
and is further adjusted by the dilution employed for matri-
ces in the study. For vegetable oil, which was tested neat
(without dilution), the LOQ was revised to 0.05%. For all 1:4
diluted matrices, the LOQ was 0.16%. The results of the
control must be within the concentration range provided
on the package insert for the control.

(a) Methodology.—The SafTest Free Fatty Acid Test method was

calibrated once a week during the Independent Laboratory
Validation for seven weeks. A control test solution was an-
alyzed with every run and was required to be within the
range for the free fatty acid control.
(b) Results.—Calibration curves determined for six weekly ca-
librations during the Independent Laboratory Validation
demonstrated stability and robustness of the method.
Calibration curves were stable and followed a pattern of
polynomial regression (second order) with regression coef-
ficients r2 from 0.9635 to 0.9785, and averaging 0.9695.
However, the SafTest Free Fatty Acid Test is programed
for a piece wise polygonal calibration, and this exercise
by the independent laboratory simply confirmed the sta-
bility of the calibration over the six-week period with
multiple kits.
Matrix Studies
(a) Methodology.—The MP Biomedical SafTest Free Fatty Acid
Test was validated against the AOCS Ca 5a-40 method for
free fatty acids in olive oil, soybean oil, clarified butter, beef
tallow, fried potato chips, crackers, chicken meal, meat
and bone meal and fish meal. Each of the listed ten sam-
ples were prepared according to the procedures described
above. Results for the oils and fats were determined di-
rectly using the AOCS Ca 5-40. Fat was extracted from the
snack products and the meat meals using the AOCS Am 5-
04 (3) method and fish meal was extracted using the AOAC
948.16 method (4). The extracted fat was then analyzed us-
ing AOCS Ca 5a-40. Free fatty acid test results were
expressed on both the as-is basis and a fat basis. The fat
basis was obtained by dividing the as-is result by the % fat
 310 | Gordon et al.: Journal of AOAC INTERNATIONAL Vol. 104, No. 2, 2021
using Equation 1. Eight blind coded test portions of each
material were generated at the same time for both the
SafTest Free Fatty Acid Test method and the AOACS
Ca 5a-40.
(b) Results.—The mean, standard deviation(s), RSDr, and per-
cent recovery were calculated for each product. The results
are presented in Table 11. Crackers, baked corn scoop
chips, and potato chips exhibited SafTest Free Fatty Acid
Test results below the LOQ of 0.04% times the dilution of
1:8 shown or 0.32%. These results were confirmed with
AOCS Ca 5a-40 results below 0.4%. The meat and bone
meal and fish meal presented difficulties and are discussed
in the spike recovery section below. The methods exhibited
similar precision of analysis when free fatty acid could be
quantitated by both methods. Precision of the SafTest Free
Fatty Acid Test method was found to be in the range 2.6 to
13% RSDr, which is satisfactory for this type of assay.
Biases, expressed as a percent recovery from AOCS Ca 5a-
40, across all matrices ranged from 91.8 to 114.4% (with re-
moval of one chicken meal outlier), and averaged 103.8%.
Spike Recovery—Meat Meals and Fish Meals
(a) Methodology.—Due to insurmountable problems with col-
ored extracts with AOCS Ca 5a-40 in meat & bone meal, ti-
tration endpoints could not be observed, and the reference
method results were unavailable. Upon agreement with
the reviewers, a free fatty acid (oleic acid) spike recovery
study for all meat meals (chicken meal, meat and bone
meal, and fish meal) was performed for comparability to
the reference method. Spikes were performed at a level of
about 0.4% fatty acids using an oleic acid solution. Eight
test portions of each of the meat meals were spiked with
free fatty acid and analyzed by the SafTest Free Fatty Acid
Test method. Non spike test portions were also analyzed in
the batch for calculation of spike recoveries.
(b) Results.—The results are presented in Table 13. The spike
recovery of free fatty acid in all of the three meat meals
evaluated was found to be satisfactory and in the range of
89 to101.6% with RSDr values in the range of 2.65 to 8.12%.
