Five new AOCS methods

By Laura Cassiday

In This Section

June 2017

Method Book

  • The Official Methods and Recommended Practices of the AOCS, 7th edition, released in March 2017, adds five new analytical methods to more than 450 existing methods.
  • The new methods include three Official Methods, one Recommended Practice, and one Standard Procedure.
  • The new methods, which vary widely in topic, fill critical analytical needs for the fats and oils community.

The year 2016 witnessed Britain’s exit from the European Union, businessman Donald Trump’s election as US President, the announcement of several corporate mega-mergers—and the approval of five new AOCS methods. Although perhaps not as momentous to the world-at-large as the other events, the new AOCS methods fill critical gaps in the analysis of edible oils and oilseeds. Since the 1920s, AOCS has identified and validated analytical methods crucial for the processing, trade, utilization, and evaluation of fats, oils, lipids, and related products. The new methods join more than 450 other AOCS Official Methods, Recommended Practices, and Standard Procedures in the Official Methods and Recommended Practices of the AOCS, 7th edition, released in March of this year (

Becoming official

To join their esteemed colleagues in in the Official Methods and Recommended Practices of the AOCS, 7th edition, the five newest methods followed a well-established procedure. The process begins with the submission of a proposal. “Anybody can propose a new method, even the AOCS itself,” says Richard Cantrill, chief science officer at AOCS. “We identify methods mainly from industry need. We also pick them up from journal articles, or collect them in harmonization activities with other organizations.” According to Cantrill, the proposer of a new method is asked to make a presentation in one of the methods sessions at the AOCS Annual Meeting and Industry Showcases. “It’s a bit gladiatorial,” says Cantrill. “The audience basically gives thumbs up or thumbs down, or asks for more detail. It can be a bit unnerving, so anyone making a presentation has to have enough data to show that they’ve actually worked the method out.”

The proposal is then submitted to an expert panel or a subcommittee of the AOCS Uniform Methods Committee, which decides whether the method has been sufficiently validated to warrant a collaborative study. International guidelines require collaborative studies to include a minimum of eight expert laboratories, preferably domestic and international. The Uniform Methods Committee and AOCS staff select labs that have expertise in method development and in the particular topic to participate in a collaborative study.

Samples that have been sourced and prepared to ensure homogeneity are then delivered to participating labs, with a deadline for returning results. After receiving data from all labs involved in the collaborative study, AOCS staff run a statistical analysis on the data. For each sample, an overall mean of laboratory values, repeatability standard deviation [s(r)], reproducibility standard deviation [s(R)], and other parameters are calculated. Repeatability refers to the ability of the same lab to obtain similar values for replicate samples using the same instrument, under the same conditions, during a short period of time. In contrast, reproducibility compares results for the same samples between labs.

“Repeatability tells you how well you’re doing within your lab, while reproducibility tells you how well a whole cohort of labs is doing when they perform that method,” says Cantrill. “Generally the reproducibility standard deviation is larger than the repeatability standard deviation.” The repeatability relative standard deviation [RSD(r)] and reproducibility relative standard deviation [RSD(R)] express the standard deviation as a percentage of the mean value. “You’re looking for a 1–2% spread, but as you get down toward the limit of detection, you might end up with a 30% spread,” says Cantrill. And finally, the repeatability value (r) and the reproducibility value (R) reflect the 99% confidence interval for the data.

The data and statistical analysis are then presented to the Uniform Methods Committee, which can reject the method, or approve it by a two-thirds majority vote. If the method is rejected, the committee may make recommendations to change parts of the method and conduct another collaborative study. According to Cantrill, the data evaluation is left to the committee’s discretion, with no specified cutoff values for reproducibility or repeatability. “If the method is loosely written and not very easy to follow, you usually get really bizarre results back, so it’s sort of self-limiting,” he says.

