In the European Union, extra virgin olive oils must be labeled with their geographical place of origin. The provenance of olive oil can now be verified with newly developed method involving the analysis of extracted sesquiterpene hydrocarbons via gas chromatography and mass spectrometry. The method is highly precise and at the same time inexpensive. Sesquiterpene hydrocarbons are found in many live organisms and show characteristics based on olive tree cultivars and where the trees are grown, leading to a precise olive oil origin fingerprint.
Canola oil, sunflower oil or soybean oil, colorants and low-quality olive oil, anyone? Olive oil, especially extra virgin olive oil adulteration is rampant, since the risk of getting caught is low and the profits are huge. A new expert-reviewed Laboratory Guidance Document on olive oil, published by the Botanical Adulterants Prevention Program (BAPP), lists a variety of laboratory methods at different levels of complexity, as well as the most common methods of adulteration. This Laboratory Guidance Document is an indispensable guide for regulatory and research personnel in the food, supplement and cosmetics industries.
Due to health benefits, grape seed extract has become more and more popular. Cheaper plant extracts, for example peanut skin extract, show very similar results with chromatographic methods, and therefore adulteration of grape seed extract may remain undetected. The American Botanical Council’s Botanical Adulterants Prevention Program released a laboratory guidance document that reviews analytical methods for detecting adulteration of grape seed extract with proanthocyanidin-rich extracts from other botanical sources.
Honey is defined as “the natural sweet substance produced by honey bees” from the nectar of plants. However, there is not currently an FDA standard of identity for honey in the United States, which would further define and specify the allowed methods of producing, manufacturing and labeling honey (there is, however, a nonbinding guidance document for honey). Some of the details of honey production that a standard of identity might address include allowable timing and levels of supplemental feeding of bees with sugar syrups and the appropriate use of antibiotics for disease treatment.
In circumstances where strict regulatory standards for foods are not available, they may be created by other organizations.
What Is a Food Standard?
A food standard is “a set of criteria that a food must meet if it is to be suitable for human consumption, such as source, composition, appearance, freshness, permissible additives, and maximum bacterial content.”1
To ensure quality, facilitate trade, and reduce fraud, everyone in the supply chain must have a shared expectation of what each food or ingredient should be. Public standards set those expectations and allow them to be shared. They help ensure that stakeholders have a common definition of quality and purity, as well as the test methods and specifications used to demonstrate that quality and purity. Public standards help ensure fair trade, quality and integrity in food supply chains.
How Is a Standard Different from a Method?
A method is generally an analytical technique to assess a particular property of the content or safety of a food or food ingredient. For example, methods for detection of nitrates in meat products or baby food, coliforms in nut products, or high fructose syrups in honey. Methods are an important component of food standards.
A food standard goes a step further and provides an integrated set of components to define a substance and enable verification of that substance. Standards generally include a description of the substance and its function, one or more identification tests and assays (along with acceptance criteria) to appropriately characterize the substance and ensure its quality, a description of possible impurities and limits for those impurities (if applicable), and other information as needed (see Figure 1).
Figure 1. The Anatomy of an FCC Standard (Source: Food Science Program, Food Chemicals Codex, USP)
A standard defines both what a food or food ingredient should be and documents how to demonstrate compliance with that definition.
Public Standards and Food Fraud Prevention
Many of the foods prone to fraud are those that are not simple food ingredients, but agricultural products that can be more complex to characterize and identify (such as honey, extra virgin olive oil, spices, etc.). Milk products are an example of a commodity that is prone to fraud with a wide range of adulterants (for example, fluid cow’s milk is associated with 155 adulterants in the Food Fraud Database). Ensuring the quality and purity of a product link milk requires implementation of multiple analytical techniques or the development of non-targeted methods.
The creation of effective public standards with input by a range of stakeholders will be particularly important for ensuring the quality, safety and accurate labeling of these high value commodities in the future.
A Dictionary of Food and Nutrition 2005, Oxford University Press.
