The Codex Alimentarius, introduced by the World Health Organization (WHO) and the Food and Agriculture Organization of the United Nations (FAO) in May 1963, serves the international food industry as the most significant reference point in food quality.3 In effect, the Codex Alimentarius is a collection of food standards aimed at protecting consumers, facilitating trade and ensuring fair trading practices. The standards cover of all types of food and any materials used in the production and processing of food products. Being an integral part of agricultural production, pesticides are also covered in the standards, which specify 2930 maximum residue levels (MRLs) for a total of 218 pesticides.
Further to the Codex Alimentarius, global governments may specify their own MRLs. The U.S. Environmental Protection Agency (EPA), the chief pesticide regulatory body in the U.S., sets MRLs for that may remain in or on foods sold in the country. These MRLs are established based on the potential risks to human health posed by each pesticide.4 The U.S. Department of Agriculture (USDA) enforces MRLs established for meat, poultry and some egg products, while those specified for all other foods are implemented by the U.S. Food and Drug Administration (FDA). In 1996, the U.S. EPA introduced the breakthrough Food Quality Protection Act (FQPA) mandating a single, health-based safety standard for all pesticide residues in foods.5 The regulation places a special focus on infant and children protection, accelerates approval of safer pesticides and creates incentives for the development and maintenance of effective crop protection tools. To ensure the scientific data supporting pesticide registrations is always up to date, the Act requires periodic re-evaluation of pesticide registrations and MRLs.
Since 1976, the EU has introduced several directives establishing more than 45,000 MRLs for 245 pesticides in a wide range of commodities, including cereals (Directive 86/362/EEC), foodstuffs of animal origin (Directive 86/363/EEC) and fruit, vegetables and other plant products (Directives 76/895/EEC and 90/642/EEC). During these years, Member States were allowed to set MRLs at the national level for the tens of thousands of pesticide/commodity combinations for which no official MRLs existed.6 Directive EC 396/2005, introduced on September 1, 2008, harmonizes all MRLs for pesticides within the EU Member States.
The new regulation specifies a default limit of 0.01 mg/kg for all pesticide/commodity combinations for which no MRLs have been set, unless MRLs are not required or different defaults have been set.7
These different national regulations result in considerable diversity in the level of food protection on a global basis. An international study compared MRLs for 40 pesticide/food combinations and found that the EU implements the most stringent standards, followed by Australia, the U.S. and Canada.8
Analytical challenges
A particular challenge in the detection and quantification of multiple pesticides in a single run derives from the complex elution situation. Because many compounds coelute partially or completely, the SRM transition speed must be fast enough to monitor many coeluting components while generating an adequate number of data points for qualitative and quantitative analysis in addition to reliable integration of overlapping chromatographic peaks.
Analysis should include as many substances as possible, requiring a method capable of acquiring several hundred SRM transitions in a single run. Given that small changes in retention time may occur in cases of heavy matrix samples, the method of choice should also require a minimal number of acquisition segments for multiple targets. In other words, the method must be able to measure as many transitions as possible in one segment window without any sensitivity loss.
To analyze hundreds of pesticides in one run, short dwell times and interscan times have to be used. As a consequence, a memory effect of the collision cell, called "cross talk," can occur on some triple quadrupole instruments.
Available techniques
Table 1: Selected instrument conditions for the GC, autosampler and MS. Click to enlarge |
Multi-residue pesticide analysis requires a method with the inherent capability of separating analytes from one another to facilitate individual identification and measurement. The Codex Guidelines on Good Laboratory Practice in Pesticide Residue Analysis specify mass spectrometry (MS) as the most appropriate technique for multi-residue pesticide analysis being capable of providing both quantitative and qualitative data.9 In general, MS is coupled with a chromatographic separation method to achieve simultaneous generation of retention time, ion mass/charge ratio and ion abundance data.
Liquid chromatography-mass spectrometry (LC-MS) provides good supporting evidence, but offers incomplete results since the generated spectra demonstrates little characteristic fragmentation. LC-MS is also associated with matrix effects, requiring the use of standards to overcome them. High performance liquid chromatography-mass spectrometry (HPLC-MS) is an improved liquid chromatography method. However, in combination with UV absorption, it produces similar spectra for compounds possessing similar functional groups or structures diminishing the quality of results.
Gas chromatography-mass spectrometry (GC-MS) overcomes these limitations, matching retention time and library spectra to provide unequivocal confirmation of the pesticides identified during analysis. Of all GC-MS configurations, triple quadrupole gas chromatography tandem mass spectrometry (GC-MSn) is the most suitable for multi-residue pesticide applications requiring more stringent selectivity.
Triple quadrupole GC-MSn
Triple quadrupole GC-MSn is the only method capable of removing all but the ions of interest from the mass spectrum, thereby eliminating false positives and elevated concentration values due to matrix interferences. The technique can acquire several hundred SRM transitions in a single run, allowing for rapid simultaneous analysis of multi-residue pesticides including organochlorine, organophosphorus, carbamates, pyrethroids and triazine pesticide classes. Achieving short SRM dwell times and a very short interscan time, triple quadrupole GC-MSn eliminates the cross talk mentioned above.
Being an overly powerful technique, triple quadrupole GC-MSn considerably enhances analytical productivity, sensitivity, selectivity and flexibility for multi-residue pesticide analysis, enabling more dependable results and full regulatory compliance. An experiment, detailed below, was developed to demonstrate the unique multi-residue pesticide analytical capability of triple quadrupole GC-MSn.
