TY - JOUR
AU - Studmire, Willainia C
AB - Abstract Background The Official American Oil Chemists Society (AOCS) Standard Procedure Aa 4–38, AOCS Standard Procedure Am 5–04, ISO 11085:2015, and similar methods are based on extraction of fats from food products using organic solvents. These methods require large volumes of organic solvents and up to 18-hour extractions. The SafTest Percent Fat measures fat content as “total triglycerides” in solubilized snack products and meals using micro-analytical and membrane separation principles. Objective The study objective is to validate the SafTest Percent Fat in an internal study and an independent laboratory study. Method Comparability to AOCS Standard Procedure Aa 4–38 or AOCS Standard Procedure Am 5–04, LOQ, selectivity, robustness, stability, and consistency were determined. Results Mean recoveries from replicate analyses of SafTest control samples at three concentrations yielded 101 to 103%. The relative standard deviations of repeatability (RSDr) were below 5%. Studies of meat meals and snacks analyzed using the SafTest Percent Fat demonstrated RSDr of 2.3 to 5.6% compared to 2.6 to 4.9% for AOCS Aa 4–38. The method demonstrated average recoveries ranging from 88.8 to 116.2% compared to the AOCS methods results performed in two contract laboratory studies and an independent laboratory validation. Conclusions The SafTest Percent Fat test can determine fat content in meals and snack matrices with accuracy and precision comparable to results from AOCS Aa 4–38 and AOCS Am 5–04. Highlights The SafTest Percent Fat test uses small reagent volumes, instrumental analysis, easy-to-use standardized procedures, and rapid analysis times for fat content determination. General Information Lipids include a broad category of non-polar molecules, including phospholipids, waxes, sterols, tocopherols, carotenoids, cholesterol, and similar compounds. Triglycerides (glycerol with backbone with three fatty acids) are the main storage form of lipids in plants and animal. Triglycerides include fats and oils (1). Crude fat methods are empirical in nature, estimating lipid content by solubility in a given solvent (e.g., diethyl ether, petroleum ether, benzene, chloroform, hexane, methanol) under a given set of conditions (e.g., time, temperature, particle size, ratio of solvent to test portion). Being empirical methods or “Defining Methods (Type 1)”, the crude fat results obtained by a given method can only be arrived at in terms of the method per se (2). Due to their empirical nature, each method produces a somewhat different result for the same product. In addition to lipids, crude fat methods co-extract any compounds soluble under the conditions of the method and the compounds co-extracted vary by the choice of the solvent. Co-extracted compounds include residual moisture, residual ethanol, pigments, carotenes, urea, and other compounds. Crude fat methods are not specific to lipids, nor do the extraction conditions ensure that 100% of the lipid material will be extracted. Peroxide and free fatty acid values are often converted from an as-is basis to a percent fat basis to compare them to results of other methods or to compare them to historic data. The SafTest Percent Fat test provides a rapid analysis of fat content for conversion of the free fatty acid and peroxide values in a food matrix to a percent fat basis. The SafTest Percent Fat test is specific to triglycerides, and this makes it suitable as a Reference Method (Type II) for estimation of fat as percent triglycerides when triglycerides can be demonstrated to be completely extracted. While it does not measure phospholipids, waxes, sterols, tocopherols, carotenoids, cholesterol, and similar compounds, the SafTest Percent Fat test demonstrates no interference from non-lipid materials that are co-extracted with traditional empirical Type I crude fat methods. The SafTest Percent Fat test is appropriate for quality control monitoring but is not intended to replace fatty acids methods used for nutritional labeling. The SafTest Percent Fat test is a component of the SafTest System, a series of test kits developed to assess lipid content and quality. Scope of Method Analyte.—Fat content as % fat in grams (g) determined as triglyceride per 100 g of test matrix. Matrices.—Potato chips and grain-based snack products; meat meal and fishmeal products. Summary of validated performance claims.—The practical dynamic range of the determination was established as 0.04 to 0.33 g fat determined as triglycerides in 100 g of these matrices. Higher fat content levels of up to 50% fat can be tested with an appropriate dilution. The LOQ was determined as 0.06% fat for low-fat lipid solutions run neat (0.06% fat is the low calibrant) and 0.24% for most materials diluted 1:4 during solubilization (fish meal and crackers were 0.28 and 0.36%, respectively). Mean recoveries from replicate analyses of triglyceride solutions at three concentrations were 101 to 103% with relative standard deviation of repeatability (RSDr) below 5%. Spike recoveries of triglycerides from liquid egg whites, crackers, pretzels, and chicken meals ranged from 99.8 to 108%. Studies are underway to determine if an acid hydrolysis step prior to testing is necessary for complete recovery of fat from fish meal. Glycerol produces interference with this method. Glycerol content below 5% can be corrected by subtraction. Intended Users The SafTest Percent Fat test can be used by the pet food, rendering, and snack industries to test incoming raw materials, materials in process, and finished products for fat content. The test can be used in a laboratory environment as well as a lab station on a production or on a receiving floor. The test is easy to perform and requires sample preparation and a 10 min incubation to produce an analytical result. The SafTest System includes a desktop workstation and disposable kits containing membrane units and reagents. This workstation, shown in Figure 1, accommodates up to six samples. Figure 1. Open in new tabDownload slide The SafTest system analytical workstation. Figure 1. Open in new tabDownload slide The SafTest system analytical workstation. Principle of the Method The SafTest System is a series of micro-analytical techniques based on membrane separation technology as an alternative to sensory and traditional analytical methods. One test of the series, the SafTest Percent Fat test, measures fat content. Fat content is used to convert free fatty acid and peroxide values to a fat basis as well as monitoring raw material ingredient contents. The Percent Fat test is designed to measure the fat content in prepared test portions using enzymatic hydrolysis of triglycerides to glycerol, followed by an enzymatic and colorimetric determination of the glycerol released. For liquid materials, test portions are solubilized in the proprietary preparation reagent, reacted with reagent, and read with an optical reader. For dry materials, test portions are solubilized, separated through the membrane separation pack, reacted with reagent, and read with an optical reader. This test procedure is intended to accurately measure percent fat levels above the LOQ of 0.06% for liquid materials run neat and 0.24% for dried food products diluted 1:4. However, food products with higher levels such as nut butters can be easily tested with a dilution step prior to analysis. The SafTest Percent Fat test produces reliable results with large dilutions of up to 1:100. Materials and Methods Test Kit Information, Equipment, and Additional Supplies Kit name.—Percent Fat test kit. Catalog number.—KTPF2000. Ordering information.—Worldwide MP Biomedicals, 29525 Fountain Parkway, Solon, OH 44139. Phone: 800-854-0530. Web: www.mpbio.com/ 07 KTPF-2000-Percent Fat Kit Test Kit Components Preparation reagent.—Part no. PREP2000. Stabilized isopropanol to solubilize the analyte. Fat reagent A.—Part no. PF1010A. A pH buffered solution of enzymes used for hydrolysis of triglycerides to glycerol and colorimetric determination of the released glycerol. Reagent blank.—Part no. PF1010RB, 0.00 g/100 mL triglyceride calibrant in preparation reagent. Calibrator 1.—Part no. CAL1. PF1010 Cal 1, 0.06 g/100 mL triglyceride calibrant in preparation reagent. Calibrator 2.—Part no. CAL2. PF1010 Cal 2, 0.08 g/100 mL triglyceride calibrant in preparation reagent. Calibrator 3.—Part no. CAL3. PF1010 Cal 3, 0.12 g/100 mL triglyceride calibrant in preparation reagent. Calibrator 4.—Part no. CAL4. PF1010 Cal 4, 0.16 g/100 mL triglyceride calibrant in preparation reagent. Calibrator 5.—Part no. CAL5. PF1010 Cal 5, 0.33 g/100 mL triglyceride calibrant in preparation reagent. Control.—Part no. CON PF1010 Con, 0.1 g/100 mL triglyceride control in preparation reagent. Apparatus SafTest workstation.—Part no. 07LI2000. The workstation (Figure 1) includes an analyzer, heat block, membrane separation unit, pipettors, an adjustable volume dispenser, and one fixed volume dispenser. Additional Supplies and Materials Required Membranes.—Part no. 07DF1000. High non-specific adsorption membranes are used for prefiltration and consist of polypropylene or polyethenesulfame or other polyethylene structures. Reference materials.—Triolein: Sigma T7140. 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 solvents. Protective equipment that becomes contaminated must be removed and replaced as necessary. Additional health and safety information is provided in the Safety Data Sheets (SDSs), which are included on the website (MpBio.com under SafTest) Test Method Initial Preparation Preheat the heat block to 37–44°C for all materials except those containing saturated fats (solid and semi-solid tallows and fats). For materials with saturated fats, preheat to 46–50°C. Remove the preparation reagent from the refrigerator and allow it to stand at room temperature (18–25°C) for 1–2 h. Attach the preparation reagent dispenser to the bottle. Set the volume on the dispenser as needed (Table 1). To eliminate air bubbles and oxidized reagent, dispense 4–5 aliquots of the reagent into a waste container immediately before use. Turn on SafTest analyzer. Table 1. Preparation instructions Matrix . Preparation instructions . Dilution factor . Block conditions . Meat meals and fish meal Reduce particle size of >100 g analytical sample with appropriate mill. Weigh 1.0 g of test portion (±0.05 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 ± 0.2 min. Warm in heat block. See “Block conditions”. 