After a method is officially adopted, it is named according to AOCS conventions and included in the Official Methods and Recommended Practices of the AOCS. There are three types of AOCS methods: Official Methods, Recommended Practices, and Standard Procedures. An Official Method has been validated and approved by the process described above. A Standard Procedure is a method that relies on a specific apparatus in accordance with the manufacturer’s instructions. Unlike Official Methods, Standard Procedures can be vendor-specific. Standard Procedures are validated and approved by the same procedure as that for Official Methods. On the other hand, Recommended Practices are methods that may be of interest or value, but they do not have enough validation data to qualify as an Official Method. A Recommended Practice may or may not have been subjected to a collaborative study. In some cases, a collaborative study may reveal data variation that is unacceptable for an Official Method, but the method may still be of value for simple, rapid, or qualitative analyses.

What’s in a name?

Have you ever wondered how an AOCS method gets its name (Ac 6-16, Cd 39-15, etc.)?

  • The capital letter refers to the section of the Official Methods and Recommended Practices of the AOCS in which the method appears:
    Vegetable oil source materials (Section A)
    Oilseed by-products (Section B)
    Commercial fats and oils (Section C)
    Soap and Synthetic Detergents (Section D)
    Glycerin (Section E)
    Sulfonated and Sulfated Oils (Section F)
    Soap stocks (Section G)
    Specifications for Reagents, and Solvents and Apparatus (Section H) Lecithin (Section J)
    Evaluation and Design of Test Methods (Section M)
    Analytical Guidelines for Testing Industrial Oils and Derivatives (Section S)
    Test Methods for Industrial Oils and Derivatives (Section T)
  • The lower-case letter designates a group of related methodologies within a section. For example, “Ab” methods all involve the analysis of peanuts as vegetable oil source materials.
  • The first number (before the dash) refers to the number of the method within the group of related methodologies. For example, Ab 1, Ab 2, Ab 3, etc.
  • The second number (after the dash) indicates the year the method was first published. For example, Ac 6-16 was published in 2016, whereas Aa 1-38 was introduced in 1938.

To order the Official Methods and Recommended Practices of the AOCS, 7th Edition, or to purchase individual methods, visit the Methods section of the AOCS website (

Official Method Ac 6-16: Extraction and indirect enzyme-linked-lectin-assay (ELLA) analysis of soybean agglutinin in soybean grain

Soybean agglutinin (SBA) is a carbohydrate-binding protein, or lectin, that decreases the growth rate of monogastric animals, such as chickens and swine, that consume raw soybean seeds. “Soybean agglutinin is considered an anti-nutrient, and it is measured in all soybean biotech products as part of the safety assessment for regulatory approvals,” says Elisa Leyva-Guerrero, a plant biochemist at Monsanto (St. Louis, MO, USA) who helped develop Official Method Ac 6-16. Heat from cooking or other processing destroys most of the SBA in raw soybeans.

Since the 1950s, scientists have quantified SBA with a hemagglutination technique that requires rabbit red blood cells. As a lectin, SBA can bind to polysaccharides on the surfaces of red blood cells, causing the cells to clump together, or agglutinate. However, the hemagglutination method is costly, time-consuming, not very accurate, and has arbitrary units (hemagglutinating units), says Leyva-Guerrero.

The new method Ac 6-16 uses an enzyme-linked lectin assay (ELLA) to quantify SBA. The technique is analogous to the well-known enzyme-linked immunosorbent assay (ELISA), but uses carbohydrates to capture and detect SBA, rather than antibodies. Specifically, the carbohydrate N-acetylgalactosamine (GalNAc) is linked to polyacrylamide (PAA) to immobilize the carbohydrate within the wells of a microtiter plate. When soybean extract is added to a well, SBA in the extract binds to the immobilized carbohydrate. Then, a biotinylated version of GalNAc-PAA (GalNAc-PAA-Biotin) is added, which also binds to SBA, forming a “sandwich.” Washing steps remove any unbound GalNAc-PAA-Biotin. To detect the immobilized GalNAc-PAA-Biotin (and associated SBA), researchers add a conjugate of neutravidin (a protein that binds specifically to biotin) and horseradish peroxidase (HRP). Upon addition of 3,3′,5,5′-tetramethylbenzidine (TMB), a chromogenic substrate for HRP, a yellow pigment is produced that absorbs light at 450 nm. When the microtiter plate is placed in a plate reader, the absorbance at 450 nm of multiple samples and standards can be measured simultaneously. By comparison to a standard curve of known SBA values, the researchers can calculate the mg of SBA per g of soybean tissue.