Experimental
Despite the large number of pesticides covered in this application, a short capillary column was used to speed up the chromatography analysis for high sample throughput. The data acquisition method included a total of 175 SRM transitions in a single 20-minute run using a Thermo Scientific TSQ Quantum GC triple quadrupole mass spectrometer. The run was divided into 11 retention time windows (acquisition segments) with up to 25 SRM transitions in each. Dwell times of 25 ms were used to obtain good sensitivity and data rate for the quantification of the targets.
The company's TRACE GC Ultra gas chromatograph was used to provide fast chromatography, while the TriPlus autosampler, configured for liquid injection, was employed for automated, productive sample introduction. A TRACE TR-5MS analytical column was used for chromatographic separation. Table 1 lists selected instrument parameters for the GC, autosampler and MS.
To streamline the data review and reporting process for multiple pesticides analysis, Thermo Scientific QuanLab Forms 2.5 software provided productivity enhancements tailored to the workflow demands of the pesticides analytical laboratory. The software includes an integrated user interface for the management of the total list of pesticides, automated data review, customizable views and versatile reporting formats.
Results and discussion
Figure 2: Cross talk is absent in the fast SRM acquisition sequence from Monocrotophos (m/z 127 109) at 8.09 min (bottom trace) to Fonofos (m/z 137 109) (upper trace) even at high concentration. Click to enlarge |
A complete chromatogram demonstrating the complexity of the elution profile of 170 pesticides at 50 pg/µL is shown in Figure 1, with the last peak eluted at 20 minutes. The pesticides covered multiple compound classes, including organochlorine, organophosphorus, carbamates, pyrethroids and triazine, which were analyzed using 175 corresponding SRM transitions.
The SRM acquisition segments were primarily chosen to ensure that each window provided a comfortable setup of the compound list, with each component having a transition dwell time of 25 ms. With a total of 25 SRM transitions for 27 pesticides in the segment window from 5.97 to 8.27 min, the dwell and interscan times were very short, requiring a fast scanning sequence with active cleaning of the collision cell before acquiring the next data set.
Cross talk was not observed, even in the window containing 25 SRM transitions. Zero cross talk was demonstrated by acquiring two different SRM transitions of Monocrotophos (m/z 127 109) and Fonofos (m/z 137 109), which yielded the same product ion of m/z 109 in the same segment window (Figure 2). A similar situation appeared in the case of BHC (m/z 219 183) and pyrimethanil (m/z 198 183). Triple quadrupole GC-MSn showed the clear absence of cross talk, providing absolute certainty in the data with no false positive results.
The short, 15 m column length of the triple quadrupole GC-MSn method contributed to the fast run time and improved sample throughput. Overall, considerable improvement in analytical productivity was observed in terms of increased sample throughput, as well as through reduction in the time required to review and report for multiple pesticides analysis.
Conclusion
Triple quadrupole GC-MSn has emerged as the technique of choice for multi-residue pesticide analysis, facilitating effective control at the international MRL levels and helping to safeguard the health of consumers worldwide. The method provides high productivity, increased sample throughput and excellent sensitivity, selectivity and flexibility. False positive results from cross talk are eliminated, even under fast acquisition cycles.
Weiguo Zhang, Lin Lu is based in Shanghai, China, while Hans-Joachim Huebschmann is based in Bremen, Germany. Both work for Thermo Fisher Scientific.
References
1. Environmental Protection Agency (EPA) Web site; Pesticides, Health and Safety, Pesticides and Food, Why Children May Be Especially Sensitive to Pesticides: http://www.epa.gov/opp00001/food/pest.htm
2. Steady Health Web site; Pesticides – how dangerous is our nutrition?: http://www.steadyhealth.com/articles/Pesticides__how_dangerous_is_our_nutrition__a99_f0.html
3. Codex Alimentarius Web site; About Codex, Understanding Codex: ftp://ftp.fao.org/codex/Publications/understanding/Understanding_EN.pdf
4. Environmental Protection Agency (EPA) Web site; Laws and Regulations, Regulatory Information by Business Sector, Pesticides, Regulating Pesticides, Pesticide Tolerances: http://www.epa.gov/pesticides/regulating/tolerances.htm
5. Environmental Protection Agency (EPA) Web site; Laws and Regulations, Regulatory Information by Business Sector, Pesticides, Regulating Pesticides, Laws and Regulations, The Food Quality Protection Act (FQPA) of 1996, The Food Quality Protection Act (FQPA) Background" http://www.epa.gov/pesticides/regulating/laws/fqpa/backgrnd.htm
6. Europa, European Commission, Directorate General for Health and Consumers Web site; A-Z, Food Safety, Plant Protection, Pesticide Residues, Regulation EC No 396/2005: http://ec.europa.eu/food/plant/protection/pesticides/regulation_ec_396_2005_en.htm
7. Food Standards Agency, Safety and Hygiene, Chemical Safety, Pesticides, How will EC regulation 396/2005 affect pesticide regulation in the UK?: http://www.food.gov.uk/safereating/chemsafe/pesticides/pesticidesmainqa/ecregulation
8. Boyd, David R. The Food We Eat: An International Comparison of Pesticide Regulations, Healthy Environment, Healthy Canadians Series, A Report prepared for the David Suzuki Foundation, October 2006, http://www.polisproject.org/PDFs/Food%20we%20eat.pdf
9. Codex Alimentarius Web site, Official Standards, Official Codex Standards, List, Analysis of Pesticide Residues, Guidelines on Good Laboratory Practice in Pesticide Residue Analysis, CAC/GL 40-1993, Rev.1-2003: http://www.codexalimentarius.net/web/standard_list.do?lang=en