1:4 37–44°C for 10 min Potato chips and grain-based snack products Using a coffee grinder, grind >100 g analytical sample for 1 min unless fat content is >20%. If >20%, then grind for 30 s. Weigh 1.0 g of test portion (±0.05 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 ± 0.2 min. Warm in heat block. See “Block conditions”. 1:4 37–44°C for 10 min Matrix . Preparation instructions . Dilution factor . Block conditions . Meat meals and fish meal Reduce particle size of >100 g analytical sample with appropriate mill. Weigh 1.0 g of test portion (±0.05 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 ± 0.2 min. Warm in heat block. See “Block conditions”. 1:4 37–44°C for 10 min Potato chips and grain-based snack products Using a coffee grinder, grind >100 g analytical sample for 1 min unless fat content is >20%. If >20%, then grind for 30 s. Weigh 1.0 g of test portion (±0.05 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 ± 0.2 min. Warm in heat block. See “Block conditions”. 1:4 37–44°C for 10 min Open in new tab Table 1. Preparation instructions Matrix . Preparation instructions . Dilution factor . Block conditions . Meat meals and fish meal Reduce particle size of >100 g analytical sample with appropriate mill. Weigh 1.0 g of test portion (±0.05 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 ± 0.2 min. Warm in heat block. See “Block conditions”. 1:4 37–44°C for 10 min Potato chips and grain-based snack products Using a coffee grinder, grind >100 g analytical sample for 1 min unless fat content is >20%. If >20%, then grind for 30 s. Weigh 1.0 g of test portion (±0.05 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 ± 0.2 min. Warm in heat block. See “Block conditions”. 1:4 37–44°C for 10 min Matrix . Preparation instructions . Dilution factor . Block conditions . Meat meals and fish meal Reduce particle size of >100 g analytical sample with appropriate mill. Weigh 1.0 g of test portion (±0.05 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 ± 0.2 min. Warm in heat block. See “Block conditions”. 1:4 37–44°C for 10 min Potato chips and grain-based snack products Using a coffee grinder, grind >100 g analytical sample for 1 min unless fat content is >20%. If >20%, then grind for 30 s. Weigh 1.0 g of test portion (±0.05 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 ± 0.2 min. Warm in heat block. See “Block conditions”. 1:4 37–44°C for 10 min Open in new tab Sample Preparation Laboratory samples should be stored cold and in sealed containers. This test uses small test portion masses (1 g), therefore materials should be comminuted to a small, uniform particle size prior to test portion selection. See Table 1 for specific instructions for solubilizing and filtering the specific matrices. Weigh 1 g ± 0.05 g test portion into conical tube. Add ten glass beads and 3 mL of preparation reagent. Cap conical tubes and vortex the test solutions at the fastest dial setting for 1 ± 0.2 min. Place prepared test solutions in the heat block set at 37–44°C (or 46–50°C for test materials with saturated fats) for 10 min. While solutions are being heated, label a new glass test tube for each unknown and place the new tubes in the acrylic base of the SafTest filtration unit. Position the membrane holder (with membranes attached) onto the acrylic base. Remove tubes from the heat block. Pour the test solutions into the membranes, filling each membrane to the top. Cap membranes to seal them. Turn on the filtration unit and slowly increase the vacuum pressure to 5–10 in Hg. Filter until the solution is uniform and clear. Materials with high fat content may appear slightly cloudy and may need further dilution to completely solubilize the fat. Note: Improper filtration or too high of a fat content is indicated by the formation of two layers. Should this occur, dilute, prepare, and filter the samples again using new membranes. When filtering is complete, reduce the pressure to 0 and turn off the filtration unit. Discard the membrane holder when all membranes have been used. Do not reuse membranes. Remove the test tubes containing the filtered test solutions. Cap the tubes and place them in the heat block to keep warm until you perform the test. Perform the test within 2 h. Calibration (Weekly) Label five new 12 mm glass test tubes to correspond to the reagent blank and calibrators 1 (0.06% triglyceride) , 2 (0.08% triglyceride), 3 (0.12% triglyceride), 4 (0.16% triglyceride), and 5 (0.33% triglyceride); and one new 12 mm tube for the control. Using the positive displacement pipette, transfer 25 μL of the calibrators and control into the corresponding labeled test tube. Before dispensing reagents, gently swirl the contents of the reagent bottle to eliminate air bubbles and dispense 4–5 aliquots of the reagent into a waste container immediately before use. Dispense one aliquot (fixed volume 1.82 mL) of fat reagent A into every calibrator and control test tube. Start the timer for 10 ± 1 min. Cap the test tubes and invert them six times. 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 tubes are not read within 2 min, the tubes must be discarded. Check to be sure the SafTest Analyzer is turned on. To run up the calibration press [RUN] on screen. Press the [FAT] icon; when highlighted yellow, press the [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. Then insert the reagent blank when prompted. The optical density (OD) of the reagent blank must be less than 0.20 OD. 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 fat control ranges that can be found on the fat control package insert. If they do not, rerun the calibration. Measure the Test Sample on the SafTest Analyzer Label one new 12 mm glass test tube per solution or two new glass test tubes if preparing duplicates. Use a positive displacement pipette to transfer 25 μL of each prepared test solution into the corresponding labeled test tube. Dispense one aliquot (1.82 mL) of fat reagent A into each test tube. Start the timer for 10 ± 1 min. Cap the test tubes and invert them five times. Place the test tubes in the heat block at 37–44°C for the remaining time. When Analyzing Samples Press [STAT] icon and the Press [FAT] icon are highlighted yellow, press [NEXT] icon. At the “Blank Tube” prompt, insert a test tube of distilled water in the sample compartment. Use the “+” or “-” arrows to select the number of replicate tubes (1–5) for each unknown (replicate unknowns are recommended to ensure results are consistent). Normally, two replicates should be analyzed to ensure that results are consistent. 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]. The SafTest analyzer will use the calibrators to calculate the fat content as percent fat in the test solution. Calculations The SafTest analyzer uses the triglyceride calibrants to calculate the percent fat in the test solution. To convert the result from the SafTest analyzer to the fat content in the test portion, multiply by the appropriate dilution factor as shown in Table 2. Table 2. Applying the dilution factor Dilution . Dilution factor . SafTest analyzer result, % . Result, % × dilution factor . Final calculated result, %a . 1:4 4 0.19 0.19 × 4 0.76 1:10 10 0.19 0.19 × 10 1.9 Dilution . Dilution factor . SafTest analyzer result, % . Result, % × dilution factor . Final calculated result, %a . 1:4 4 0.19 0.19 × 4 0.76 1:10 10 0.19 0.19 × 10 1.9 a Final Percent Fat result, in test portion. Open in new tab Table 2. Applying the dilution factor Dilution . Dilution factor . SafTest analyzer result, % . Result, % × dilution factor . Final calculated result, %a . 1:4 4 0.19 0.19 × 4 0.76 1:10 10 0.19 0.19 × 10 1.9 Dilution . Dilution factor . SafTest analyzer result, % . Result, % × dilution factor . Final calculated result, %a . 1:4 4 0.19 0.19 × 4 0.76 1:10 10 0.19 0.19 × 10 1.9 a Final Percent Fat result, in test portion. Open in new tab Validation Study This validation study was conducted under the AOAC Research Institute (RI) Performance Tested Method SM program. The SafTest Percent Fat test was validated for potato chips, crackers, and other grain-based snack products and meat meals and fish meals. The method developer studies included calibration, LOD and LOQ, matrix studies, selectivity, stability, robustness, and bias. The studies included comparison to the AOCS and AOAC reference methods (2–4) for crude fat content. The independent validation study included calibration, matrix studies, LOQ, and bias. Reference Method Lipids include a broad category of non-polar molecules, including phospholipids, waxes, sterols, tocopherols, carotenoids, cholesterol, and similar compounds. Triglycerides (including fats and oils) are the main storage form of lipids in plants and animal (1). Choice of a reference method was difficult because there are many methods, each is designed to estimate lipid content differently, each is limited to specific matrices, and none of them measure true lipid content. Reference methods were often chosen/approved because of the availability of equipment in the contract and independent laboratories and their familiarity with the method. For these studies, reference methods AOCS Standard Procedure Aa 4–38 (3), AOCS Standard Procedure Am 5–04 (4), and AOAC Official Method 948.16 (5) were used for crude fat determinations. AOAC Official Method 948.16 was specifically used for fish meal in the independent validation study. AOCS Standard Procedure Aa 4–38 estimates lipid content as substances extracted by petroleum ether and was used by Medallion Laboratories and Pilot Laboratory (POS) for crude fat extraction since this was a method choice in contract laboratories. AOCS Standard Procedure Am 5–04 estimates lipids indirectly by weighing the test portion before and after high temperature petroleum ether extraction and assumes that the fraction removed is predominately triglycerides. This method was used in the independent laboratory study because the method scope (oilseeds, meats, feeds, and foods) was consistent for the intended use. Neither AOCS Standard Procedure Aa 4–38 nor AOCS Standard Procedure Am 5–04 provides for a water prewash step (common to other solvent extraction methods) which is critical for some matrices to remove water soluble components that can co-extract with the lipids and result in over-estimation of lipid content. AOAC Official Method 948.16 estimates lipids in fish meal as substances in two steps: those initially extracted by acetone plus those extracted after digestion with 4 M HCl, followed by rinsing, and re-extraction with acetone. The scope of the method is specific to fish meal and was used in the independent laboratory study. Method Developer Studies Calibration and Control Study Methodology.