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From: Food Quality & Safety magazine, February/March 2009

Dioxin Detection Offers Protection

High-tech testing methods can ensure your meat supply is safe

by Dirk Krumwiede and Hans-Joachim Huebschmann

In recent years, several high-profile contamination crises have raised concerns over dioxin levels in a variety of food products. In December 2008, the Irish government recalled all pork products made in the Republic of Ireland after the discovery of dioxins in slaughtered pigs. Tests on some of the Irish pork products showed dioxin levels up to 200 times the recognized safety limit. This was followed by the Chinese government’s December 9th announcement banning all imports of pork from Ireland after some of the meat was found to be contaminated with elevated dioxin levels. These crises dealt a massive blow to consumer confidence in these regions; government agencies and food processors needed to source instrumentation that could accurately identify and contain the spread of dioxins in the global food supply.

Polychlorinated biphenyls (PCBs) and dioxins are suspected carcinogens. They are fat soluble, and more than 90% of human exposure to them comes though our daily food intake, specifically through the consumption of meat, fish, and dairy products.1 Because dioxins, which build up primarily in fatty tissues over time, are neither readily metabolized nor excreted, even low levels of dioxin exposure can mean accumulation to dangerous levels over time.

Dioxin’s effects on human health have been difficult to determine, due to a lack of controlled dose experiment data; however, tests on animals have demonstrated strong links between dioxins and birth defects, cancer, liver toxicity, immunosuppression, and endocrine disruption. The chemical is also believed to have a dramatic impact on the nervous, immune, and reproductive systems.

Regulations and Food Testing

The findings on the adverse health affects of dioxins led regulatory bodies such as the U.S. Food and Drug Administration (FDA), the U.S. Environmental Protection Agency (EPA), and the Association of Official Analytical Chemists to produce detailed reviews and testing guidelines on dioxins and dioxin-like substances, while the World Heath Organization charted tolerable daily, weekly, and annual dioxin intake levels. The EPA set maximum levels for dioxins in food; its directives require limits of quantitation 80% lower than the lowest reported level in EPA Method 1613 Rev. B. These new guidelines mean that much more demanding detection, selectivity, and sensitivity levels are required during the testing process in order to confirm the presence of dioxins.

The FDA developed its own dioxin strategy, aimed at obtaining data on base levels of dioxin in food and animal feed, identifying dioxin contamination sources that can be eliminated or significantly reduced, and estimating dietary dioxin exposure.3

The high-resolution gas chromatography and high-resolution mass spectrometry (HRGC/HRMS) method, which is capable of increased levels of sensitivity, selectivity, and detection, has been cited as the most effective analytical technique for this application. The EPA and many European regulatory bodies require HRGC/HRMS dioxin testing methods as standard. To demonstrate the effectiveness of HRGC/HRMS in low-level analysis of the polychlorinated dioxins, the following experiment was performed utilizing the Thermo Scientific DFS High Resolution GC/MS system.

Sample Analysis

An experiment was devised to test the capabilities of HRGC/HRMS instruments in the analysis of dirty matrix samples. Measurements were carried out on the Thermo Fisher Scientific DFS High Resolution GC/MS system coupled to a Thermo Fisher Scientific TRACE GC Ultra gas chromatograph equipped with a split/split-less injector.

Samples were injected using the company’s TriPlus Autosampler. The injection volume was 2mL of each sample measured. A TRACE TR-5MS GC column with dimensions 60m length, 0.25mm ID, and 0.1mm film thickness was used for the analysis.

Injection was performed using the “hot needle technique,” in which the empty needle is heated up in the injector for two to three seconds before injecting the sample, thus eliminating any discrimination of higher boiling congeners. The mass spectrometer was set up in the multiple ion detection (MID) mode at a resolution of 10,000 (10% valley definition).

FC43 was used as a reference compound to provide the inherent lock and calibration masses. These reference masses are monitored scan to scan to ensure the highest mass precision, stability, and ruggedness necessary for routine target compound analysis on an HRMS. For all native dioxin congeners, as well as for their specific 13C labeled internal standards, one quantification mass and one ratio mass were implemented in the MID setup.

The effective resolution is constantly monitored on the reference masses and documented in the data files for each MID window. Modifications of the MID descriptor used in this application might be necessary for different applications. For example, the EPA method 1613 standards typically do not contain the octa-furan 13C labeled internal standard. To set the boundaries of the MID retention time windows for each individual congener group, a window defining standard such as a fly ash must be used to properly set the MID time windows.