With a 2-hour incubation procedure, ELLA exhibits sensitivity in the μg/mL range—more than sufficient to detect SBA in soybean, which is expressed in the mg/mL range (Breeze, M. L., et al.,, 2015). The new method demonstrates a linear response to purified SBA over one order of magnitude. Another advantage is that all reagents required for the method, including GalNAc-PAA and GalNAc-PAA-Biotin) are commercially available. Leyva-Guerrero and her colleagues used the validated ELLA method to quantify SBA in nine commercial soybean varieties introduced between 1972 and 2008 (Breeze, M. L., et al.,, 2015). The study revealed that the concentration of SBA ranged from 2.03 to 2.92 mg/g and varied with soybean genotype and environment.

“The ELLA method can measure SBA with increased accuracy, non-arbitrary units (mg lectin/g seed), and decreased cost and time in comparison to the hemagglutination method,” says Leyva-Guerrero. She notes that Official Method Ac 6-16 was developed and validated with the support of the Analytical Excellence through Industry Collaboration (AEIC; Composition Working Group, whose members represent major agricultural biotech companies and contract research organizations.

Official Method Cd 30-15: Analysis of 2- and 3-MCPD fatty acid esters and glycidyl esters in oil-based emulsions

Monochloropropane-1,2-diol (MCPD) esters and glycidyl esters are process contaminants formed during the high-temperature deodorization step of edible oil refining (Cassiday, L., Inform 27, 6–11, 2016) (Fig. 1). In recent years, these contaminants have come under increased scrutiny by food safety organizations such as Germany’s Federal Institute for Risk Assessment (BfR) and the European Food Safety Authority (EFSA) after free MCPD and glycidol were linked to cancer, infertility, and other health problems in animal studies. 2- and 3-MCPD esters and glycidyl esters have been detected in refined oils (mainly palm), as well as in oil-containing foods such as bread, margarine, French fries, baby food, and infant formula.

Figure 1

FIG. 1. Chemical structures of a) 3-MCPD monoesters, b) 3-MCPD diesters, and c) glycidyl esters.

Official Method Cd 30-15 joins three other AOCS Official Methods for the simultaneous analysis of 2- and 3-MCPD esters and glycidyl esters (Cd 29a-13, Cd 29b-13, and Cd 29c-13). “The original methods were only for fats and oils¬, mainly vegetable oils,” says Cantrill. “Cd 30-15 is AOCS’s first method for analyzing 2- and 3-MCPD esters and glycidyl esters in fatty foods.” Cd 30-15 is an extraction procedure for isolating 2- and 3-MCPD and glycidyl esters from oil-based emulsions such as spreads, margarines, dressings, and mayonnaise. After extraction, any of the previously published methods (Cd 29a-13, Cd 29b-13, or Cd 29c-13) may be used to quantify these process contaminants.

In Cd 30-15, the food sample is first homogenized in a solvent consisting of heptane and methyl tert-butyl ether, and then incubated in an ultrasonic bath. Next, a liquid-liquid extraction is performed to purify lipophilic substances in the sample. After evaporation of organic solvents, the lipophilic substances (including 2- and 3-MCPD and glycidyl esters) can be analyzed by one of the previously published AOCS Official Methods. Although the methods differ in experimental details, they all involve the chemical cleavage of esters from 2- and 3-MCPD and measurement of the free MCPD by gas chromatography/mass spectrometry (GC/MS). Two of the methods (Cd 29a-13 and Cd 29b-13) convert glycidyl esters to 3-monobromopropanediol (3-MBPD) prior to GC/MS analysis. The third (Cd 29c-13) involves converting 2- and 3-MCPD and glycidyl esters to 3-MCPD in the presence and absence of chloride (glycidol cannot form 3-MCPD without chloride).

The extraction procedure showed good recovery of 2- and 3-MCPD and glycidyl esters and high sensitivity (limit of detection, 0.04 and 0.05 mg/kg for MCPD and glycidyl esters, respectively), in addition to satisfactory repeatability and reproducibility (Ermacora, A., and Hrnčiřík, K.,, 2014).