—Multiple calibration curves of five calibrators (0.06, 0.08, 0.12, 0.16, and 0.32% triglycerides) and a reagent blank were used to create the calibration curve and establish the range of the method (Figure 2). In addition, the calibration curves were generated on seven analyzers to evaluate the repeatability of the linear calibration curves across analyzers. Results.—Linearity of the three calibration curves were 0.999, 0.998, and 0.999 (see Figure 2). Comparison of calibration curves on seven analyzers (Figure 3) exhibited excellent linearity and repeatability. A low control read against each of the seven curves averaged 0.103% with an RSDr of 0.004%. Figure 2. Open in new tabDownload slide Percent fat linear calibration curve with 0.999 correlation. Figure 2. Open in new tabDownload slide Percent fat linear calibration curve with 0.999 correlation. Figure 3. Open in new tabDownload slide Calibration curves for 7 different analyzers. Figure 3. Open in new tabDownload slide Calibration curves for 7 different analyzers. LOD and LOQ Methodology.—The LOD and LOQ were empirically determined by 10 replicate analyses of 0.10% triolein solution in preparation reagent. The LOD was calculated as 3 × the standard deviation of the 10 results. In another set of studies, seven products representing the matrices of scope were studied to determine the LOQ of the specific matrix with dilution during solubilization. Products were obtained with as low a fat content as could be obtained. Results.—The empirically determined LOD was calculated as 0.012% (3 × 0.004). The empirical LOQ was determined as 0.04%. (10 × 0.004). The empirical LOQ was less than the low calibrant concentration. So, the low calibrant concentration was determined to be a more practical LOQ. In the LOQ studies on seven products containing low levels of fat, the LOQ was determined as 10 × the standard deviation of the result, as follows: chicken meal 0.12%, meat meal 0.20%, fish meal 0.28%, potato chips 0.06%. baked chips 0.24%, cracker (saturated fat) 0.36%, and sweet cracker 0.16%. In general, LOQ for matrices is 0.12 to 0.36%. Since the low calibrant multiplied by the dilution factor represents a concentration of 0.24%, the LOQs below 0.24% are not practical. So, 0.24% fat is an appropriate LOQ, except for fish meal and crackers, which are 0.28 and 0.36%, respectively. Robustness Methodology.—Three critical parameters were evaluated for effect on the fat content test results: (1) reaction time, (2) temperature of sample preparation, and (3) grinding time of dry matrices. See Table 3 for a list of conditions and parameters studied. Results.— Reaction times for Percent Fat tests were tested at 10 ± 1 min and 20 ± 1 min using a 0.120% triolein solution. The results at 10 min average 0.121 ± 0.02% fat (n = 3) which is a 100.8% recovery. The results at 20 min averaged 0.102 ± 0.05% fat (n = 3) which is an 85% recovery. Time must be controlled to 10 ±1 min to achieve complete recovery of fat. Three portions of crackers with saturated fat were prepared at the recommended temperature of 46–50°C and at 37–44°C, the temperature used for materials without saturated fats. At a preparation temperature of 37–44°C, the Percent Fat result was 25.5 ± 3.7%. At a preparation temperature of 46–50°C the Percent Fat result was 27.9 ± 2.4%. The second value is in good agreement with the AOCS Standard Procedure Aa 4–38 result of 27.4%. Saturated fat products require the higher preparation temperature (46–50°C) for full recovery. Potato chips (high fat) were prepared with the recommended grinding time of 30 ± 5 s and a grinding time used for lower fat test portions of 60 ± 5 s. Fat content of the potato chips was 32.2% with RSDr of 3.5% after 60 s of grinding and 35.1% with RSDr of 2.2% with 30 s of grinding. The second Percent Fat test results are in good agreement with the AOCS Standard Procedure Aa 4–38 result of 36.1%. Grinding time for high fat samples must be controlled at 30 s. The test time, the preparation temperature, and grinding time must be carefully controlled as instructed in the kit instructions (see Table 3). Table 3. Robustness parameters Matrix . Parameter . . n . Condition 1 . Condition 2 . Triolein solution (0.120%) Reaction time Parameter condition 10 min 20 min SafTest Fat % ± sr 3 0.121 ± 0.02% 0.102 ± 0.05% Saturated fat cracker (27.4% by AOCS Aa 4–38) Temperature Parameter condition 46− 50°C 37 − 44°C SafTest Fat % ± sr 3 27.9 ± 2.4% 25.5 ± 3.7% High fat potato chip (36.1% by AOCS Aa 4–38) Grinding time Parameter condition 30 s 60 s SafTest Fat % ± sr 3 35.1 ± 2.2% 32.2 ± 3.5% Matrix . Parameter . . n . Condition 1 . Condition 2 . Triolein solution (0.120%) Reaction time Parameter condition 10 min 20 min SafTest Fat % ± sr 3 0.121 ± 0.02% 0.102 ± 0.05% Saturated fat cracker (27.4% by AOCS Aa 4–38) Temperature Parameter condition 46− 50°C 37 − 44°C SafTest Fat % ± sr 3 27.9 ± 2.4% 25.5 ± 3.7% High fat potato chip (36.1% by AOCS Aa 4–38) Grinding time Parameter condition 30 s 60 s SafTest Fat % ± sr 3 35.1 ± 2.2% 32.2 ± 3.5% Open in new tab Table 3. Robustness parameters Matrix . Parameter . . n . Condition 1 . Condition 2 . Triolein solution (0.120%) Reaction time Parameter condition 10 min 20 min SafTest Fat % ± sr 3 0.121 ± 0.02% 0.102 ± 0.05% Saturated fat cracker (27.4% by AOCS Aa 4–38) Temperature Parameter condition 46− 50°C 37 − 44°C SafTest Fat % ± sr 3 27.9 ± 2.4% 25.5 ± 3.7% High fat potato chip (36.1% by AOCS Aa 4–38) Grinding time Parameter condition 30 s 60 s SafTest Fat % ± sr 3 35.1 ± 2.2% 32.2 ± 3.5% Matrix . Parameter . . n . Condition 1 . Condition 2 . Triolein solution (0.120%) Reaction time Parameter condition 10 min 20 min SafTest Fat % ± sr 3 0.121 ± 0.02% 0.102 ± 0.05% Saturated fat cracker (27.4% by AOCS Aa 4–38) Temperature Parameter condition 46− 50°C 37 − 44°C SafTest Fat % ± sr 3 27.9 ± 2.4% 25.5 ± 3.7% High fat potato chip (36.1% by AOCS Aa 4–38) Grinding time Parameter condition 30 s 60 s SafTest Fat % ± sr 3 35.1 ± 2.2% 32.2 ± 3.5% Open in new tab Assessment of Triglyceride Solutions Methodology.—The low (0.10%), medium (0.60%), and high(0.80%) triglyceride test solutions were analyzed ten times and the concentration compared to the true value. Results.—The results are provided in Table 4. Triglyceride recoveries ranged from 101 to 103% and within-laboratory precision (RSDr%) ranged from 1.2 to 4.9%. Table 4. Assessment of triglyceride solutions True value fat, % . No. replicates . Measured fat, %a . RSDr, % . Recovery, % . 0.100 10 0.102 1.2 102 0.600 10 0.603 4.9 101 0.800 10 0.824 1.9 103 True value fat, % . No. replicates . Measured fat, %a . RSDr, % . Recovery, % . 0.100 10 0.102 1.2 102 0.600 10 0.603 4.9 101 0.800 10 0.824 1.9 103 a Mean of SafTest Percent Fat test results. Open in new tab Table 4. Assessment of triglyceride solutions True value fat, % . No. replicates . Measured fat, %a . RSDr, % . Recovery, % . 0.100 10 0.102 1.2 102 0.600 10 0.603 4.9 101 0.800 10 0.824 1.9 103 True value fat, % . No. replicates . Measured fat, %a . RSDr, % . Recovery, % . 0.100 10 0.102 1.2 102 0.600 10 0.603 4.9 101 0.800 10 0.824 1.9 103 a Mean of SafTest Percent Fat test results. Open in new tab Stability Methodology.—The stability of the Percent Fat kit was studied at months 2, 6, 12, and 18 after production by testing 10 replicates of a 0.10% triglyceride solution at each point in the time interval. Results.—Mean results ranged from 0.102% fat at 12 months to 0.104% fat at 6 and 18 months. No trends in means were observed. RSDrs ranged from 2.9 to 3.8% with no trend for increasing variability with time. Results are reported in Table 5. Shelf life of the kit was confirmed to be at least eighteen months when stored properly at 2–8°C. Table 5. SafTest Percent Fat test shelf life stability study SafTest Percent Fat kit age, months . Control, SafTest Percent Fata . sr . RSD, % . 2 0.103 0.003 2.9 6 0.104 0.004 3.8 12 0.102 0.003 2.9 18 0.104 0.004 3.8 SafTest Percent Fat kit age, months . Control, SafTest Percent Fata . sr . RSD, % . 2 0.103 0.003 2.9 6 0.104 0.004 3.8 12 0.102 0.003 2.9 18 0.104 0.004 3.8 a Mean of ten replicate results in SafTest Percent Fat test. Open in new tab Table 5. SafTest Percent Fat test shelf life stability study SafTest Percent Fat kit age, months . Control, SafTest Percent Fata . sr . RSD, % . 2 0.103 0.003 2.9 6 0.104 0.004 3.8 12 0.102 0.003 2.9 18 0.104 0.004 3.8 SafTest Percent Fat kit age, months . Control, SafTest Percent Fata . sr . RSD, % . 2 0.103 0.003 2.9 6 0.104 0.004 3.8 12 0.102 0.003 2.9 18 0.104 0.004 3.8 a Mean of ten replicate results in SafTest Percent Fat test. Open in new tab Selectivity Methodology.