The Food Chemicals Codex is a source of public standards for foods and food ingredients. It was created by the U.S. FDA and the National Institute of Medicine in 1966 and is currently published by the nonprofit organization USP. The FCC contains 1250 standards for food ingredients, which are developed by expert volunteers and posted for public comment before publication.
The Decernis Food Fraud Database is a continuously updated collection of food fraud records curated specifically to support vulnerability assessments. Information is gathered from global sources and is searchable by ingredient, adulterant, country, and hazard classification. Decernis also partners with standards bodies to provide information about fraudulent adulterants to support standards development.
Dioxins are highly toxic organic compounds that can remain in the environment for extended periods. These persistent organic pollutants (POPs), which include polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs), are mainly generated by the combustion or manufacture of chlorine-containing materials such as plastics. Dioxins and other closely related POPs, such as polychlorinated biphenyls (PCBs), are classed as carcinogenic by the United States Environmental Protection Agency, and present a significant threat to human health even at low levels.
Dioxins and PCBs can enter the food chain when livestock consume contaminated animal feed, and can accumulate in the fatty tissues of animals due to their high fat-solubility. As a result, over 90% of human exposure to dioxins and PCBs is through the consumption of meat, fish, dairy and other foods of animal origin.1 Given the health risks posed by dioxins and PCBs, effective food testing workflows are essential to ensure products do not exceed regulatory-defined safe levels.
GC-MS/MS: A Robust Technique for Analyzing Dioxins and PCBs in Food and Animal Feed
To control human exposure to PCDDs, PCDFs and PCBs from the food chain, global regulatory bodies have established maximum levels (MLs) and action levels (ALs) for various POPs in food products, as well as approved analytical methods for food testing laboratories to enforce these standards. In the European Union (EU), for example, European Commission regulations 2017/644 and 2017/771 outline sampling, sample preparation and analysis protocols for the detection of dioxins and other dioxin-like compounds in food and animal feedstuffs.2,3
With food testing laboratories tasked with handling potentially hundreds of samples every day, these workflows must be supported by robust and reliable analytical technologies that can confidently identify and accurately quantify dioxins and PCBs with minimal maintenance requirements in order to minimize downtime and maximize throughput.
Thanks to ongoing improvements in the robustness and sensitivity of gas chromatography-triple quadrupole mass spectrometry (GC-MS/MS) systems, regulations were updated in 2014 to permit this technique as an alternative to gas chromatography-high resolution mass spectrometry (GC-HRMS) for confirmatory analysis and for the control of MLs and ALs. The latest GC-MS/MS systems are capable of exceptionally reliable performance for the routine analysis of dioxins and PCBs, providing accurate and sensitive quantification of these compounds even at trace levels.
Case Study: Sensitive and Reliable Determination of Dioxins Using GC-MS/MS
The performance of modern GC-MS/MS systems was evaluated in a recent study involving the confirmatory analysis and quantification of 17 PCDDs and PCDFs, and 18 dioxin-like and non-dioxin-like PCBs in solvent standards and various food and feedstuff samples. The samples were analyzed using a triple quadrupole GC-MS/MS system equipped with the advanced electron ionization source (AEI) and a TG-Dioxin capillary GC column. Two identical GC-MS/MS systems in two separate laboratories were used to assess the reproducibility of the method.
Extraction was performed by Twisselmann hot extraction or pressurized liquid extraction. The automated clean-up of the extracts was performed using a three-column setup, comprising multi-layered acidic silica, alumina and carbon columns. Two fractions were generated per sample (the first containing non-ortho PCBs, PCDDs and PCDFs, and the second containing mono-ortho and di-ortho PCBs and indicator PCBs) and these were analyzed separately. The analytical method gave excellent separation of all the PCDD, PCDF and PCB congeners in less than 45 minutes.