Official Method Ce 12-16: Sterols and stanols in foods and dietary supplements containing added phytosterols

Plant sterols and stanols, collectively known as phytosterols, are cholesterol-like molecules in plants that have been shown to reduce serum total and low-density lipoprotein (LDL) cholesterol levels in humans who consume them. Because of their potential to reduce the risk of cardiovascular disease, phytosterols are added to many foods such as margarines and other spreads, salad dressings, and snack bars, as well as dietary supplements. The US Food and Drug Administration (FDA) allows food and supplement manufacturers to make health claims on the relationship between phytosterols and a reduced risk of coronary heart disease, provided that the products contain specified amounts of the five major phytosterols that have shown beneficial effects (campesterol, campestanol, stigmasterol, β-sitosterol, and sitostanol; Fig. 2).

Figure 2

FIG. 2. Chemical structures of the five phytosterols for which beneficial health effects have been reported. Reprinted from Srigley, C. T., and Haile, E. A. (2015) "Quantification of plant sterols/stanols in foods and dietary supplements containing added phytosterols." J. Food Comp. Anal. 40, 163-176, with permission from Elsevier.

According to Cantrill, Official Method Ce 12-16 arose from a collaboration between Cargill and the FDA. “Sterols and stanols are commonly included in margarines and dietary supplements, so Official Method Ce 12-16 is a test to find out whether they have the correct amounts of sterols and stanols as claimed on the label,” he says. Previous methods for phytosterol analysis were limited in various aspects, such as a lack of validation for stanol quantification, limited range or accuracy, or unsuitability for the analysis of dietary supplements (Srigley, C. T., and Haile, E. A,, 2015).

Method Ce 12-16 can determine total free sterols/stanols and total steryl/stanol esters, as well as quantify each of the five major phytosterols that are the subject of the FDA’s health claim. Ce 12-16 provides three protocols for extracting phytosterols from different matrices (sterol/stanol concentrates, steryl/stanol ester concentrates, foods, and dietary supplements). Each protocol derivatizes the phytosterols to trimethylsilyl (TMS) ethers so that they may be separated on a capillary GC column, detected by a flame ionization detector (FID), and identified by their retention times. The method uses eicoprostanol as an internal standard. Using the method, researchers determined that 25 analyzed samples, including spreads, beverages, baked goods, and dietary chews, had total phytosterol contents that varied from 0.2 to 55.2 g/100 g (Srigley, C. T., and Haile, E. A,, 2015). Total phytosterol contents ranged from 83% to 137% of the amounts declared on labels. The limit of detection (LOD) and limit of quantitation (LOQ) for an individual phytosterol were 0.3 mg/100 g and 1 mg/ 100 g, respectively.

Recommended Practice Ce 13-16: Determination of cyclopropenoic and nutritional fatty acids in cottonseed and cottonseed oil by gas chromatography

Cotton plants of the genus Gossypium are cultivated primarily for their textile fibers. “However, cotton products are also consumed by humans and animals,” says Barb Mitchell, staff scientist at Covance Labs, Inc. (Madison, Wisconsin, USA), who helped develop Recommended Practice Ce 13-16. Cottonseed oil has been used as a cooking oil and in foods such as mayonnaise and salad dressing, and cottonseed meal is included in animal feed. “As new cottonseed varieties are developed through biotechnology, it is necessary to assess their safety,” says Mitchell.

According to Mitchell, the Organization for Economic Co-operation and Development (OECD) recommends the analysis of fatty acid profiles in cottonseed—both nutritional fatty acids and the anti-nutritive cyclopropenoid fatty acids. “Cyclopropenoid fatty acids in cottonseed include malvalic acid, sterculic acid, and dihydrosterculic acid, which have been shown to have unfavorable health effects in livestock,” says Mitchell. Most cyclopropenoid fatty acids are removed during the deodorization step of oil refining, but the fatty acids can be a problem for producers of cold-pressed cottonseed oil.

Historically, the determination of nutritional fatty acids and of cyclopropenoid fatty acids required two separate analyses. Recommended Practice Ce 13-16 combines the two analyses into a single GC procedure. First, the triacylglycerols from cottonseed or cottonseed oil are converted to fatty acid methyl esters by base transesterification using sodium methoxide. Then, the individual esters are analyzed by GC using a polyethylene glycol stationary phase with an FID.