—Two experiments were conducted to test selectivity of the SafTest Percent Fat test. A selection of 22 compounds similar in structure to triglycerides (e.g., fatty amides, amines, alcohols, aldehydes, esters, anhydrides, and acetates and other salts and smaller molecular structures such as glycerol) were tested by the SafTest Percent Fat test to determine response (positive interference) in the method. See Table 6 for a listing of the compounds tested. The responses to five commercially available lipid products were tested using the SafTest Percent Fat test. The three commercial mixtures of triglycerides were Sigma 7810, Sigma T2630, and Sigma T9517; the mixture of monoglycerides, diglycerides, and triglycerides was Sigma 1787; and the phospholipid liposome mixture was Sigma L 4398. All were tested for recovery with the SafTest Percent Fat test and compared to recoveries obtained with AOCS Standard Procedure Aa 4–38. All were analyzed using the kit instructions for liquids, with dilutions to achieve an approximately 0.1% by weight basis. Results are reported in Table 7 as a percent recovery of the AOCS Aa 4–38 results. Water-soluble co-extractants are known to interfere with solvent extraction fat methods and must be removed with a water extraction step prior to the solvent extraction of fat (6). Theoretically, water-soluble components should have no interference with the SafTest Percent Fat test. To verify the absence of interference, cracker and fried potato chip products were tested after spiking with 5% added sucrose, and chicken meal was tested after spiking with 5% urea. Sucrose-spiked portions were prepared by thoroughly blending 9.5 g of ground chip and 9.5 g of cracker each with 0.5 g of sucrose. Urea spiked portions were prepared by thoroughly blending 9.5 g chicken meal with 0.5 g urea. Controls were prepared by thoroughly blending 9.5 g of the matrix with 0.5 g of a nonfat protein, albumin. Four replicate test portions of each preparation were then analyzed using the SafTest Percent Fat procedure described above. Results.— Results of the 22 potential interferents are reported in Table 6. No notable interferences were observed except for glycerol which produced significant cross reactivity (93%) in the SafTest Percent Fat test method. Products with added glycerol will produce interferences with this test. If the glycerol content is below 5%, a subtraction can be made for the glycerol interference. While not a part of this selectivity study, strongly acid food matrices (pH <5) and strongly basic food matrices (pH >8) cannot be tested using this method due to inactivation of the enzymes in fat reagent A. Results for the three mixtures of triglycerides, the mixture of monoglycerides, diglycerides, and triglycerides, and the phospholipid are reported in Table 7. Recoveries of the four glyceride solutions ranged from 99.89 to 99.90% (normalized to 100% recovery for results by AOCS Standard Procedure Aa 4–38). There was no response to the liposome by either method, indicating that neither method was responsive to a phospholipid. Results for the test portions with sugar or urea are presented in Table 8. Recovery of fat from sugar spiked crackers and potato chips compared to the control with added albumin was 96.6% for crackers and 98.3% for potato chips. Fat recoveries from chicken meal with added urea was 100.8% of the control chicken meal. Table 6. SafTest free fatty acid test method selectivity for potential chemical interferents MPBio ID . Customer ID . Constituent . CAS No. . Free fatty acid . SafTest Percent Fata . AOCS Aa 4–38 . Biasb . 2844 O-4500 1-Octanol 111-87-5 0.00 0.0 0.00 2845 D-7388 n-Decylamine 2016-57-1 0.00 0.0 0.00 2846 D-7384 n-Decyl aldehyde (a.k.a caprinaldehyde) 112-31-2 0.00 0.0 0.00 2847 D-0634 n-Decyl alcohol (a.k.a 1-decanol) 112-30-1 0.00 0.0 0.00 2848 G-7757 Glycerol 56-81-5 0.93 0.02 0.91 2849 C-3642 Capric anhydride 2082-76-0 0.00 0.0 0.00 2850 H-13303 1-Hexanol 111-27-3 0.01 0.0 0.01 2851 H-9008 Hexanal 66-25-1 0.00 0.0 0.00 2852 C-2125 Caproic ccid methyl ester 106-70-7 0.00 0.0 0.00 2853 C-5049 Capric acid methyl ester 110-42-9 0.00 0.0 0.00 2854 O-9875 Oleyl acetate 693-80-1 0.00 0.0 0.00 2855 O-8880 Oleyl alcohol 143-28-2 0.00 0.0 0.00 2856 O-7251 Oleic anhydride 24909-72-6 0.00 0.0 0.00 2857 O-9500 Oleic ethyl ester (a.k.a oleic acid ethyl ester or ethyl oleate) 111-62-6 0.00 0.0 0.00 2863 H-7883 2-Heptanone 110-43-0 0.00 0.0 0.00 2873 H-0255 n-Hexadecane 544-76-3 0.00 0.0 0.00 2874 H-2381 1,6-Hexanediamine 124-09-4 0.00 0.0 0.00 2875 02160 Adipic acid diethyl ester (a.k.a diethyl adipate) 141-28-6 0.00 0.0 0.00 2877 L-4003 Lauryl acetate 112-66-3 0.00 0.0 0.00 2878 L-5503 Lauryl chloride 112-16-3 0.00 0.0 0.00 2879 L-7272 Lauric acid methyl ester 111-82-0 0.00 0.0 0.00 2880 Sigma L-5901 Lauryl sulfate lithium 2044-56-6 0.00 0.0 0.00 MPBio ID . Customer ID . Constituent . CAS No. . Free fatty acid . SafTest Percent Fata . AOCS Aa 4–38 . Biasb . 2844 O-4500 1-Octanol 111-87-5 0.00 0.0 0.00 2845 D-7388 n-Decylamine 2016-57-1 0.00 0.0 0.00 2846 D-7384 n-Decyl aldehyde (a.k.a caprinaldehyde) 112-31-2 0.00 0.0 0.00 2847 D-0634 n-Decyl alcohol (a.k.a 1-decanol) 112-30-1 0.00 0.0 0.00 2848 G-7757 Glycerol 56-81-5 0.93 0.02 0.91 2849 C-3642 Capric anhydride 2082-76-0 0.00 0.0 0.00 2850 H-13303 1-Hexanol 111-27-3 0.01 0.0 0.01 2851 H-9008 Hexanal 66-25-1 0.00 0.0 0.00 2852 C-2125 Caproic ccid methyl ester 106-70-7 0.00 0.0 0.00 2853 C-5049 Capric acid methyl ester 110-42-9 0.00 0.0 0.00 2854 O-9875 Oleyl acetate 693-80-1 0.00 0.0 0.00 2855 O-8880 Oleyl alcohol 143-28-2 0.00 0.0 0.00 2856 O-7251 Oleic anhydride 24909-72-6 0.00 0.0 0.00 2857 O-9500 Oleic ethyl ester (a.k.a oleic acid ethyl ester or ethyl oleate) 111-62-6 0.00 0.0 0.00 2863 H-7883 2-Heptanone 110-43-0 0.00 0.0 0.00 2873 H-0255 n-Hexadecane 544-76-3 0.00 0.0 0.00 2874 H-2381 1,6-Hexanediamine 124-09-4 0.00 0.0 0.00 2875 02160 Adipic acid diethyl ester (a.k.a diethyl adipate) 141-28-6 0.00 0.0 0.00 2877 L-4003 Lauryl acetate 112-66-3 0.00 0.0 0.00 2878 L-5503 Lauryl chloride 112-16-3 0.00 0.0 0.00 2879 L-7272 Lauric acid methyl ester 111-82-0 0.00 0.0 0.00 2880 Sigma L-5901 Lauryl sulfate lithium 2044-56-6 0.00 0.0 0.00 a SafTest Free Fatty Acid test. b Bias calculated as SafTest Free Fatty Acid test result minus AOCS Ca 5a–40 result. Open in new tab Table 6. SafTest free fatty acid test method selectivity for potential chemical interferents MPBio ID . Customer ID . Constituent . CAS No. . Free fatty acid . SafTest Percent Fata . AOCS Aa 4–38 . Biasb . 2844 O-4500 1-Octanol 111-87-5 0.00 0.0 0.00 2845 D-7388 n-Decylamine 2016-57-1 0.00 0.0 0.00 2846 D-7384 n-Decyl aldehyde (a.k.a caprinaldehyde) 112-31-2 0.00 0.0 0.00 2847 D-0634 n-Decyl alcohol (a.k.a 1-decanol) 112-30-1 0.00 0.0 0.00 2848 G-7757 Glycerol 56-81-5 0.93 0.02 0.91 2849 C-3642 Capric anhydride 2082-76-0 0.00 0.0 0.00 2850 H-13303 1-Hexanol 111-27-3 0.01 0.0 0.01 2851 H-9008 Hexanal 66-25-1 0.00 0.0 0.00 2852 C-2125 Caproic ccid methyl ester 106-70-7 0.00 0.0 0.00 2853 C-5049 Capric acid methyl ester 110-42-9 0.00 0.0 0.00 2854 O-9875 Oleyl acetate 693-80-1 0.00 0.0 0.00 2855 O-8880 Oleyl alcohol 143-28-2 0.00 0.0 0.00 2856 O-7251 Oleic anhydride 24909-72-6 0.00 0.0 0.00 2857 O-9500 Oleic ethyl ester (a.k.a oleic acid ethyl ester or ethyl oleate) 111-62-6 0.00 0.0 0.00 2863 H-7883 2-Heptanone 110-43-0 0.00 0.0 0.00 2873 H-0255 n-Hexadecane 544-76-3 0.00 0.0 0.00 2874 H-2381 1,6-Hexanediamine 124-09-4 0.00 0.0 0.00 2875 02160 Adipic acid diethyl ester (a.k.a diethyl adipate) 141-28-6 0.00 0.0 0.00 2877 L-4003 Lauryl acetate 112-66-3 0.00 0.0 0.00 2878 L-5503 Lauryl chloride 112-16-3 0.00 0.0 0.00 2879 L-7272 Lauric acid methyl ester 111-82-0 0.00 0.0 0.00 2880 Sigma L-5901 Lauryl sulfate lithium 2044-56-6 0.00 0.0 0.00 MPBio ID . Customer ID . Constituent . CAS No. . Free fatty acid . SafTest Percent Fata . AOCS Aa 4–38 . Biasb . 2844 O-4500 1-Octanol 111-87-5 0.00 0.0 0.00 2845 D-7388 n-Decylamine 2016-57-1 0.00 0.0 0.00 2846 D-7384 n-Decyl aldehyde (a.k.a caprinaldehyde) 112-31-2 0.00 0.0 0.00 2847 D-0634 n-Decyl alcohol (a.k.a 1-decanol) 112-30-1 0.00 0.0 0.00 2848 G-7757 Glycerol 56-81-5 0.93 0.02 0.91 2849 C-3642 Capric anhydride 2082-76-0 0.00 0.0 0.00 2850 H-13303 1-Hexanol 111-27-3 0.01 0.0 0.01 2851 H-9008 Hexanal 66-25-1 0.00 0.0 0.00 2852 C-2125 Caproic ccid methyl ester 106-70-7 0.00 0.0 0.00 2853 C-5049 Capric acid methyl ester 110-42-9 0.00 0.0 0.00 2854 O-9875 Oleyl acetate 693-80-1 0.00 0.0 0.00 2855 O-8880 Oleyl alcohol 143-28-2 0.00 0.0 0.00 2856 O-7251 Oleic anhydride 24909-72-6 0.00 0.0 0.00 2857 O-9500 Oleic ethyl ester (a.k.a oleic acid ethyl ester or ethyl oleate) 111-62-6 0.00 0.0 0.00 2863 H-7883 2-Heptanone 110-43-0 0.00 0.0 0.00 2873 H-0255 n-Hexadecane 544-76-3 0.00 0.0 0.00 2874 H-2381 1,6-Hexanediamine 124-09-4 0.00 0.0 0.00 2875 02160 Adipic acid diethyl ester (a.k.a diethyl adipate) 141-28-6 0.00 0.0 0.00 2877 L-4003 Lauryl acetate 112-66-3 0.00 0.0 0.00 2878 L-5503 Lauryl chloride 112-16-3 0.00 0.0 0.00 2879 L-7272 Lauric acid methyl ester 111-82-0 0.00 0.0 0.00 2880 Sigma L-5901 Lauryl sulfate lithium 2044-56-6 0.00 0.0 0.00 a SafTest Free Fatty Acid test. b Bias calculated as SafTest Free Fatty Acid test result minus AOCS Ca 5a–40 result. Open in new tab Table 7. Selectivity study for triglycerides, mixed glycerides, and liposomes Lab ID . Sigma Cat. No. . Lipid product . Fat, % . SafTest Percent Fat . AOCS Aa4–38 . 2881 17810 Triglyceride mixed 99.9 100.0 2882 T2630 Glyceryl tripalmitate 99.9 100.0 2883 T9517 Glyceryl trilinoleate 99.9 100.0 2884 1787 Lipid standard, mono-, di-, and triglyceride mix, certified reference material 99.9 100.0 2885 L4395 Liposome powder No response No response Lab ID . Sigma Cat. No. . Lipid product . Fat, % . SafTest Percent Fat . AOCS Aa4–38 . 2881 17810 Triglyceride mixed 99.9 100.0 2882 T2630 Glyceryl tripalmitate 99.9 100.0 2883 T9517 Glyceryl trilinoleate 99.9 100.0 2884 1787 Lipid standard, mono-, di-, and triglyceride mix, certified reference material 99.9 100.0 2885 L4395 Liposome powder No response No response Open in new tab Table 7. Selectivity study for triglycerides, mixed glycerides, and liposomes Lab ID . Sigma Cat. No. . Lipid product . Fat, % . SafTest Percent Fat . AOCS Aa4–38 . 