Over the past 15 years, the United States specialty tea market has quadrupled and is now worth over $6.8 billion annually.[1] Scientific research provides strong evidence to suggest that a number of health benefits can be attributed to the consumption of tea, specifically green tea. Green tea is made solely with the leaves of Camellia sinesis that have undergone minimal oxidation during processing. Rich in catechin polyphenols, particularly epigallocatechin gallate (EGCG), it is deemed to be a powerful antioxidant. Evidence suggests that those who drink green tea regularly may have lower chances of developing heart disease and certain types of cancer. It is also thought to be effective in lowering LDL cholesterol levels and inhibiting the abnormal formation of blood clots.

Though the benefits of green tea have been widely reported new research suggests that green tea crops are increasingly becoming contaminated with pesticide residues. Pesticides include any product, substance or organism used to control pests and their usage in agriculture has lead to concern over dietary exposure. Some samples of green tea have been found to contain dichlorodiphenyltrichloroethane (DDT); raising concerns over the negative impact green tea could have on the health of those who consume it regularly. DDT is one of the most well-known synthetic pesticides. It is classified as “moderately toxic” by the U.S. National Toxicology program and has been linked to a range of health problems in humans including diabetes, neurological problems and asthma.

Since 1972, the use of the pesticide DDT has been illegal in the United States. The decision to prohibit the use of DDT was taken following three years of intensive governmental inquiries into its usage. As a result of this examination, it was found that the continued use of DDT posed unacceptable risks to the environment and potential harm to human health.

With concerns regarding pesticides increasing, it is important that an accurate method of analysis is used to ascertain the levels of pesticides present in green tea crops. GC/MS analysis offers an effective solution as it is used to perform a specific test, positively identifying the actual presence of a particular substance in a given sample. In addition, GC/MS eliminates the need for separators, minimizes dead volumes, reduces the absorption effect and increases the speed of analysis.

Food and beverages contain traces of many different aromatic compounds some are naturally present in the raw material while others form during processing. To monitor and analyze the presence of such substances in food products, GC/MS is extensively employed. It can also be used to detect and measure contaminants from spoilage or adulteration that may be harmful and which are often controlled by governmental agencies, for example pesticides.

This article examines the food and beverage safety regulations in place in the United States and discusses the use of modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) extraction and ion trap GC/MSS© for the analysis of multi-residue pesticides in green tea.

United States Regulations
The United States Environmental Protection Agency (EPA) has taken a number of measures to regulate the use of pesticides in food and beverage products. The EPA sets limits on how much of a pesticide residue can be present on food and feed products, or commodities. These pesticide residue limits are known as tolerances. Inspectors from the Food and Drug Administration and the United States Department of Agriculture monitor food in interstate commerce to ensure that these limits are not exceeded.[3]

QuEChERS—A New Technique for Multi-residue Analysis of Pesticides
Recently formulated pesticides are quite different in their physical properties from their predecessors such as 4, 4’-DDT. Most of these newer pesticides are smaller in molecular weight and are designed to break down rapidly in the environment. To successfully identify and quantify these compounds in foods, more careful consideration must be placed on the sample preparation for extraction and the instrument parameters for analysis. This article covers the preparation of extracts and the optimization of the analytical parameters of the splitless injection, separation and detection.

The determination of pesticides in fruits, vegetables, grains and herbs has been simplified by a new sample preparation method, QuEChERS, published recently as AOAC Method 2007.01.[3] The sample preparation is simplified by using a single-step buffered acetonitrile (MeCN) extraction and liquid-liquid partitioning from water in the sample by salting out with sodium acetate and magnesium sulfate (MgS0₄).[3] QuEChERS can be used to prepare green tea samples for analysis by gas chromatography/tandem mass spectrometry (GC/MSS©) on the Thermo Scientific ITQ 700 GC-ion trap mass spectrometer.

An application was performed to determine the linear ranges, quantitation limits and detection limits for a partial list of pesticides that are commonly used on green tea crops, prepared in matrix using the QuEChERS sample preparation guidelines. A splitless injection of 22 pesticides was made in a single injection with detection in electron ionization (EI) MS/MS. Since the extracts were prepared in MeCN, a solvent exchange was made to hexane/acetone (9:1) prior to conventional splitless injection.[4] Once the calibration curve was constructed, multiple matrix spikes were analyzed at levels of 37.5, 75, 150, 225, 600 or 1,200 ng/g (ppb) and low level spikes of 7.5, 15, 37.5, 75 or 300 ng/g (ppb) to verify the precision and accuracy of the analytical method. These concentrations were chosen based on the requirements of various regulatory agencies.