Discussion
For materials analyzed neat (e.g., vegetable oils, refined oils),
the LOQ is 0.05% free fatty acids. For materials that are diluted
1:4 prior to testing (e.g., meat meals, snacks), the LOQ is 0.16%
free fatty acids. For materials that are diluted 1:10 prior to test-
ing (e.g., fats, tallows), the LOQ is 0.40% free fatty acids.
The method was demonstrated to be selective for 20 short
and long chain fatty acids. All but 5 of 22 potential interferents
produced responses below the LOQ, with 2 providing notable in-
terference (1,6-hexanediamine and lauryl sulfate lithium).
This validation study yielded good agreement between
SafTest Free Fatty Acid Test and the traditional AOCS Ca 5a-40.
Table 13. Spike recovery of fatty acids from chicken, meat and bone, and fish meals
Matrix n Mean SafTest FFA, %a
Chicken meal 8 0.364
Meat and bone meal 8 0.405
Fish meal 8 0.451
a
Mean of SafTest Free Fatty Acid Test results.
Mean recoveries from replicate analyses of free fatty acid
solutions prepared at three different concentrations yielded
recoveries ranging from 97 to 106%, well within the AOAC-
recommended accuracy level of 80 to 120%. The relative stan-
dard deviations of repeatability (RSDr) for the SafTest Free Fatty
Acid Test ranged from 1.1 to 8.1% across all method developer
matrix studies and were very similar to the RSDr obtained using
AOCS Ca 5a-40 which ranged from 0.3 to 8.7%. The biases be-
tween the SafTest Free Fatty Acid Test and AOCS Ca 5a-40,
expressed as a percent recovery from AOCS Ca 5a-40, ranged as
follows: for olive oils, from 79 to 108%, and averaged 96.5%; for
animal fats, from 80 to 102%, and averaged 94.0%; for meat
meals, ranged from 92 to 109%, and averaged 103.9% (excluding
the problematic poultry meal).
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The SafTest Free Fatty Acid Test demonstrated shelf life sta-
bility in test results of a single kit stored at 4 C over 11 months
from the date of manufacture. The SafTest Free Fatty Acid Test
demonstrated lot to lot consistency in test results from testing
an oleic acid and a caproic acid control solution in three manu-
factured lots.
An independent laboratory study performed at Merieux
NutriSciences confirmed method developer studies. Similar
results were observed in the independent laboratory study
where RSDr of the SafTest Free Fatty Acid Test method was
found to be in the range 2.6 to 13%. Biases as reported in the in-
dependent laboratory study across all matrices, expressed as a
percent recovery from AOCS Ca 5a-40, ranged from 91.8 to
114.4% (with removal of one chicken meal outlier), and averaged
103.8%. Spike recovery of free fatty acid from three meat meals
was found to be satisfactory ranging from 89 to 101.6%, with
RSDr ranging from 2.65 to 8.12%.
The MP Biomedicals SafTest Free Fatty Acid Test Kit proves
to be an acceptable alternative method to the traditional AOCS
CA 5a-40 test and very valuable for food matrices where suf-
ficient fat for reference method testing cannot be obtained.
Using direct measurements (no preliminary fat extractions re-
quired), small reagent volumes, instrumental analysis and rapid
detection times, and easy-to-use, standardized procedures, the
SafTest Free Fatty Acid Test can determine free fatty acid levels
in specific food matrices with high degree of accuracy and
precision.
During these comprehensive validation studies, several
noteworthy observations were made.
(a) Inorganic acids in food matrices can cross react with the
SafTest Free Fatty Acid Test method.
(b) Intensely colored oils interfere with the AOCS Ca 5a-40;
however, the dilution of these dark oils in the SafTest Free
Fatty Acid Test method eliminates any color interference.
(c) A limitation of this test kit is that it is not suitable for
measurements of free fatty acid in refined oils below the
matrix LOQ of 0.05% and in snack products below the ma-
trix LOQ of 0.16%. There are some applications when a
lower concentration would be of interest.
True free fatty acid, % Recovery, % RSDr, %
0.408 89.0 4.13
0.406 99.9 8.12
0.444 101.6 2.65
 Gordon et al.: Journal of AOAC INTERNATIONAL Vol. 104, No. 2, 2021
(d) Repeatability of the SafTest Free Fatty Acid Test method
was found to be in the range 2.6 to 13%, which is within
performance expectations for this assay, and comparable
to the repeatability experienced with the reference method
AOCS Ca 5a-40.