2881 17810 Triglyceride mixed 99.9 100.0 2882 T2630 Glyceryl tripalmitate 99.9 100.0 2883 T9517 Glyceryl trilinoleate 99.9 100.0 2884 1787 Lipid standard, mono-, di-, and triglyceride mix, certified reference material 99.9 100.0 2885 L4395 Liposome powder No response No response Lab ID . Sigma Cat. No. . Lipid product . Fat, % . SafTest Percent Fat . AOCS Aa4–38 . 2881 17810 Triglyceride mixed 99.9 100.0 2882 T2630 Glyceryl tripalmitate 99.9 100.0 2883 T9517 Glyceryl trilinoleate 99.9 100.0 2884 1787 Lipid standard, mono-, di-, and triglyceride mix, certified reference material 99.9 100.0 2885 L4395 Liposome powder No response No response Open in new tab Table 8. Effect of sucrose and urea on Percent Fat test results Matrix . Mass used for base, g . Spiking material . Mass spiked into base, g . Fat, % . Average fat, % . RSD fat, % . Recovery, %a . Cracker 9.5 Sucrose 0.5 31.3 32.0 2.2 96.6 31.1 31.2 32.5 Cracker 9.5 Albumin 0.5 33.3 33.1 33.1 31.1 33.1 34.8 Potato chip 9.5 Sucrose 0.5 38.4 40.3 4.5 98.3 38.0 43.8 40.2 Potato chip 9.5 Albumin 0.5 39.6 41.0 2.4 40.8 42.6 41.1 Chicken meal 9.5 Urea 0.5 12.8 12.6 2.9 100.8% 12.0 13.2 12.4 Chicken meal 9.5 Albumin 0.5 12.7 12.5 2.3 12.0 12.4 13.0 Matrix . Mass used for base, g . Spiking material . Mass spiked into base, g . Fat, % . Average fat, % . RSD fat, % . Recovery, %a . Cracker 9.5 Sucrose 0.5 31.3 32.0 2.2 96.6 31.1 31.2 32.5 Cracker 9.5 Albumin 0.5 33.3 33.1 33.1 31.1 33.1 34.8 Potato chip 9.5 Sucrose 0.5 38.4 40.3 4.5 98.3 38.0 43.8 40.2 Potato chip 9.5 Albumin 0.5 39.6 41.0 2.4 40.8 42.6 41.1 Chicken meal 9.5 Urea 0.5 12.8 12.6 2.9 100.8% 12.0 13.2 12.4 Chicken meal 9.5 Albumin 0.5 12.7 12.5 2.3 12.0 12.4 13.0 a Recovery of fat from sugar and urea spiked materials expressed as a percentage of fat from albumin spiked control materials. Open in new tab Table 8. Effect of sucrose and urea on Percent Fat test results Matrix . Mass used for base, g . Spiking material . Mass spiked into base, g . Fat, % . Average fat, % . RSD fat, % . Recovery, %a . Cracker 9.5 Sucrose 0.5 31.3 32.0 2.2 96.6 31.1 31.2 32.5 Cracker 9.5 Albumin 0.5 33.3 33.1 33.1 31.1 33.1 34.8 Potato chip 9.5 Sucrose 0.5 38.4 40.3 4.5 98.3 38.0 43.8 40.2 Potato chip 9.5 Albumin 0.5 39.6 41.0 2.4 40.8 42.6 41.1 Chicken meal 9.5 Urea 0.5 12.8 12.6 2.9 100.8% 12.0 13.2 12.4 Chicken meal 9.5 Albumin 0.5 12.7 12.5 2.3 12.0 12.4 13.0 Matrix . Mass used for base, g . Spiking material . Mass spiked into base, g . Fat, % . Average fat, % . RSD fat, % . Recovery, %a . Cracker 9.5 Sucrose 0.5 31.3 32.0 2.2 96.6 31.1 31.2 32.5 Cracker 9.5 Albumin 0.5 33.3 33.1 33.1 31.1 33.1 34.8 Potato chip 9.5 Sucrose 0.5 38.4 40.3 4.5 98.3 38.0 43.8 40.2 Potato chip 9.5 Albumin 0.5 39.6 41.0 2.4 40.8 42.6 41.1 Chicken meal 9.5 Urea 0.5 12.8 12.6 2.9 100.8% 12.0 13.2 12.4 Chicken meal 9.5 Albumin 0.5 12.7 12.5 2.3 12.0 12.4 13.0 a Recovery of fat from sugar and urea spiked materials expressed as a percentage of fat from albumin spiked control materials. Open in new tab Triglyceride Response Methodology.—To validate recoveries of a wide variety of triglycerides, a mixture of several categories of triglycerides was prepared and analyzed using the SafTest Percent Fat test (n = 10 replicates) and AOCS Standard Procedure Aa 4–38 (n = 3 replicates). Compounds were selected for this study to represent major classifications of cis- and trans-glycerides of carbon chain lengths ranging from 16 to 20 carbons with and without double bonds. Compounds represented typical triglycerides found in vegetable oils and animal fats. See Table 9 for a listing of the compounds chosen. The mixture was formulated to a theoretical fat content of 1.0% fat. Results.—A mean of 1.02 ± 0.2% fat was obtained on the 1.00% triglyceride mixture (see Table 10) resulting in a recovery of 102%. This study validates that the SafTest Percent Fat test is capable of detecting a wide range of triglycerides typically found in foods with a high degree of accuracy and precision. Table 9. Compounds for the triglyceride mixture recovery Lab ID . Constituent . CAS No. . R421500 Tripalmitin LA97189 R421510 Trimyristin LA85716 R421545 Tristearin LA97905 R475570 Triolein LA94637 R476200 Tributyrin (C4:0) LA86174 R476210 tripalmitolein (C16:1, cis-9) LA99633 R476215 Tripetroselinin (C18:1, cis-6) LA86165 R476220 Trielaidin (C18:1, trans-9) LA97164 R476225 Trilinolein (C18:2, cis-9, 12) LA86171 R476230 Trilinolenin (C18:3) LA89684 R476235 Triarachidin (C20:0) LA86176 R476240 Tri-11-Eicosenoin (C20:1) LA99225 Lab ID . Constituent . CAS No. . R421500 Tripalmitin LA97189 R421510 Trimyristin LA85716 R421545 Tristearin LA97905 R475570 Triolein LA94637 R476200 Tributyrin (C4:0) LA86174 R476210 tripalmitolein (C16:1, cis-9) LA99633 R476215 Tripetroselinin (C18:1, cis-6) LA86165 R476220 Trielaidin (C18:1, trans-9) LA97164 R476225 Trilinolein (C18:2, cis-9, 12) LA86171 R476230 Trilinolenin (C18:3) LA89684 R476235 Triarachidin (C20:0) LA86176 R476240 Tri-11-Eicosenoin (C20:1) LA99225 Open in new tab Table 9. Compounds for the triglyceride mixture recovery Lab ID . Constituent . CAS No. . R421500 Tripalmitin LA97189 R421510 Trimyristin LA85716 R421545 Tristearin LA97905 R475570 Triolein LA94637 R476200 Tributyrin (C4:0) LA86174 R476210 tripalmitolein (C16:1, cis-9) LA99633 R476215 Tripetroselinin (C18:1, cis-6) LA86165 R476220 Trielaidin (C18:1, trans-9) LA97164 R476225 Trilinolein (C18:2, cis-9, 12) LA86171 R476230 Trilinolenin (C18:3) LA89684 R476235 Triarachidin (C20:0) LA86176 R476240 Tri-11-Eicosenoin (C20:1) LA99225 Lab ID . Constituent . CAS No. . R421500 Tripalmitin LA97189 R421510 Trimyristin LA85716 R421545 Tristearin LA97905 R475570 Triolein LA94637 R476200 Tributyrin (C4:0) LA86174 R476210 tripalmitolein (C16:1, cis-9) LA99633 R476215 Tripetroselinin (C18:1, cis-6) LA86165 R476220 Trielaidin (C18:1, trans-9) LA97164 R476225 Trilinolein (C18:2, cis-9, 12) LA86171 R476230 Trilinolenin (C18:3) LA89684 R476235 Triarachidin (C20:0) LA86176 R476240 Tri-11-Eicosenoin (C20:1) LA99225 Open in new tab Table 10. Triglyceride recovery Method . n . Fat, % . Recovery, % . Theoretical . Measured mean . sr . SafTest Percent Fat 10 1.00 1.02 0.02 102 AOCS Aa 4–38 3 1.00 1.00 0.07 100 Method . n . Fat, % . Recovery, % . Theoretical . Measured mean . sr . SafTest Percent Fat 10 1.00 1.02 0.02 102 AOCS Aa 4–38 3 1.00 1.00 0.07 100 Open in new tab Table 10. Triglyceride recovery Method . n . Fat, % . Recovery, % . Theoretical . Measured mean . sr . SafTest Percent Fat 10 1.00 1.02 0.02 102 AOCS Aa 4–38 3 1.00 1.00 0.07 100 Method . n . Fat, % . Recovery, % . Theoretical . Measured mean . sr . SafTest Percent Fat 10 1.00 1.02 0.02 102 AOCS Aa 4–38 3 1.00 1.00 0.07 100 Open in new tab Spike and Recovery Methodology.—To evaluate the recovery of fat from different food matrices, spike recovery studies were performed on liquid egg white, crackers, pretzels, and chicken meals. Materials were spiked with 10% triolein [obtained from Sigma; reference materials (b)] at a volume to achieve the final desired spiked concentration. Liquid egg white was tested immediately after spiking and aged one day and retested. The egg white was tested undiluted (the fat content is very low). The crackers, pretzels, and chicken meal were dried and powdered and spiked during the initial 1:4 dilution. Chicken meals were further diluted to 1:10. Crackers and pretzels were further diluted to 1:40. See Table 11 for respective spiking concentrations. Results.—Original fat content, amount spiked, theoretical fat content, SafTest result on the spiked test portion, and percent recovery are reported in Table 11. Spike recoveries ranged from 99.8 to 108%. Table 11. Spike recovery study of triglycerides from food matrices Matrix . Fat, % . Dilution . Recovery, %b . Initial . Spike . Theoretical, with spike . Actual, SafTest Percent Fata (n = 3) . Egg white, fresh 0.003 0.034 0.037 0.040 neat 108 Egg white, aged 0.003 0.034 0.037 0.037 neat 100 Chicken meal 1 1.22 1.42 2.64 0.269 1:10 102 Chicken meal 2 1.36 1.42 2.78 0.278 1:10 100 Crackers 9.65 1.84 11.49 0.299 1:40 104 Pretzels 1.24 1.84 3.08 0.306 1:40 99.8 Matrix . Fat, % . Dilution . Recovery, %b . Initial . Spike . Theoretical, with spike . Actual, SafTest Percent Fata (n = 3) . Egg white, fresh 0.003 0.034 0.037 0.040 neat 108 Egg white, aged 0.003 0.034 0.037 0.037 neat 100 Chicken meal 1 1.22 1.42 2.64 0.269 1:10 102 Chicken meal 2 1.36 1.42 2.78 0.278 1:10 100 Crackers 9.65 1.84 11.49 0.299 1:40 104 Pretzels 1.24 1.84 3.08 0.306 1:40 99.8 a Mean of SafTest Percent Fat test results (n = 3). b Recovery SafTest Percent Fat expressed as a percentage of theoretical. Open in new tab Table 11. Spike recovery study of triglycerides from food matrices Matrix . Fat, % . Dilution . Recovery, %b . Initial . Spike . Theoretical, with spike . Actual, SafTest Percent Fata (n = 3) . Egg white, fresh 0.003 0.034 0.037 0.040 neat 108 Egg white, aged 0.003 0.034 0.037 0.037 neat 100 Chicken meal 1 1.22 1.42 2.64 0.269 1:10 102 Chicken meal 2 1.36 1.42 2.78 0.278 1:10 100 Crackers 9.65 1.84 11.49 0.299 1:40 104 Pretzels 1.24 1.84 3.08 0.306 1:40 99.8 Matrix . Fat, % . Dilution . Recovery, %b . Initial . Spike . Theoretical, with spike . Actual, SafTest Percent Fata (n = 3) . Egg white, fresh 0.003 0.034 0.037 0.040 neat 108 Egg white, aged 0.003 0.034 0.037 0.037 neat 100 Chicken meal 1 1.22 1.42 2.64 0.269 1:10 102 Chicken meal 2 1.36 1.42 2.78 0.278 1:10 100 Crackers 9.65 1.84 11.49 0.299 1:40 104 Pretzels 1.24 1.84 3.08 0.306 1:40 99.8 a Mean of SafTest Percent Fat test results (n = 3). b Recovery SafTest Percent Fat expressed as a percentage of theoretical. Open in new tab Matrix Study: Potato Chips, Crackers, and Processed Grain-Based Snack Products, Study 1 Methodology.