Phthalates are an emerging class of contaminants due to their widespread applications across industry. For many years, phthalates have been used in the manufacturing of food packaging, and today also can be found in many plastic kitchen tools. Recent cases of phthalate contamination in certain food products and ongoing health concerns over the consumption of phthalates have led some regulators to set new phthalate limits in food contact materials (FCMs) and foodstuffs.
To support adherence to these regulatory limits, robust and sensitive analytical methods are required. While gas chromatography-mass spectrometry (GC-MS) is widely used for this purpose, the reliable identification and quantitation of phthalates can be challenging, particularly when it comes to analyzing fatty food matrices such as cooking oils. Here, we look at a novel GC-MS workflow for phthalate testing that overcomes these challenges to enable accurate determination in foods.
Phthalates in the Food Industry
Phthalates are a family of man-made chemicals that are commonly used across a number of industries such as plasticizers to soften plastics such as polyvinylchloride (PVC). Diethylhexyl phthalate (DEHP) (Figure 1) is one of the most widely used, accounting for almost 40 percent of global phthalate consumption.
In the food and beverage industry, phthalates are often used to increase the flexibility and durability of film packaging and plastic materials. Because they are weakly bound to the polymeric matrix, however, phthalates can potentially leach out into food, especially in the presence of heat or solvents. Due to the lipophilic nature of phthalates, leaching into fatty foods is of particular concern.
Phthalates can also enter food items during processing due to the use of PVC in food production and processing systems, as well as from other environmental sources, such as indoor air dust. In some countries, phthalates are intentionally added as a clouding agent to a variety of foods and beverages, including sports drinks, fruit juice, and tea-based drinks.
In the United States and Europe, contaminated food has been identified as the main source of human exposure to phthalates. Cream-based dairy products and vegetable oils in U.S. and EU consumer markets have been found to contain high concentrations of DEHP, and consumption of these products has been linked to increases in DEHP urinary metabolite levels.
Phthalate Consumption Health Risks
Phthalates have been used as plasticizers in the food industry for more than 50 years, but only relatively recently have they been understood to pose a risk to our health. Epidemiological studies link high phthalate metabolite levels to endometriosis in women and decreased male reproductive hormones, while prenatal exposure to phthalates is associated with reduced masculinization in newborn boys. Phthalate exposure also has been linked with autism development, although this has recently been disputed. DEHP is also listed as “reasonably anticipated to be a human carcinogen” according to the U.S. National Toxicology Program.
Given these health risks, phthalate residues in foods and beverages are regulated internationally, and several expert panels, mostly in the EU and U.S., have carried out risk assessments on these compounds. For example, a special EU Food Safety Authority panel (the Food Contact Materials, Enzymes, and Processing Aids Panel) recently released an updated draft opinion stipulating a group tolerable daily intake of 50 µg/kg body weight per day for four phthalates, including DEHP. This corresponds to a limit of 0.1 percent of phthalates in FCMs.
Challenges with Detecting Phthalates in Food
Given the potential risk to human health, phthalate testing is necessary to ensure foods adhere to regulatory guidelines. Since phthalate compounds need to be detected in food at low concentrations, GC-MS is widely used to determine the phthalate content of foodstuffs due to its inherently high separation efficiency and the selectivity of quadrupole MS. This analysis method may conceal several challenges, however.
The first issue concerns the risk of sample contamination during GC-MS analysis, which affects the reliability of the resulting data. Phthalates are ubiquitous in the environment so they can easily contaminate samples during preparation and analysis, and potentially be carried over from injection to injection. The use of clean glassware, correct GC consumables, high purity standards, and solvents are crucial for producing data that analysts can have confidence in. Moreover, poorly optimized experimental conditions can cause phthalates to persist in instrument inlets, transfer lines, and ion sources, causing contamination over extended analyses and resulting in false positive results.