Experimental Conditions
The sample preparation involves careful homogenization of the sample. Extraction solvents must be buffered and the powdered reagents measured at appropriate amounts for the size of the sample prepared. Some reagents cause an exothermic reaction when mixed with water, which can adversely affect the recoveries of target compounds. The recommended consumables required for sample preparation and analysis were rigorously tested. A list of the pesticides to be studied was created that would address all of the various functional groups and different physical properties of most pesticides. MSS© was optimized with the use of a variable buffer gas, the testing of the isolation efficiency and adjustment of the collision-induced dissociation (CID) voltage. A surge splitless injection was made into a Thermo Scientific TRACE TR-Pesticide III 35% diphenyl/65% dimethyl polysiloxane column, (0.25 mm × 30 meter and a film thickness of 0.25 μm with a 5-m guard column).

Sample Extraction and Clean Up
The QuEChERS sample preparation procedure consists of three parts: extraction, clean up and solvent exchange. The solvent exchange provides a final solvent that is more amenable to splitless injection and concentrates the analytes to reach lower detection limits. In addition, the solvent exchange and final clean up removed caffeine and polyphenols from the sample before injection. These compounds readily dissolve in acetonitrile. However, they are not readily soluble in hexane:acetone (9:10). This helps keep the analytical system clean.

Care must be taken to adequately and thoroughly homogenize the sample. A large amount of water must be added during the homogenization step when preparing the tea for extraction. This must be taken into consideration in the final calculations of spikes and standards. A total of 1,200 mL water was added to 200 g green tea in this experiment. An observation was made during the extraction phase of the sample preparation. If the MeCN extract was poured into the MgSO₄, poor spike recoveries were observed. This was due to an exothermic reaction of any water in the sample and the MgSO₄. Although many vendors offer the pre-measured powder reagents in a separate capped centrifuge tube, it is recommended not to add the sample to these tubes. Instead reagents from these tubes should be added directly to the sample containing the acidified MeCN. Therefore, an empty 50-mL FEP extraction tube was included in the list of consumables for sample preparation. A thoroughly homogenized 15 g sample of green tea and water were weighed into the FEP extraction tube. Then 15 mL of 1% glacial acetic acid MeCN extraction solvent was poured into the tube on top of the sample. The surrogate and the pesticide solutions were spiked into this MeCN layer for the method validation (MVD) and method detection limit (MDL) samples.

The tube was capped and vortexed for 30 seconds. The cap was removed and the powder reagents were poured slowly into the MeCN layer. The cap was tightened securely on the 50-mL extraction tube and was vortexed for 30 seconds until all of the powder reagents were mixed with the liquid layers. The tube was placed on a mechanical shaker for 5 min and then centrifuged for 5 min at 3,000 rpm. Next, 11 mL of the top MeCN layer was removed and transferred to a 15-mL clean-up tube. This tube was capped and vortexed for 30 sec and centrifuged for 5 min at 3,000 rpm. A 5-mL aliquot of the top layer was transferred into a clean test tube for solvent exchange.

Solvent Exchange
The 5-mL aliquot of cleaned-up extract was evaporated to dryness with a gentle stream of nitrogen at 40 °C in about two hours. A film formed on top of the solvent layer and samples required mixing to break the film and continue the evaporation process. Care was taken to remove the tube immediately when dried. Approximately 1 mL of the extracted compounds from the tea remained in the tubes after evaporation. A 900-μL aliquot of hexane/acetone (9:1) was added and 100 μL of the internal standard, d10-parathion, was spiked into the organic solution. The tube was capped and vortexed for 15 sec. The 1-mL extract was transferred to a 2-mL clean-up tube, capped tightly and vortexed for 30 sec. After centrifuging for 5 min at 3,000 rpm, 200 μL of the lightly colored extract was transferred to an autosampler vial with a small glass insert for injection on the ITQ 700™. The individual calibration levels were spiked into each extract for the calibration curve in matrix before the final cleanup step.

Injection
The ITQ 700 is paired with the Thermo Scientific FOCUS GC gas chromatograph, which is a single-channel GC with a standard split/splitless (SSL) injection port. The SSL inlet temperature was set to 250 °C. A 5-mm ID splitless liner with a volume of 1.6 mL was selected for the surged pressure injection. For the surge splitless injection, the inlet pressure was held at an elevated pressure of 250 kPa for the 0.5 minute injection (splitless) time. This technique reduces the vapor cloud of a 2-μL injection from 0.37 mL to 0.19 mL. At an elevated injection flow rate of 4.6 mL/min, the liner was swept several times during the injection. The target compounds moved through the inlet so rapidly that they had less time to interact with the inside walls of the liner. This minimized the amount of breakdown of the more fragile pesticides.