—Each of eight snack products were ground at MP Biomedicals to reduce particle size and portioned into eight blind-coded sealed glass containers (64 total portions) for in-house testing using the SafTest. Percent Fat test. Additional portions of each of the eight products were placed into three blind-coded sealed glass jars (24 total portions) and shipped to Medallion Labs for testing with AOCS Aa 4–38 (3). Results.—For each product, the mean, SDs, and RSDrs were calculated for each method as reported in Table 12. The bias was calculated as SafTest Percent Fat test result minus AOCS Aa 4–38 result and percent recovery was calculated as percent of the AOCS Aa 4–38 test result, reported in Table 12. Recoveries compared to AOCS Aa 4–38 ranged from 94.2 to 114.9% with an average of 104.5%, meaning the AOCS Aa 4–38 results tended to be slightly lower than those using the SafTest Percent Fat test method. RSDr for the SafTest Percent Fat test ranged from 2.9 to 7.2% compared to 1.1 to 6.9% for AOCS Aa 4–38. A comparison of the SafTest Percent Fat test and the AOCS Aa 4–38 are presented graphically in Figure 4 (r2 = 0.986). Figure 4. Open in new tabDownload slide Comparisons of the SafTest percent fat test and the AOCS 4–38 test on snack products. Figure 4. Open in new tabDownload slide Comparisons of the SafTest percent fat test and the AOCS 4–38 test on snack products. Table 12. Method developer matrix study on potato chips, crackers, and other grain-based snacks study 1 Matrix . Method . n . Mean fat, % . sr . RSDr, % . Biasa . Recovery, %b . Potato chip AOCS Aa 4–38 3 33.8 0.53 1.6 −1.22 94.2 SafTest Percent Fat 8 32.85 1.58 4.9 Kettle potato chip AOCS Aa 4–38 3 21.9 1.3 5.9 1.60 107.2 SafTest Percent Fat 8 23.50 0.98 3.4 Graham cracker AOCS Aa 4–38 3 14.4 0.10 6.9 1.45 110.5 SafTest Percent Fat 8 15.85 0.56 3.5 Wheat cracker AOCS Aa 4–38 3 12.33 0.24 1.1 0.56 104.9 SafTest Percent Fat 8 12.89 0.61 4.7 Round cracker AOCS Aa 4–38 3 27.17 0.32 1.2 3.74 109.9 SafTest Percent Fat 8 30.91 0.97 3.1 Tortilla chip AOCS Aa 4–38 3 23.05 0.55 2.3 −1.82 95.4 SafTest Percent Fat 8 21.17 1.45 6.8 Baked potato chip AOCS Aa 4–38 3 9.70 0.29 3.0 1.46 114.9 SafTest Percent Fat 8 11.16 0.81 7.2 Sweet cracker AOCS Aa 4–38 3 10.93 0.12 1.1 2.47 113.7 SafTest Percent Fat 8 12.40 0.36 2.9 Matrix . Method . n . Mean fat, % . sr . RSDr, % . Biasa . Recovery, %b . Potato chip AOCS Aa 4–38 3 33.8 0.53 1.6 −1.22 94.2 SafTest Percent Fat 8 32.85 1.58 4.9 Kettle potato chip AOCS Aa 4–38 3 21.9 1.3 5.9 1.60 107.2 SafTest Percent Fat 8 23.50 0.98 3.4 Graham cracker AOCS Aa 4–38 3 14.4 0.10 6.9 1.45 110.5 SafTest Percent Fat 8 15.85 0.56 3.5 Wheat cracker AOCS Aa 4–38 3 12.33 0.24 1.1 0.56 104.9 SafTest Percent Fat 8 12.89 0.61 4.7 Round cracker AOCS Aa 4–38 3 27.17 0.32 1.2 3.74 109.9 SafTest Percent Fat 8 30.91 0.97 3.1 Tortilla chip AOCS Aa 4–38 3 23.05 0.55 2.3 −1.82 95.4 SafTest Percent Fat 8 21.17 1.45 6.8 Baked potato chip AOCS Aa 4–38 3 9.70 0.29 3.0 1.46 114.9 SafTest Percent Fat 8 11.16 0.81 7.2 Sweet cracker AOCS Aa 4–38 3 10.93 0.12 1.1 2.47 113.7 SafTest Percent Fat 8 12.40 0.36 2.9 a Bias calculated as Saftest Percent Fat test result minus AOCS Aa 4–38 result. b Recovery of the Saftest Percent Fat test expressed as a percentage of the AOCS Aa 4–38 result. Open in new tab Table 12. Method developer matrix study on potato chips, crackers, and other grain-based snacks study 1 Matrix . Method . n . Mean fat, % . sr . RSDr, % . Biasa . Recovery, %b . Potato chip AOCS Aa 4–38 3 33.8 0.53 1.6 −1.22 94.2 SafTest Percent Fat 8 32.85 1.58 4.9 Kettle potato chip AOCS Aa 4–38 3 21.9 1.3 5.9 1.60 107.2 SafTest Percent Fat 8 23.50 0.98 3.4 Graham cracker AOCS Aa 4–38 3 14.4 0.10 6.9 1.45 110.5 SafTest Percent Fat 8 15.85 0.56 3.5 Wheat cracker AOCS Aa 4–38 3 12.33 0.24 1.1 0.56 104.9 SafTest Percent Fat 8 12.89 0.61 4.7 Round cracker AOCS Aa 4–38 3 27.17 0.32 1.2 3.74 109.9 SafTest Percent Fat 8 30.91 0.97 3.1 Tortilla chip AOCS Aa 4–38 3 23.05 0.55 2.3 −1.82 95.4 SafTest Percent Fat 8 21.17 1.45 6.8 Baked potato chip AOCS Aa 4–38 3 9.70 0.29 3.0 1.46 114.9 SafTest Percent Fat 8 11.16 0.81 7.2 Sweet cracker AOCS Aa 4–38 3 10.93 0.12 1.1 2.47 113.7 SafTest Percent Fat 8 12.40 0.36 2.9 Matrix . Method . n . Mean fat, % . sr . RSDr, % . Biasa . Recovery, %b . Potato chip AOCS Aa 4–38 3 33.8 0.53 1.6 −1.22 94.2 SafTest Percent Fat 8 32.85 1.58 4.9 Kettle potato chip AOCS Aa 4–38 3 21.9 1.3 5.9 1.60 107.2 SafTest Percent Fat 8 23.50 0.98 3.4 Graham cracker AOCS Aa 4–38 3 14.4 0.10 6.9 1.45 110.5 SafTest Percent Fat 8 15.85 0.56 3.5 Wheat cracker AOCS Aa 4–38 3 12.33 0.24 1.1 0.56 104.9 SafTest Percent Fat 8 12.89 0.61 4.7 Round cracker AOCS Aa 4–38 3 27.17 0.32 1.2 3.74 109.9 SafTest Percent Fat 8 30.91 0.97 3.1 Tortilla chip AOCS Aa 4–38 3 23.05 0.55 2.3 −1.82 95.4 SafTest Percent Fat 8 21.17 1.45 6.8 Baked potato chip AOCS Aa 4–38 3 9.70 0.29 3.0 1.46 114.9 SafTest Percent Fat 8 11.16 0.81 7.2 Sweet cracker AOCS Aa 4–38 3 10.93 0.12 1.1 2.47 113.7 SafTest Percent Fat 8 12.40 0.36 2.9 a Bias calculated as Saftest Percent Fat test result minus AOCS Aa 4–38 result. b Recovery of the Saftest Percent Fat test expressed as a percentage of the AOCS Aa 4–38 result. Open in new tab Matrix Study: Potato Chip, Crackers, and Processed Grain-Based Snacks, Study 2 Methodology.—Each of six snack products were ground at MP Biomedicals to reduce particle size and portioned into eight blind-coded sealed glass containers (48 total portions) for in-house testing using the SafTest Percent Fat test. Additional portions of each of the eight products were placed into eight blind-coded sealed glass jars (48 total portions) and shipped to POS for testing with AOCS Aa 4–38 (3). The snack products included crackers, pretzels, tortillas, tortilla chips, baked potato chips, and corn chips ranging from 2 to 20% fat. Results.—For each product, the mean, SDs, and RSDs were calculated for each method as reported in Table 13. The bias was calculated as SafTest Percent Fat test result minus AOCS Aa 4–38 result and percent recovery was calculated as percent of the AOCS Aa 4-38 test result, reported in Table 13. RSDr for the SafTest Percent Fat test ranged from 2.3 to 5.6% compared to 1.7 to 21.5% for AOCS Aa 4–38. This validation demonstrates that the SafTest Percent Fat is capable of producing results at least as precise, and better, than AOCS Aa 4–38. It was observed during this study that the AOCS 4–38 (2) yielded slightly lower fat content results compared to those of Percent Fat test and recoveries ranged from 95.6 to 131.2% with an average of 116.2%. The snack products have largely triglyceride fats and the SafTest Percent Fat test optimizes the release and recovery of triglycerides. Table 13. Method developer matrix study on potato chips, crackers, and other grain-based snacks, study 2 Matrix/Lab ID . Method . n . Mean fat ,% . sr . RSD, % . Biasa . Recovery, %b . Baked chips/#111S–118S AOCS Aa 4–38 8 9.51 0.16 1.7 0.59 131.2 SafTest Percent Fat 7 13.08 0.40 3.1 Pretzels/ #121S–128S AOCS Aa 4–38 8 2.34 0.15 6.4 0.1 95.6 SafTest Percent Fat 8 2.24 0.09 4.0 Crackers/#131S–138S AOCS Aa 4–38 8 7.35 0.21 2.9 1.31 118.1 SafTest Percent Fat 8 8.66 0.33 3.8 Corn tortillas/#141S–148S AOCS Aa 4–38 8 1.81 0.26 14.4c 0.35 120.0 c SafTest Percent Fat 8 2.16 0.12 5.6 Flour tortillas/#151S–158S AOCS Aa 4–38 8 10.31 2.22 21.5c 4.21 NCd SafTest Percent Fat 8 14.52 0.50 3.4 Corn strips/#161S–168S AOCS Aa 4–38 8 16.89 0.61 3.6 3.52 120.0 SafTest Percent Fat 8 20.41 0.47 2.3 Matrix/Lab ID . Method . n . Mean fat ,% . sr . RSD, % . Biasa . Recovery, %b . Baked chips/#111S–118S AOCS Aa 4–38 8 9.51 0.16 1.7 0.59 131.2 SafTest Percent Fat 7 13.08 0.40 3.1 Pretzels/ #121S–128S AOCS Aa 4–38 8 2.34 0.15 6.4 0.1 95.6 SafTest Percent Fat 8 2.24 0.09 4.0 Crackers/#131S–138S AOCS Aa 4–38 8 7.35 0.21 2.9 1.31 118.1 SafTest Percent Fat 8 8.66 0.33 3.8 Corn tortillas/#141S–148S AOCS Aa 4–38 8 1.81 0.26 14.4c 0.35 120.0 c SafTest Percent Fat 8 2.16 0.12 5.6 Flour tortillas/#151S–158S AOCS Aa 4–38 8 10.31 2.22 21.5c 4.21 NCd SafTest Percent Fat 8 14.52 0.50 3.4 Corn strips/#161S–168S AOCS Aa 4–38 8 16.89 0.61 3.6 3.52 120.0 SafTest Percent Fat 8 20.41 0.47 2.3 a Bias calculated as SafTest Percent Fat test result minus AOCS Aa 4–38 result. b Recovery of the SafTest Percent Fat test expressed as a percentage of the AOCS Aa 4–38 result. c Reference method RSD > 20% so not included in recovery. d NC = Not calculated. Open in new tab Table 13. Method developer matrix study on potato chips, crackers, and other grain-based snacks, study 2 Matrix/Lab ID . Method . n . Mean fat ,% . sr . RSD, % . Biasa . Recovery, %b . Baked chips/#111S–118S AOCS Aa 4–38 8 9.51 0.16 1.7 0.59 131.2 SafTest Percent Fat 7 13.08 0.40 3.1 Pretzels/ #121S–128S AOCS Aa 4–38 8 2.34 0.15 6.4 0.1 95.6 SafTest Percent Fat 8 2.24 0.09 4.0 Crackers/#131S–138S AOCS Aa 4–38 8 7.35 0.21 2.9 1.31 118.1 SafTest Percent Fat 8 8.66 0.33 3.8 Corn tortillas/#141S–148S AOCS Aa 4–38 8 1.81 0.26 14.4c 0.35 120.0 c SafTest Percent Fat 8 2.16 0.12 5.6 Flour tortillas/#151S–158S AOCS Aa 4–38 8 10.31 2.22 21.5c 4.21 NCd SafTest Percent Fat 8 14.52 0.50 3.4 Corn strips/#161S–168S AOCS Aa 4–38 8 16.89 0.61 3.6 3.52 120.0 SafTest Percent Fat 8 20.41 0.47 2.3 Matrix/Lab ID . Method . n . Mean fat ,% . sr . RSD, % . Biasa . Recovery, %b . Baked chips/#111S–118S AOCS Aa 4–38 8 9.51 0.16 1.7 0.59 131.2 SafTest Percent Fat 7 13.08 0.40 3.1 Pretzels/ #121S–128S AOCS Aa 4–38 8 2.34 0.15 6.4 0.1 95.6 SafTest Percent Fat 8 2.24 0.09 4.0 Crackers/#131S–138S AOCS Aa 4–38 8 7.35 0.21 2.9 1.31 118.1 SafTest Percent Fat 8 8.66 0.33 3.8 Corn tortillas/#141S–148S AOCS Aa 4–38 8 1.81 0.26 14.4c 0.35 120.0 c SafTest Percent Fat 8 2.16 0.12 5.6 Flour tortillas/#151S–158S AOCS Aa 4–38 8 10.31 2.22 21.5c 4.21 NCd SafTest Percent Fat 8 14.52 0.50 3.4 Corn strips/#161S–168S AOCS Aa 4–38 8 16.89 0.61 3.6 3.52 120.0 SafTest Percent Fat 8 20.41 0.47 2.3 a Bias calculated as SafTest Percent Fat test result minus AOCS Aa 4–38 result. b Recovery of the SafTest Percent Fat test expressed as a percentage of the AOCS Aa 4–38 result. c Reference method RSD > 20% so not included in recovery. d NC = Not calculated. Open in new tab Matrix Study: Meat Meals, Including Fish Meals Methodology.