A second key challenge is related to the complexity of fatty matrices, such as cooking oils, which are difficult to analyze directly using GC-MS and often require extensive sample clean up procedures prior to injection. Additionally, heavier fractions, like triacylglycerols, can be difficult to elute from the chromatographic column due to their high boiling points. Given these challenges, more robust GC-MS workflows are required to ensure the reliable detection of phthalate contaminants in complex fatty foods.
Lastly, phthalates are characterized by similar molecular structures and physical properties. Many of them produce similar fragment ions and can co-elute if the chromatographic separation is not optimized. The correct choice of capillary column and MS quantification ions are important for the reliable identification of phthalates.
An Advanced GC-MS Workflow
A novel approach has recently been developed that overcomes the challenges of detecting phthalates in fatty foods. The new workflow, which makes use of the Thermo Scientific ISQ 7000 GC-MS system configured with the sensitive Advanced Electron Ionization (AEI) source, has been successfully used for the detection of 13 phthalates in vegetable oil, offering a fast, sensitive, and robust method for phthalates quantification.
To assess the linearity, limit of detection (LOD), and limit of quantification (LOQ) of the new method, vegetable oil samples were spiked with phthalates at three concentration levels (5, 25, and 50 µg/kg). The spiked vegetable oil samples were added to acetonitrile, vortexed, and sonicated before being centrifuged. The supernatant was collected and extracted to dryness, reconstituted into hexane, and subsequently analyzed for phthalates by GC-MS. To minimize the risk of contamination and to handle the high boiling nature of the analytes and the matrix, low bleed and highly inert consumables combined with optimized instrument conditions were used. The method employed polytetrafluoroethylene and siloxane vial closures, bleed-temperature-optimized inlet septa, and used optimized syringe washes, inlet, and MS temperature conditions. The results showed no heavier compound carryover, highlighting the robustness of the method.
Using timed selective ion monitoring (timed-SIM) mode enabled a significant improvement in analytical selectivity and sensitivity over full-scan acquisition (Figure 2), as only data on masses of interest were collected, rather than the full mass range. Thermo Scientific Chromeleon Chromatography Data System software was used to automatically optimize scan rate and dwell time for faster experimental setup and analysis. The system demonstrated selective and sensitive detection of phthalates in complex vegetable oil matrices.
A Better Analytical Approach for Phthalate Testing
The new timed-SIM GC-MS workflow achieved estimated LOQs ranging from 5 to 25 µg/kg, and all 13 phthalates showed excellent linear responses, with an average R²=0.999. An assessment of the recoveries of the pre- and post-spiked vegetable oil samples (across the three 5, 25, and 50 µg/kg spiking levels) returned average recovery values between 80 and 102 percent, well within the required method performance limits. These results highlight the ideal limits of detection achieved by the method, even when studying challenging food samples.
The robustness of the AEI ion source over time was demonstrated by the ion ratio stability being within ± 10 percent over 100 repeated injections of the 50 ng/mL spiked vegetable oil extract. The improved geometry of the AEI source enhanced the ionization efficiency while generating a highly focused ion beam, reducing the risk of source contamination. Thanks to the enhanced sensitivity of the AEI source, the oil extract can be further diluted before injection, or a higher split ratio can be used, maintaining sub-ppb limits of detection, giving more flexibility in sample preparation and lowering the risk of contamination to the GC flow path.
To support the strict limits for phthalate levels in FCMs and food products set by international food safety regulators, robust analytical methods for the reliable detection of phthalates in foods are required. However, the high risk of contamination, critical chromatographic separation, and low vapor pressure of phthalates and triacylglycerols make it challenging to reliably detect phthalates in complex fatty matrices. A new phthalate testing workflow, based on a timed-SIM GC-MS approach and making use of a highly sensitive ion source, shows enhanced sensitivity, selectivity, and routine grade robustness in phthalate analysis. This workflow has been shown to be helpful for laboratories providing food testing to better protect consumers against potentially harmful phthalate exposure, while maintaining adherence to regulatory limits.
Dr. Cavagnino is product marketing manager of gas chromatography, chromatography and mass spectrometry at Thermo Fisher Scientific. Reach her at firstname.lastname@example.org.