Selecting the dioxin confirmation masses requires special attention and may vary according to different analysis methods. In this setup, the ratio mass at m/z 371.82300 (52%) was used for the native hexa-furan instead of the normal furan ratio mass trace at m/z 375.81723 (81%). This was done because the FC43 reference mass at m/z 375.980170 is too close in mass to the analyte and may interfere in the signal of the analyte ion. This alternate ion selection decreases the background noise and increases the signal-to-noise value of the analyte mass trace. A similar situation can be seen for the hepta-dioxin ratio mass at m/z 425.77317. Here, the FC43 mass m/z at 425.976977 is close and could cause interferences.

In spite of these effects, FC43 offers practical advantages over perfluorokerosin (PFK) as an alternative reference compound. For dioxin analysis, FC43 provides reference masses with good intensity for all MID windows, even at reduced reference gas flows into the ion source. In addition to having a lower boiling point, FC43 contaminates the ion source less than PFK. The optimization of the electron energy on the instrument is critical in obtaining the best results. On the DFS instrument used for the demonstrated measurements, an electron energy of 48eV provided optimum sensitivity.

This parameter should be determined once for a given instrument; typical optimum values are generally found between 40 and 50eV. During the optimized procedure, the best instrument performance was achieved by autotuning the ion source on the FC43 reference mass m/z 414 with a resolution setting of 10,000.

Sample Measurements

Two types of experiments were conducted to prove instrument sensitivity, stability, and robustness. First, a sequence of 72 repeated injections measuring 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) masses of a 17fg/mL 2,3,7,8-TCDD standard (diluted from a 100fg/mL TCDD standard; Wellington Laboratories Inc., Guelph, Ontario, Canada) was performed. Next, a real-life sample series was analyzed by measuring the full set of dioxin/furan masses of a blood pool sample that contained low concentrations of dioxins/furans quantified for 2,3,7,8-TCDD at 20fg/mL.

Repeat injections were made of several analytical sequences. A method 1613 CS1 calibration standard (1:10 diluted = 50fg/mL tetras; 250fg/mL pentas to heptas; 500fg/mL octas; Cambridge Isotope Laboratories Inc., Andover, Mass.) was used to check the chromatographic separation performance of the system.

A typical separation using the GC parameters of an EPA 1613 CS1 dioxin standard at 50fg/mL of TCDD and 2,3,7,8- tetrachlorodibzofuran (TCDF). These GC parameters were also employed for the blood sample. Instrument sensitivity is demonstrated by the injection of a 20fg/mL TCDD standard. To demonstrate how well the system performs using low dioxin concentrations in dirty matrices, repeated injections were made of a challenging real-life sample, a pooled blood sample extract.

The confirmation ratios (relative areas of quantification and ratio masses) for all dioxins/furans in repeated injections of 17fg/mL were evaluated for the standard and the blood pool sample. All 2,3,7,8-TCDD results in the standard and the blood sample series gave results within the required ±15% window at the lowest detection levels and provided the confirmation.

It is clear that a HRGC/ HRMS system can test food samples, including meats, for the presence of dioxins in the low femtogram range. Even difficult sample types with heavy matrix effects can be successfully analyzed. The reliability, sensitivity, and long-term robustness of the GC/MS system were demonstrated with a series of repeated injections of a “dirty” matrix blood sample.

Using HRGC/HRMS, the system delivers confirmatory analyses that ensure compliance with worldwide regulations, making it an ideal test method to ensure that public health is safeguarded from the threat of dioxin contamination. Identifying foods that do not contain dioxins reduces the number of samples that must be analyzed using HRGC/HRMS, which significantly lowers the cost. n

Krumwiede is product specialist, trace MS, and Huebschmann is GC/MS technology manager at Thermo Fisher Scientific. Reach Huebschmann at +49-421-5493-0 or hans-joachim.huebschmann@thermofisher.com and Krumwiede at dirk.krumwiede@thermofisher.com.

References

  1. ActionPA. Dioxin homepage. ActionPA Web site. Available at: http://www.ejnet.org/dioxin/. Accessed February 3, 2009.
  2. United States Environmental Protection Agency. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD). U.S. EPA Technology Transfer Network Air Toxics Web site. Available at: http://www.epa.gov/ttn/atw/hlthef/dioxin.html. Accessed February 3, 2009.
  3. United States Food and Drug Administration. Dioxins: FDA strategy for monitoring, method development, and reducing human exposure. FDA Center for Food Safety and Applied Nutrition. Available at: http://www.cfsan.fda.gov/ ~lrd/dioxstra.html. Accessed February 3, 2009.

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