—Each of seven meat meal products were portioned into eight blind-coded sealed glass containers (56 total portions) for in-house testing using the SafTest Percent Fat test. Additional portions of each of the eight products were placed into three blind-coded sealed glass jars (56 total portions) and shipped to POS for testing with AOCS Aa 4–38 (3). The meat meal samples included poultry meal, chicken meat and bone meal, lamb meal, and fishmeal ranging from 6 to 15% fat. Results.—For each product, the mean, SDs, and RSDs were calculated for each method as reported in Table 14. The bias was calculated as SafTest Percent Fat test result minus AOCS Aa 4–38 result and percent recovery was calculated as percent of the AOCS Aa 4–38 test result, reported in Table 14. The recoveries as percent of the AOCS Aa 4–38 results were 71.2 to 105.3% with an average of 88.8%. The means averaged 12.48% for the SafTest Percent Fat test and 13.93% for AOCS Aa 4–38, indicating that the SafTest Percent Fat test had lower recoveries of “fat”. The RSDrs ranged from to 1.4 to 3.4%, and averaged 2.6% for the SafTest Percent Fat test; and ranged from 2.5 to 21.6%, and averaged 9.7% for AOCS Aa 4–38. In these studies, the AOCS Aa 4–38 method demonstrated inherently higher RSDrs in comparison to SafTest Percent Fat test. Table 14. Method developer matrix study on meat meals, including fish meal Matrix/Lab ID . Method . n . Mean fat ,% . sr . RSD, % . Biasa . Recovery, %b . Poultry meal/#111–118M AOCS Aa 4–38 8 16.30 2.84 17.4 −1.20 92.4 SafTest Percent Fat 8 15.10 0.35 2.3 Chicken meal/ #121M–128M AOCS Aa 4–38 8 15.81 3.42 21.6 −3.79 79.2 SafTest Percent Fat 8 12.02 0.25 2.0 Pork meal/#131M–138M AOCS Aa 4–38 8 15.32 1.60 14.4 −1.85 88.5 SafTest Percent Fat 8 13.47 0.19 1.4 Chicken meal/ #141M–148M AOCS Aa 4–38 8 13.86 0.61 4.5 −0.90 93.5 SafTest Percent Fat 8 12.96 0.35 2.4 Animal meal/ #151M–158M AOCS Aa 4–38 8 13.86 0.61 4.3 −0.86 93.5 SafTest Percent Fat 8 12.92 0.44 3.4 Fish meal/#161M–168M AOCS Aa 4–38 8 9.07 0.66 6.2 −2.63 71.2 SafTest Percent Fat 8 6.47 0.24 2.8 Lamb meal/#171M–178M AOCS Aa 4–38 8 13.55 0.34 2.5 0.99 105.3 SafTest Percent Fat 8 14.44 0.39 2.7 Matrix/Lab ID . Method . n . Mean fat ,% . sr . RSD, % . Biasa . Recovery, %b . Poultry meal/#111–118M AOCS Aa 4–38 8 16.30 2.84 17.4 −1.20 92.4 SafTest Percent Fat 8 15.10 0.35 2.3 Chicken meal/ #121M–128M AOCS Aa 4–38 8 15.81 3.42 21.6 −3.79 79.2 SafTest Percent Fat 8 12.02 0.25 2.0 Pork meal/#131M–138M AOCS Aa 4–38 8 15.32 1.60 14.4 −1.85 88.5 SafTest Percent Fat 8 13.47 0.19 1.4 Chicken meal/ #141M–148M AOCS Aa 4–38 8 13.86 0.61 4.5 −0.90 93.5 SafTest Percent Fat 8 12.96 0.35 2.4 Animal meal/ #151M–158M AOCS Aa 4–38 8 13.86 0.61 4.3 −0.86 93.5 SafTest Percent Fat 8 12.92 0.44 3.4 Fish meal/#161M–168M AOCS Aa 4–38 8 9.07 0.66 6.2 −2.63 71.2 SafTest Percent Fat 8 6.47 0.24 2.8 Lamb meal/#171M–178M AOCS Aa 4–38 8 13.55 0.34 2.5 0.99 105.3 SafTest Percent Fat 8 14.44 0.39 2.7 a Bias calculated as Saftest Percent Fat test result minus AOCS Aa 4–38 result. b Recovery of the Saftest Percent Fat test expressed as a percentage. Open in new tab Table 14. Method developer matrix study on meat meals, including fish meal Matrix/Lab ID . Method . n . Mean fat ,% . sr . RSD, % . Biasa . Recovery, %b . Poultry meal/#111–118M AOCS Aa 4–38 8 16.30 2.84 17.4 −1.20 92.4 SafTest Percent Fat 8 15.10 0.35 2.3 Chicken meal/ #121M–128M AOCS Aa 4–38 8 15.81 3.42 21.6 −3.79 79.2 SafTest Percent Fat 8 12.02 0.25 2.0 Pork meal/#131M–138M AOCS Aa 4–38 8 15.32 1.60 14.4 −1.85 88.5 SafTest Percent Fat 8 13.47 0.19 1.4 Chicken meal/ #141M–148M AOCS Aa 4–38 8 13.86 0.61 4.5 −0.90 93.5 SafTest Percent Fat 8 12.96 0.35 2.4 Animal meal/ #151M–158M AOCS Aa 4–38 8 13.86 0.61 4.3 −0.86 93.5 SafTest Percent Fat 8 12.92 0.44 3.4 Fish meal/#161M–168M AOCS Aa 4–38 8 9.07 0.66 6.2 −2.63 71.2 SafTest Percent Fat 8 6.47 0.24 2.8 Lamb meal/#171M–178M AOCS Aa 4–38 8 13.55 0.34 2.5 0.99 105.3 SafTest Percent Fat 8 14.44 0.39 2.7 Matrix/Lab ID . Method . n . Mean fat ,% . sr . RSD, % . Biasa . Recovery, %b . Poultry meal/#111–118M AOCS Aa 4–38 8 16.30 2.84 17.4 −1.20 92.4 SafTest Percent Fat 8 15.10 0.35 2.3 Chicken meal/ #121M–128M AOCS Aa 4–38 8 15.81 3.42 21.6 −3.79 79.2 SafTest Percent Fat 8 12.02 0.25 2.0 Pork meal/#131M–138M AOCS Aa 4–38 8 15.32 1.60 14.4 −1.85 88.5 SafTest Percent Fat 8 13.47 0.19 1.4 Chicken meal/ #141M–148M AOCS Aa 4–38 8 13.86 0.61 4.5 −0.90 93.5 SafTest Percent Fat 8 12.96 0.35 2.4 Animal meal/ #151M–158M AOCS Aa 4–38 8 13.86 0.61 4.3 −0.86 93.5 SafTest Percent Fat 8 12.92 0.44 3.4 Fish meal/#161M–168M AOCS Aa 4–38 8 9.07 0.66 6.2 −2.63 71.2 SafTest Percent Fat 8 6.47 0.24 2.8 Lamb meal/#171M–178M AOCS Aa 4–38 8 13.55 0.34 2.5 0.99 105.3 SafTest Percent Fat 8 14.44 0.39 2.7 a Bias calculated as Saftest Percent Fat test result minus AOCS Aa 4–38 result. b Recovery of the Saftest Percent Fat test expressed as a percentage. Open in new tab Independent Laboratory Study The independent laboratory validation study was conducted by Merieux Nutrisciences Silliker Food Science Center (FSC) under the guidance of the AOAC-RI. Training Study Prior to the start of the validation study and after a training exercise, an interlaboratory comparison was made between the SafTest Percent Fat results in the independent and MP Biomedicals laboratories to qualify training effectiveness of the independent laboratory personnel by MP Biomedical personnel. Methodology.—Chicken meal, a wheat cracker, and a potato chip were analyzed in both laboratories in triplicate. Materials were provided by MP Biomedicals but prepared at each laboratory independently. Results.—Means on each material from each laboratory are reported in Table 15. Excellent agreement was observed between the results obtained in the two laboratories and training was deemed effective. It should be noted that means for potato chips of 35.5% (MP Biomedicals) and 36.1% (independent laboratory) were quite acceptable because this was a noted problem later in the matrix study. Table 15. Training study Matrix . Fat, %a . MP SafTest . Silliker . Chicken meal 9.6 9.9 Cracker wheat 28.5 29.9 Potato chip 35.5 36.1 Matrix . Fat, %a . MP SafTest . Silliker . Chicken meal 9.6 9.9 Cracker wheat 28.5 29.9 Potato chip 35.5 36.1 a Mean of three replicate results by SafTest Percent Fat test in both labs. Open in new tab Table 15. Training study Matrix . Fat, %a . MP SafTest . Silliker . Chicken meal 9.6 9.9 Cracker wheat 28.5 29.9 Potato chip 35.5 36.1 Matrix . Fat, %a . MP SafTest . Silliker . Chicken meal 9.6 9.9 Cracker wheat 28.5 29.9 Potato chip 35.5 36.1 a Mean of three replicate results by SafTest Percent Fat test in both labs. Open in new tab LOQ Determination Methodology.—The LOQ of the SafTest Percent Fat test was calculated mathematically by the relationship between the SD (s) of the calibration curve and its slope (S) using the multiplier (10) suggested by International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use as follows: LOQ = (10 × s/S). Results.—The average LOQ based on five calibration curves was determined as 0.072 g/100 g (0.072%), assuming a test portion of 1g and no dilution. Accounting for a 1:4 dilution of dry materials, this would equate to a LOQ of 0.287 g/100 g (0.287%) in a diluted material. These results are slightly higher, but close to those obtained by MP Biomedicals which are reported elsewhere in this paper. Matrix Studies Methodology.—The MP Biomedical SafTest Percent Fat test was validated against AOCS Standard Procedure Am 5–04 for fat in fried potato chips, baked corn chips, crackers, chicken meal, meat and bone meal, and against AOAC Official Method 948.16 in fish meal. Eight blind-coded portions of each material were generated for both the SafTest Percent Fat (48 portions) and the respective reference method (48 portions). Each material was prepared and tested according to the procedures described above with one exception. The exception was that all materials were ground for 60 s. This was because the procedure used for bind-coding did not identify high materials which should be ground for 30 s. Results.—The mean, RSDr percent recovery, and t-test comparing means were calculated for each product. The results are presented in Table 16. RSDrs for the SafTest Percent Fat test ranged from 2.6 to 4.9%. Similarly, RSDrs for the reference methods ranged from 1.2 to 4.7%. Recoveries (based on the reference method result) ranged from 74.8 to 100.8% with an average of 88.3%. Excluding meat and bone meal and fish meal, the recoveries ranged from 90.3 to 100.8%. Due to the differences observed between the SafTest Percent Fat test and the reference methods, t-tests were performed to compare means. The means for crackers and chicken meal were found to be not statistically different (P < 0.05) by the t-test. The mean results for corn scoops were statistically different due to tight repeatability for both methods, but not practically different, at 7.48 ± 0.20% for the SafTest Percent Fat test and 8.01 ± 0.26% for AOCS Am 5–04. The mean results for potato chips was lower by the SafTest Percent Fat test than those obtained by the AOCS Am 5–04 (P < 0.05). This appears to be an anomaly to the independent laboratory matrix study compared to other matrix studies. This could be due to (1) over grinding of this high fat material (all materials were ground for 60 s by the independent lab); (2) choice of a different reference method from other matrix studies (the reference methods are empirical, so each produces a different test result). The SafTest Percent Fat results for meat and bone meal and fish meal were lower than corresponding results by the AOCS Am 5–04 and AOAC Method 948.16 methods (<0.05), with fish meal being the most notable. For meat meal, the discrepancy could be related to the lack of a water prewash step with AOCS Am 5–04 to remove water soluble co-extractables (resulting in high bias). For fishmeal, this is most likely due to the incorporation of an acid predigestion step