Given the high sensitivity of modern GC-MS/MS instruments, a calibration-based approach was used to determine limits of quantitation (LOQs) of the analytical system. Using calibration standards at the LOQ and subsequent check standards at this level enabled the performance of the method to be assessed throughout the analytical sequence. This also allowed LOQs for the individual congeners to be determined, assuming a fixed sample weight. Individual congener LOQs could be applied to upper-bound, middle-bound and lower-bound toxicity equivalence (TEQ) results by substituting the result of any congener that fell below the lowest calibration point with this value multiplied by the toxicity equivalence factor (TEF) of the congener.
To evaluate the response factor deviation over the course of the analytical sequences, standards at the specified LOQ were analyzed at the start, during and end of each run. Using a nominal weight of 2 g, and assuming 100% 13C-labeled standard recovery and all natives were less than the LOQ in the sample, a minimum upper-bound value of 0.152 pg/g WHO-PCDD/F-TEQ was determined. This met regulatory requirements for reporting at 1/5th of the ML upper-bound sum TEQ for all food and feedstuffs with a nominal intake of 2 g, with the exception of guidance associated with liver of terrestrial animals and food for infants or young children, which both have legal limits defined on a fresh weight basis. In these cases, either a larger sample intake or a magnetic sector instrument would be required. All of the calibration sequences demonstrated response factor %RSDs within EU regulations, highlighting the suitability of the method.
To demonstrate the performance of the GC-MS/MS system, six replicate extractions of a mixed fat quality control sample (QK1) were prepared, split between the two sites and analyzed at regular intervals throughout the analytical sequences (14 injections in total). The measured WHO-PCDD/F-TEQ values for congener were in excellent agreement with the reference value provided by the EU Reference Laboratory for Halogenated POPs in Feed and Food, and the upper bound WHO-PCDD/F-TEQ value did not deviate by more than 6% from the reference value for all 14 measurements (Figure 1). The deviation between the upper-bound and lower-bound WHO-PCDD/F-TEQ for each measurement was consistently less than 1.2%, well below the maximum limit of 20% necessary to support compliance with EU regulations.
Robust Routine Analysis of Dioxin and Dioxin-like Compounds
To assess the robustness of the GC-MS/MS system, the PCDD, PCDF and non-ortho PCB extracts were pooled into a mixed matrix sample and analyzed more than 161 injection sequences across a period of approximately two weeks. Each sequence consisted of 40 matrix injections and 40 LOQ standards, interspersed with nonane blanks. No system maintenance, tuning or user intervention was undertaken throughout the two-week study. Figure 2 highlights the exceptional peak area stability achieved for selected PCDD and PCDF congeners.
These results highlight the exceptional levels of day-to-day measurement repeatability offered by the latest GC-MS/MS systems. By delivering consistently high performance without the need for extensive maintenance steps, modern GC-MS/MS systems are maximizing instrument uptime and increasing sample throughput for routine POP analysis workflows.
Developments in GC-MS/MS technology, namely the advanced electron ionization source, are pushing the limits of measurement sensitivity, repeatability and robustness to support the needs of routine dioxin and PCBs analysis in food and feed samples. By minimizing instrument downtime while maintaining exceptional levels of analytical performance, these advanced systems are helping high-throughput food testing laboratories to analyze more samples and ultimately better protect consumers from these harmful pollutants.
Malisch, R. and Kotz, A. (2014) Dioxins and PCBs in feed and food – Review from European perspective. Sci Total Environ, 491, 2-10.
European Commission. Commission Regulation (EU) 2017/644, Off J Eur Union, 2017, L92 9-34.
European Commission. Commission Regulation (EU) 2017/771, Off J Eur Union, 2017, L115 22-42.
This article is based on research by Richard Law and Cristian Cojocariu (Thermo Fisher Scientific, Runcorn, UK), Alexander Schaechtele (EU Reference Laboratory for Halogenated POPs in Feed and Food, Freiburg, Germany), Amit Gujar (Thermo Fisher Scientific, Austin, US), and Jiangtao Xing (Thermo Fisher Scientific, Beijing, China).
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