“The biggest challenge was to adjust the GC conditions to get the best resolution between both malvalic and stearic acids and dihydrosterculic and α-linolenic acids,” says Mitchell. “What worked better for one pair was worse for the other pair.” However, the researchers eventually determined GC conditions that allowed an adequate separation of all compounds. The LOQ for various nutritional and cyclopropenoid fatty acids ranged from 0.001 to 0.012 mg/mL (Mitchell, B., et al.,, 2015).

Standard Procedure Cd 12c-16: Accelerated oxidation test for the determination of the oxidation stability of foods, oils, and fats using the Oxitest Oxidation Test Reactor

Lipid oxidation is a major factor that limits the shelf life of foods containing fats and oils (Cassiday, L., I, 406–411, 2015). Various methods exist for assessing the rate of lipid oxidation in foods. However, these techniques require the fat to be extracted from food samples before oxidation tests can be performed. In contrast, the Oxitest instrument (VELP Scientifica; Usmate, Italy) can analyze fat oxidation in whole food samples, providing a simpler and more rapid method (Fig. 3).

Figure 3

FIG. 3. Sample loading in the Oxitest chambers

Standard Procedure Cd 12c-16 details how to use the Oxitest Oxidation Test Reactor to analyze the oxidative stability of whole food samples. Two samples can be analyzed simultaneously on the same instrument. A food sample, which can be liquid, solid, or doughy, is placed in one of two oxidation chambers, where it is subjected to accelerated oxidation conditions of high temperature (up to 110 ºC) and high oxygen pressure (up to 8 bar). In this way, lipid oxidation can be observed over a shortened time period (hours) compared with the days, weeks, or months required for the food to naturally become rancid. By monitoring changes in absolute pressure within the chamber, the Oxitest instrument measures the oxygen uptake of reactive components in the food. The instrument generates a value called the Induction Period (IP), which refers to the time required for a sample to show a sudden increase in the rate of oxidation. The longer the IP, the more resistant the sample is to oxidation. Cd 12c-16 can be used for a wide range of sample types with at least 2–4% fat content, including meat, oils, mayonnaise, and baked goods.

Researchers used the Oxitest method to analyze the oxidative stability of several extra virgin olive oils that came from two regions of Italy (Caruso, M. C., et al., I, 26–29, 2017). They found a strong correlation between the total content of polyphenols (which are natural antioxidants) in the olive oil and oxidation stability, as measured by IP. The IP values for all of the investigated oils ranged from 20 to 78 hours. The data did not indicate a direct correlation between geographical origin of the olive oil and IP value.

Although the five new AOCS methods may not have made the top news headlines of 2016, they will certainly be appreciated by members of the fats and oils community. The availability of reliable, accurate, validated methods will simplify and accelerate research on fats, oils, and the foods that contain them.

Laura Cassiday is an associate editor of INFORM at AOCS. She can be contacted at


  • Breeze, M.L., “Validation of a method for quantitation of soybean lectin in commercial varieties,” J. Am. Oil Chem. Soc. 92: 1085–1092, 2015,
  • Caruso, M.C., et al., “Accelerated shelf life studies of extra virgin olive oils using the Oxitest method,” Inform 28: 26–29, January 2017.
  • Cassiday, L., “Minimizing process contaminants in edible oils.” Inform 27, 6–11, March 2016.
  • Ermacora, A. and K. Hrnčiřík, “Development of an analytical method for the simultaneous analysis of MCPD esters and glycidyl esters in oil-based foodstuffs,” Food Addit. Contam. Part A 31: 985–994, 2014,
  • Mitchell, B., et al., “Determination of nutritional and cyclopropenoid fatty acids in cottonseed by a single GC analysis,” J. Am. Oil Chem. Soc. 92: 947–956, 2015,
  • Srigley, C.T. and E.A. Haile, “Quantification of plant sterols/stanols in foods and dietary supplements containing added phytosterols,” J. Food Comp. Anal. 40: 163–176, 2015,