(e) In the Independent Laboratory Validation, the end point
detection using AOCS Ca 5a-40 was problematic when
extracted fat was colored and the titration endpoint could
not be observed. For meat meals where AOCS Ca 5a-40 was
not dependable, spike recovery was used to evaluate accu-
racy of the SafTest Free Fatty Acid Test. Recoveries ranged
from 89 to 102% for meat and fish meals, and repeatability
ranged from 4 to 8%, both of which meet acceptance crite-
ria. Based on these experiences, meat meals, particularly
meat and bone meal, should be removed from the scope of
AOCS Ca 5a-40.
Conclusions
The SafTest Free Fatty Acid Test Kit is a rapid method designed
to measure the free fatty acid content of vegetable oils; fish oil;
animal fats (tallows); meat meal and fish meal products; and
crackers, chips, and other processed grain-based snack products
using micro-analytical and membrane separation principles.
Mean recoveries from replicate analyses of control samples pre-
pared at three different concentrations yielded 97 to 106%, well
within the AOAC-recommended accuracy limits of 80 to 120%.
The relative standard deviations of repeatability (RSDr) for the
SafTest Free Fatty Acid Test ranged from 1.1 to 8.1% across all
method developer matrix studies and were very similar to the
RSDr obtained using AOCS Ca 5a-40 which ranged from 0.3 to
8.7%. The biases between the SafTest Free Fatty Acid Test and
AOCS Ca 5a-40, expressed as a percent recovery from AOCS Ca
5a-40, ranged as follows: for olive oils, from 79 to 108%, and av-
eraged 96.5%; for animal fats, from 80 to 102%, and averaged
94.0%; for meat meals, ranged from 92 to 109%, and averaged
103.9% (excluding the problematic poultry meal). Similar results
were observed in the independent laboratory study where RSDr
of the SafTest Free Fatty Acid Test method was found to be in
the range 2.6 to 13%. Biases as reported in the independent labo-
ratory study across all matrices, expressed as a percent recovery
| 311
from AOCS Ca 5a-40, ranged from 91.8 to 114.4% (with removal
of one chicken meal outlier), and averaged 103.8%.
Using direct measurements (no preliminary fat extractions
required), small reagent volumes, instrumental analysis, and
easy-to-use, standardized procedures with rapid detection
times, SafTest Free Fatty Acid Test can detect free acid levels in
a variety of oils and fats as well as meat meal and snack matri-
ces with a high degree of accuracy and precision. The Free Fatty
Acid Test Kit are in good agreement to the AOCS Ca 5a-40.
Submitting Company
MP Biomedicals, 29525 Fountain Parkway, Solon, Ohio 44139
Independent Laboratory
Merieux NutriSciences, Siliker Food Science Center (FSC), 111
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East Wacker Dr, Chicago, IL 60601; Contract Laboratory for
Snack Products Matrix Study; Medallion Labs, 4000 Plymouth
Ave, Minneapolis, MN 55427; Contract Laboratory for Fats, Oils
and Meat Meal Matrix Studies; Canadian Grain Commission, 303
Main St, Winnipeg, Manitoba, R3C3GB.
Reviewers
Mark Collison, Director of Analytical Chemistry, ADM Research,
Decatur, IL
Richard (Rick) Dellaporta, Senior Principal Scientist, PepsiCo
Global Analytical, Plano, TX
Gina Clapper, Senior Scientific Liaison, Foods, USP (US
Pharmacopeia), Rockville, MD
References
1. Official Methods and Recommended Practices of the AOCS
(1996) 11th Ed., AOCS, Method AOCS Ca 5a-40, revised 2017
2. Official Methods and Recommended Practices of the AOCS
(1996) 11th Ed., AOCS, Method AOCS Aa 4-38, reapproved 2017
3. Official Methods and Recommended Practices of the AOCS
(1996) 11th Ed., AOCS, Method AOCS Am 5-04, reapproved
2017
4. Official Methods of Analysis (2016) 20th Ed., AOAC
INTERNATIONAL, Rockville, MD, Method 948.16.