in AOAC Method 948.16. It is suggested that MP Biomedicals investigate an acid predigestion step for fish meal for the SafTest Percent Fat test. Table 16. Independent laboratory matrix study . . . Fat, %a . . Fat, %a . . . . Matrix . n . Parameter . SafTest Percent Fat . Reference method . Reference method . Recovery, %b . P value by t-testc . Crackers 8 Mean 26.64a AOCS Am 5–04 27.45 97 0.1275 RSDr, % 4.9 1.2 Baked corn Scoops 8 Mean 7.48a AOCS Am 5–04 8.01 93.4 0.0006 RSDr, % 2.7 3.3 Potato chips, fried 8 Mean 33.29a AOCS Am 5–04 36.87 90.3 0.0004 RSDr, % 2.6 4.7 Chicken meal 8 Mean 13.95 AOCS Am 5–04 13.85 100.8 0.6997 RSDr, % 3.9 4.1 Meat & bone meal 8 Mean 8.13a AOCS Am 5–04 9.85 82.3 4.253 × 10−7 RSDr, % 4.6 1.9 Fish meal 8 Mean 8.39a AOAC 948.16 11.22 74.8 5.378 × 10−10 RSDr, % 4.2 3.6 . . . Fat, %a . . Fat, %a . . . . Matrix . n . Parameter . SafTest Percent Fat . Reference method . Reference method . Recovery, %b . P value by t-testc . Crackers 8 Mean 26.64a AOCS Am 5–04 27.45 97 0.1275 RSDr, % 4.9 1.2 Baked corn Scoops 8 Mean 7.48a AOCS Am 5–04 8.01 93.4 0.0006 RSDr, % 2.7 3.3 Potato chips, fried 8 Mean 33.29a AOCS Am 5–04 36.87 90.3 0.0004 RSDr, % 2.6 4.7 Chicken meal 8 Mean 13.95 AOCS Am 5–04 13.85 100.8 0.6997 RSDr, % 3.9 4.1 Meat & bone meal 8 Mean 8.13a AOCS Am 5–04 9.85 82.3 4.253 × 10−7 RSDr, % 4.6 1.9 Fish meal 8 Mean 8.39a AOAC 948.16 11.22 74.8 5.378 × 10−10 RSDr, % 4.2 3.6 a All values are an average of eight independent replicates. b Recovery SafTest Percent Fat expressed as a % of reference method test result. c Result statistically different from corresponding reference results (P < 0.05). Open in new tab Table 16. Independent laboratory matrix study . . . Fat, %a . . Fat, %a . . . . Matrix . n . Parameter . SafTest Percent Fat . Reference method . Reference method . Recovery, %b . P value by t-testc . Crackers 8 Mean 26.64a AOCS Am 5–04 27.45 97 0.1275 RSDr, % 4.9 1.2 Baked corn Scoops 8 Mean 7.48a AOCS Am 5–04 8.01 93.4 0.0006 RSDr, % 2.7 3.3 Potato chips, fried 8 Mean 33.29a AOCS Am 5–04 36.87 90.3 0.0004 RSDr, % 2.6 4.7 Chicken meal 8 Mean 13.95 AOCS Am 5–04 13.85 100.8 0.6997 RSDr, % 3.9 4.1 Meat & bone meal 8 Mean 8.13a AOCS Am 5–04 9.85 82.3 4.253 × 10−7 RSDr, % 4.6 1.9 Fish meal 8 Mean 8.39a AOAC 948.16 11.22 74.8 5.378 × 10−10 RSDr, % 4.2 3.6 . . . Fat, %a . . Fat, %a . . . . Matrix . n . Parameter . SafTest Percent Fat . Reference method . Reference method . Recovery, %b . P value by t-testc . Crackers 8 Mean 26.64a AOCS Am 5–04 27.45 97 0.1275 RSDr, % 4.9 1.2 Baked corn Scoops 8 Mean 7.48a AOCS Am 5–04 8.01 93.4 0.0006 RSDr, % 2.7 3.3 Potato chips, fried 8 Mean 33.29a AOCS Am 5–04 36.87 90.3 0.0004 RSDr, % 2.6 4.7 Chicken meal 8 Mean 13.95 AOCS Am 5–04 13.85 100.8 0.6997 RSDr, % 3.9 4.1 Meat & bone meal 8 Mean 8.13a AOCS Am 5–04 9.85 82.3 4.253 × 10−7 RSDr, % 4.6 1.9 Fish meal 8 Mean 8.39a AOAC 948.16 11.22 74.8 5.378 × 10−10 RSDr, % 4.2 3.6 a All values are an average of eight independent replicates. b Recovery SafTest Percent Fat expressed as a % of reference method test result. c Result statistically different from corresponding reference results (P < 0.05). Open in new tab Discussion The practical dynamic range of the determination was established as 0.04 to 0.33 g triglyceride in 100 g of these matrices. Higher fat content levels can be evaluated with a dilution. The LOQ was determined as 0.06% for low fat lipid solutions run neat and 0.24% for most materials diluted 1:4 during solubilization. The LOQs for fish meal and crackers were slightly higher at 0.28 and 0.32%, respectively. Mean recoveries from replicate analyses of triglyceride solutions at three concentrations yielded 101 to 103%, with RSDrs below 5%. The average recoveries from all matrix studies ranged from 88.8 to 116.2% when calculated as a percent of reference method results. Reference methods were often approved because of the availability of equipment in the contract and independent laboratories and their familiarity with the methods. The choice of reference method would have a large impact on recovery. Over all matrix studies, the repeatability of the SafTest Percent Fat test was found to be in the range 1.4 to 7.2%, which is well within performance expectations for this assay and overall an improvement over the reference methods which ranged from 1.1 to 21.5%. Fatty amides, amines, alcohols, aldehydes, esters, anhydrides, and acetates with the exception of glycerol produced no interferences. Glycerol was found to be almost completely recovered and interferes with the SafTest Percent Fat test method. Solutions of varying amounts and mixtures of monoglycerides, diglycerides, and triglycerides gave recoveries ranging from 99.9 to 102% relative to AOCS Aa 4–38. Recovery of a phospholipid was 0% which is also similar to AOCS Aa 4–38, which also had no recovery of phospholipid. Limitation of the method include: Inorganic acids or bases in food matrices can interfere with the SafTest Percent Fat test by inactivating the enzymes in reagent A. Materials being tested should be in the pH range of 5 to 8. There are some applications when concentrations below the LOQ of 0.24% in solubilized food materials would be of interest. Recoveries were inconsistent for meat meals and appear to be product dependent. It is critical that the preparation of meals with saturated fat is performed at the higher temperature (46–50°C), and if the fat composition is unknown the higher temperature should be used. Low recoveries were observed for fish meals compared to the reference methods. The differences seen in the results on potato chips in the independent laboratory study compared to the earlier training study results demonstrates the importance of handling high fat matrices as recommended. The fishmeal results using SafTest Percent Fat test were lower than AOAC Method 948.16 that was specifically adapted for fishmeal. It is evident that a preparation step should be included in the Percent Fat test to digest the fishmeal adequately for membrane separation as has been done in AOAC Method 948.16 (3) and this update is in progress. Conclusions The results of the percent fat in a matrix may vary considerably between the various empirical conventional gravimetric solvent extraction methods due to differences in solvents used, matrix interference, extraction temperature, number of re-extractions performed, acid or base conditions, lipid components not recovered, and co-extractables which may be recovered along with the lipids. It is difficult to achieve complete or accurate recovery by any known method of extraction (1, 5, 6). Therefore, it is not surprising that existing methods can vary considerably in the results they produce, especially inter-laboratories where method modifications get introduced, inherently changing the results generated. In addition, solvent extraction methods are labor intensive and involve the use of toxic extracting solvents. The SafTest Percent Fat test described in this report measures fat content (0.06 to 50%) in solubilized matrices by extracting fat using stabilized isopropanol with mechanical disruption, mixing, and warming techniques. The SafTest Percent Fat test is a quick and sensitive tool for the analysis of fat content in snack products and meat meals. The method shows excellent repeatability and recoveries averaging 88.8 to 116.2% compared to the AOCS methods. An LOQ of 0.06% has been determined for a liquid lipid solution run neat and an LOQ of 0.24% for most dried food matrices solubilized with a 1:4 dilution. The method demonstrated excellent repeatability and compares well with numerous AOCS Standard Procedures. The method utilizes small reagent volumes, instrumental analysis, rapid detection times, and easy-to-use, standardized procedures. The SafTest Percent Fat test eliminates the need for large volumes of solvents, the high observed laboratory-to-laboratory variability, and relatively longer turnaround times related to solvent extraction methods. Acknowledgment Independent Laboratories Merieux NutriSciences, Siliker Food Science Center (FSC),111 East Wacker Dr., Chicago, IL 60601 Contract Laboratory for Method Developer Snack Study Medallion Labs, 4000 Plymouth Avenue, Minneapolis, MN 55427 Contract Laboratory for Method Developer Meal and Snack Study Pilot Laboratory (POS), 118 Veterinary Rd, Saskatoon, SK S7N2R4, Canada Reviewers Sneh D. Bhandari, Independent Consultant, Crete, IL. Richard (Rick) Dellaporta, Senior Principal Scientist, PepsiCo, Global Analytical, Plano, TX. Gina Clapper, Senior Scientific Liaison, Foods Chemical Codex (US Pharmacopeia), Rockville, MD. References 1 AAFCO Lab Methods and Services Committee, Crude Fat Best Practices Working Group. Association of American Feed Control Officials , 2014 . Crude Fat Methods – Considerations. From https://www.aafco.org/O/SiteContent/Laboratory/Fat_Best_Practices_Working_GroupCrude_Fat_Methods_Considerations.pdf (accessed2020) 2 Codex Alimentarius Commission ( 2015 ) Procedural Manual , 24th Ed., FAO, Rome 3 Official Methods and Recommended Practices of the AOCS ( 1996 ) 11th Ed., AOCS, Method Aa 4 – 38 4 Official Methods and Recommended Practices of the AOCS ( 1996 ) 11th Ed., AOCS, Method Am 5 – 04 5 Official Methods of Analysis ( 2016 ) 20th Ed., AOAC INTERNATIONAL, Rockville, MD, Method948.16 6 Thiex N.J. , Anderson S. , Gildemeister B. ( 2003 ) J. AOAC Int. 86 , 899 – 908 Crossref Search ADS PubMed © AOAC INTERNATIONAL 2020. All rights reserved. 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TI - Validation of the SafTest Percent Fat for the Measurement of the Fat Content of Meat Meals, Fish Meal, and Potato Chips, Crackers, and Other and Grain-Based Snack Products: AOAC Performance Tested Method  SM #082004
JF - Journal of AOAC INTERNATIONAL
DO - 10.1093/jaoacint/qsaa129
DA - 2020-09-26
UR - https://www.deepdyve.com/lp/oxford-university-press/validation-of-the-saftest-percent-fat-for-the-measurement-of-the-fat-0yV2b80Gmf
SP - 1
EP - 1
VL - Advance Article
IS - 
DP - DeepDyve
ER -