Tag Archives: GC-MS/MS

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Advances in GC-MS/MS Enhance Routine Detection of Dioxins and Dioxin-like Compounds in Food and Animal Feed

By Richard Law
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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.

pper- and lower-bound WHO-PCDD/F-TEQ values
Figure 1. Upper- and lower-bound WHO-PCDD/F-TEQ values for all 14 measurements of the QK1 mixed animal fat quality control sample, for six replicate extractions.

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.

Peak area repeatability
Figure 2. Absolute peak area repeatability over two weeks of analysis for selected PCDD and PCDF congeners in a pooled matrix sample (%RSD and amounts on column are shown for each congener).

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.

Conclusion

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.

References

  1. Malisch, R. and Kotz, A. (2014) Dioxins and PCBs in feed and food – Review from European perspective. Sci Total Environ, 491, 2-10.
  2. European Commission. Commission Regulation (EU) 2017/644, Off J Eur Union, 2017, L92 9-34.
  3. European Commission. Commission Regulation (EU) 2017/771, Off J Eur Union, 2017, L115 22-42.

Acknowledgements

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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Keeping Baby Food Safe: Sensitive Pesticide Residue Quantitation Beyond Maximum Residue Levels Using GC-MS/MS

By Paul Silcock
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There are more than 1000 different pesticides in use around the world. While these chemicals are designed to target insects, weeds and other pests, residual amounts can remain on food that is subsequently eaten by consumers. The effects of pesticides on the population can be acute or chronic depending on the exposure. Acute over-exposure can cause poisoning and result in long-term effects such as cancer or reproductive issues. Chronic, lower dose exposure to pesticides has been associated with health issues such as respiratory problems, skin conditions, depression, birth defects, cancer and neurological disorders such as Parkinson’s disease.

People who face the greatest risk for adverse health outcomes from pesticide exposure are those in agricultural roles, who are more likely to come into direct contact with these chemicals. However, developing fetuses, infants and children, as well as pregnant and nursing mothers and women of childbearing age are at increased risk of experiencing negative health effects due to the presence of unsafe levels of pesticides in food. Exposure throughout a child’s development¬–including in the womb, infancy, early childhood, and puberty–can be particularly dangerous, affecting hormone regulation and brain development.

To minimize adverse health effects, the United States Environmental Protection Agency (EPA) and the European Union (EU) impose strict regulations on the amount of pesticides that can be applied to a crop, in order to limit the residue exposure downstream. Pesticides are assigned maximum residue levels (MRLs) depending on their toxicity, with the majority typically set at 10 µg/kg. However, due to the greater risk of certain compounds affecting the healthy development of infants and young children, some pesticides are controlled further: For instance, in the EU, specific pesticides are restricted in baby foods with MRLs of between 3–8 µg/kg.

Triple Quadrupole GC-MS/MS: Meeting the Needs of Pesticide Analysis

In order to test foods for pesticide residues at these very low levels, food safety laboratories require sophisticated analyte detection technologies. Gas chromatography-tandem mass spectrometry (GC-MS/MS) is a powerful analytical technique that offers the sensitivity and selectivity required to detect and identify pesticide residues at levels that often go beyond those mandated by regulatory authorities, even in complex sample matrices such as baby food. Indeed, GC-MS/MS can detect multiple residues within samples at levels as low as 0.025 µg/kg, much lower than the MRLs of regulated pesticides.

The sensitivity of the latest triple quadrupole GC-MS/MS systems is enabling levels of detection so low that many food testing laboratories have been able to adopt more efficient and universally-applicable sample preparation procedures based on QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) methods. Combining these modern GC-MS/MS systems with QuEChERS sample preparation techniques allows food samples to be analyzed directly, significantly reducing workflow complexity. Furthermore, the specificity of triple quadrupole GC-MS/MS can easily compensate for the additional matrix components or residual acetonitrile carried over from sample preparation.

EU SANTE Criteria for Pesticide Residue Quantitation

When it comes to the detection of pesticides in baby foods, workflows must comply with rigorous quality control and method validation standards. The EU SANTE/11813/2017 criteria outline three specific requirements that pesticide residue analysis methods must satisfy to achieve compliance.