A Performance Solution consisting of endrin and 4,4’-DDT was analyzed as a daily check to determine system activity. The analysis of endrin, DDT and their breakdown products as part of daily quality control can alert the analyst that the system has developed active sites and maintenance is needed. Without performing a breakdown analysis the laboratory may need to continually maintain the equipment and replace consumables, even when it may not be needed. Monitoring breakdown can decrease the cost of running the analysis and save significant amounts of time.

Endrin breakdown is determined by adding up the response for the two breakdown products: endrin aldehyde and endrin ketone and dividing by the total response for the breakdown products and endrin in percent. The breakdown products of DDT are DDE and DDD and are calculated similarly. The breakdown check results showed less than 15% breakdown for both compounds on a daily basis. For routine use, the liner would be changed when the breakdown of either compound reaches more than 20%. The injection port liner tested showed very good results over a long period of time without the need for maintenance.

Separation
Chromatographic separation was achieved by using a TRACE™ TR-Pesticide III 35% diphenyl/65% dimethyl polysiloxane column (0.25 mm × 30 meter, and a film thickness of 0.25 μm with a 5-m guard column). This column was chosen to improve the resolution of the more polar compounds. Some interactions within the stationary phase showed a loss of some pesticides at concentrations below 100 pg. The oven was programmed as follows: Initial Temp: 40 °C, 1.5 min, 25 °C/min to 150 °C, 0.0 min, 5 °C/min to 200 °C, 7.5 min, 25 °C /min to 290 °C with a final hold time of 12 minutes and a constant column flow rate of 1 mL/min.

Detection
The detection of the pesticides was performed using the ITQ 700 ion trap mass spectrometer with optional MSS© mode and a variable damping gas option. The MS/MS scan mode offers significantly enhanced selectivity over scanning modes such as full scan and selected ion monitoring (SIM). The ITQ 700 operated in the MS/MS mode generates unique product ion spectra by collision induced fragmentation of each of the detected pesticides. Because of the highly effective elimination of matrix interfering ions, more accurate results are produced at the lower levels.

Results
Linearity
The calibration curve was spiked into the tea matrix. Levels ranged from 1 ng/g to 1,200 ng/g, depending on the compound and its MRL in green tea. The linearity for most compounds was R² > 0.995.

Limits of Detection and Quantitation
The actual limit of detection (LOD) and limit of quantitation (LOQ) were determined by preparing matrix spikes at a level near or below the MRL. Concentrations of 7.5, 15, 37.5, 75 or 300 ng/g were analyzed in seven matrix samples and the LOD and LOQ calculated from these results by multiplying the standard deviation by 3.143 and 10, respectively. These results exhibit that this method is able to meet or exceed the MRL requirements for most of the compounds, even at the most stringent level.

Method Validation Calculations
The method validation (MVD) calculations were performed on five matrix samples spiked at a concentration of 37.5, 75, 150, 225, 600, or 1200 ng/g. Samples had an average of 104% recovery with an average % RSD of 10.8%.

Conclusion
Since the enforcement of stringent food safety regulations in the United States, it has become increasingly important for the meticulous analytical investigation of food and beverage products including green tea. The Thermo Scientific ITQ 700 GC-ion trap MS with modified QuEChERS extraction enables research scientists to perform exhaustive analysis of pesticide residues in green tea, offering the user thorough and accurate evaluation even at low concentrations. By utilizing the instrument’s MSS© functionality users can not only identify, confirm and quantify in a single analytical run, but can do so in compliance with international regulations for the control of pesticides in beverages. ♦

For more information about the Thermo Scientific GC/MS food safety applications, please call +1 866-463-6522, e-mail analyze@thermofisher.com or visit www.thermo.com/gcfoodsafety.

References

1. www.allbusiness.com/retail-trade/food-stores/4210131-1.html.
2. www.epa.gov/history/topics/ddt/01.htm.
3. epa.gov/pesticides/food/viewtols.htm.
4. Lehotay, S. 2007. AOAC Official Method 2007.01 Pesticide Residues in Foods by Acetonitrile Extraction and Partitioning with Magnesium Sulfate. J AOAC Int 90:485-520.
5. Okihashi, M. 2005. Rapid Method for the Determination of 180 Pesticide Residues in Foods by Gas Chromatography/Mass Spectrometry and Flame Photometric Detection, J Pest Sci 304:368-377.
6. Commission Decision of August 12, 2002 Implementing Council Directive 96/23/EC Concerning the Performance of Analytical Methods and the Interpretation of Results, Official Journal of European Communities. 17.8.2002.
7. www.codexalimentarius.net/mrls/pestdes/jsp/pest_q-e.jsp.