Firstly, a minimum of two product ions must be detected for each pesticide with a peak signal-to-noise ratio greater than 3 (or in case noise is absent, a signal must be present in at least five subsequent scans), and the mass resolution for precursor ion isolation must be equal to or better than unit mass resolution. Secondly, the retention time of an analyte within a sample must not differ by more than 0.1 minutes compared with standards in the same sequence. Finally, the relative ion ratio for each analyte must remain within 30% of the average of calibration standards from the same sequence.

Fortunately, modern triple quadrupole GC-MS/MS systems are ensuring food safety testing laboratories comply with these criteria. In terms of peak detection and resolution, the specificities achieved using the latest triple quadrupole instruments meet or exceed the EU SANTE requirements by providing consistent data points regardless of sample preparation approach or matrix type. Precise detection at the ultra-low concentrations required for pesticide residue quantitation is routinely achieved using modern triple quadrupole GC-MS/MS systems, with analyses offering qualitative identification of each analyte among a large group of residues. Furthermore, the latest systems deliver stable ion ratios that are well within the required 30% range at the default 10 µg/kg MRL across multiple injections.

Ultra-low-level Quantification of Pesticides Using Triple Quadrupole GC-MS/MS

In a recent study that put the capabilities of the latest triple quadrupole GC-MS/MS systems to the test, samples of baby food (carrot/potato and apple/pear/banana) spiked with a mixture of more than 200 pesticides at three concentrations (1.0, 2.5 and 10.0 μg/kg) were analyzed using the Thermo Scientific TSQ 9000 triple quadrupole GC-MS/MS system fitted with an Advanced Electron Ionization (AEI) source. Prior to injection into the instrument, the homogenized spiked samples were prepared for analysis using a QuEChERS method that included an acetonitrile extraction step, a clean-up step involving primary secondary amine (PSA) and dispersive solid phase extraction (dSPE), followed by acidification with 5% formic acid in acetonitrile.

The triple quadrupole GC-MS/MS system met all SANTE criteria at the three spiking concentrations in both food matrices. More than 97% of the target pesticide residues in the 1 μg/kg spiked sample had recoveries in the range of 70%–120%, highlighting the broad applicability of the method. The recoveries of the target pesticides from the apple/pear/banana sample spiked at 10 μg/kg are shown in Figure 1.

GC-MS/MS system, pesticide residue analysis
Figure 1. Recovery and precision data for apple/pear/banana extractions (n=6) at a concentration of 10 μg/kg, obtained using TSQ 9000 triple quadrupole GC-MS/MS system fitted with an advanced electron ionization (AEI) source.
GC-MS/MS system
(Figure 1 continued)

Triple Quadrupole GC-MS/MS: Supporting Exceptional Limits of Detection

To determine the limits of detection of the system, baby food samples prepared by the previously-described QuEChERS method were spiked with the same mixture of pesticides at 14 concentrations ranging from 0.025 to 250 μg/kg. Using the triple quadrupole GC-MS/MS system, the SANTE criteria were met for all of the pesticides targeted at the default MRL of 10 μg/kg. Additionally, more than 90% of the target compounds had a limit of identification (LOI) satisfying all SANTE requirements below 0.5 µg/kg, and more than 60% of the target residues met these criteria below 0.1 µg/kg (Figure 2).

Pesticide residue analysis
Figure 2. Number of target residues satisfying the EU SANTE requirements (carrot/potato sample matrix). IDL, instrumental detection limit; LOI, limit of identification.

Instrumental detection limits (IDLs) were also determined for each pesticide residue by performing 10 replicate injections of the lowest matrix-matched standard of carrot/potato that met all SANTE criteria. IDLs were then evaluated using one-tailed student t-tests, taking into account the concentration and absolute peak area %RSD for each compound. The evaluated IDLs ranged from approximately 5 fg (for chlorobenzilate) to 2.0 pg (for bioallethrin), with over 95% of the residues exhibiting an IDL of less than 500 fg on the column (equivalent to 0.5 µg/kg in each sample extract). These results highlight the exceptional performance of the system, offering quantitative analysis of more than 200 pesticides over up to five orders of magnitude.

Conclusion

Enforcing regulations on the amounts and types of pesticides used is essential to limit our exposure to safe levels. The latest GC-MS/MS systems are capable of detecting and identifying pesticide residues at levels far beyond those required under regulatory standards, helping food testing laboratories efficiently ensure the food our children eat is always safe to consume.