SpeedExtractor E-916 Extraction of Spices using the SpeedExtractor E-916 for the determination of Pesticides

Transcription

1 064/2011 SpeedExtractor E-916 Extraction of Spices using the SpeedExtractor E-916 for the determination of Pesticides

2 064/2011 SpeedExtractor E-916 SHORT NOTE Extraction of Spices using the SpeedExtractor E-916 for the determination of Pesticides This application note describes a fast and reliable way to extract organochlorine and other pesticides in chilli and paprika spice samples with the SpeedExtractor E-916. After the extraction the extract was cleaned up via GPC and analyzed by GC-MS/MS. Introduction Figure 1: 4,4 -DDT The adverse health effects depend on the type of pesticide. Organophosphates and carbamates for example affect the nervous system. Others may irritate the skin or eyes. Some pesticides may be carcinogens. Others may affect the hormone or endocrine system in the body [2]. Experimental Instrumentation: SpeedExtractor E-916 with 20 ml cells, GPC Gilson 233XL, Varian 3800 with MS-1200 QQQ Samples: paprika and chilli powder with incurred pesticide content. 3 g sample was weighed in a beaker and 6 ml of water was added. The mixture was placed in the refrigerator over night. The next day 3 g of diatomaceous earth was added and mixed in well. The sample was transferred into a 20 ml extraction cell. A fourfold extraction was carried out using the parameters shown in Table 1. A blank and a spiked sample were extracted as well (QC test). After the extraction a GPC clean-up was performed and the pesticide content was analysed by GC-MS/MS. For comparison the samples were also extracted with a Dionex ASE 200. Table 1: Extraction method for pesticides using the SpeedExtractor E-916 Temperature 75 C Pressure 0 bar Solvent Cyclohexane 50%, Ethyl acetate 50% Cells Vials Cycles 3 Heat-up Hold Discharge Flush with solvent Flush with gas 20 ml 60 ml 1 min 5 min 3 min 1 min 3 min Results The extraction of pesticides from a chilli and a paprika sample carried out with the SpeedExtractor E-916 showed comparable results to those conducted with the ASE 200. The results of the extractions using the SpeedExtractor showed low variations. The results of the chilli sample are displayed in Table 2. For the results of the paprika extraction please see Application note 64/2011. Table 2: Results of the pesticides extraction from a chilli sample, n = 4 Sample: Chilli SpeedExtractor E-916 Mean Mean ASE 200 gamma-hch Chlorpyrifos A pesticide is any substance or mixture of substances intended for preventing, destroying, repelling or mitigating any pest [1]. Pesticides can be classified by their chemical structure (organochlorines, organophosphates and carbamates) or by their target (herbicides, insecticides, fungicides, rodenticides, pediculocides and biocides). 4,4 -Dichlorobenzophenone Quinalphos Profenofos Ethion Triazophos Dicofol Phosalone Cypermethrin Conclusion Pesticides were extracted from a paprika and a chilli sample with the SpeedExtractor E-916 and the ASE 200. The results received are comparable (mean). The extractions conducted with the SpeedExtractor proved to be more exact (rsd). Acknowledgement We sincerely thank the Kantonales Labor Zurich, Switzerland. References [1] US Environmental (July 24, 2007), What is a pesticide? EPA Gov. retrieved on September, [2] SpeedExtractor E-916 operation manual For more details see Application note 064/ Quality in your hands

3 1 Introduction A pesticide is any substance or mixture of substances intended for preventing, destroying, repelling or mitigating any pest [1]. Pesticides can be classified by their chemical structure (organochlorines, organophosphates and carbamates) or by their target (herbicides, insecticides, fungicides, rodenticides, pediculocides and biocides). The adverse health effects of pesticides depend on the type of pesticide. Organophosphates and carbamates for example affect the nervous system. Others may irritate the skin or eyes. Some pesticides may be carcinogens. Others may affect the hormone or endocrine system in the body [2]. This application note describes the extraction of organochlorine and other pesticides in chilli and paprika spice samples with the SpeedExtractor E-916. Figure 1: Formula of 4,4 -DDT The GPC clean-up and quantification by GC-MS/MS were conducted by the Kantonales Labor Zurich, an ISO accredited laboratory. For comparison the Kantonales Labor Zurich extracted the same samples using a Dionex ASE 200 and carried out the GPC clean-up and GC-MS/MS quantification for all samples (measuring inaccuracy 30 % according to the SOP of the Kantonales Labor Zurich). 2 Equipment SpeedExtractor E-916 with 20 ml cells, 4-port mixer Analytical balance Beaker 0 ml Positive displacement pipette Volumetric flask 50 ml Centrifuge Vaudaux-Eppendorf 5804 R Flacon tube GPC Gilson 233XL (details see chapter 5.4) Syringe filter GPC vial Rotary evaporator Evaporator THERMOBIL, Ismatec SA Varian 3800 with MS-1200 QQQ (details see chapter 5.4) 3 Chemicals and Materials Extraction with SpeedExtractor E-916: Cyclohexane, Suprasolv for gas chromatography, Merck ( ) Ethyl acetate, for analysis EMSURE, Merck ( ) Diatomaceous earth, BUCHI (053201) Water, for HPLC, Fluka (95304) Spike solution: Pesticides in concentration 1 mg/l Chlorpyrifos 4,4 -Dichlorobenzophenone Application Note 064/2011 Version A, Copyright 2011 BÜCHI Labortechnik AG 3/

4 gamma-hch Quinalphos 4,4 -DDT Pesticides in concentration mg/l Dicofol Cypermethrin Ethion Phosalone Triazophos Profenofos n-hexane, 95% for Organic Residue Analysis, J. T. Baker ( , Stehelin) 4 Samples Red chilli spice Paprika Dry homogeneous powders, no reference material. Supplied by the Kantonales Labor Zurich. These samples contain incurred pesticides. The pesticide contents had already been analyzed by the Kantonales Labor Zurich the previous year. For the sake of repeatability control these samples were selected for the experiments. 5 Procedure The extraction and analysis of pesticides in paprika and chilli samples include the following steps: Preparation of the samples Preparation of the cells Extraction with SpeedExtractor E-916 Clean-up with GPC Quantification by GC-MS/MS (external standards) 5.1 Preparation of the samples Weigh in approx. 3 g of the sample into a beaker using an analytical balance Add 6 ml of pure water to swell the sample and place it in the refrigerator over night Add about 3 g of diatomaceous earth and mix in well with a spatula 5.2 Preparation of the cells Samples Fill in the mixture into the 20 ml cells using a funnel In order to completely fill the sample into the cell, gently tap the cell on the bench if necessary Spiked sample Blank Fill in half of the mixture into the 20 ml cells using a funnel Add 300 µl of spike solution using a positive displacement pipette In order to completely fill the sample into the cell, gently tap the cell on the bench if necessary. Fill the cell with diatomaceous earth Application Note 064/2011 Version A, Copyright 2011 BÜCHI Labortechnik AG 4/

5 5.3 Extraction with SpeedExtractor E-916 To carry out the extraction use the parameters of Table 1. A fourfold determination of the samples was conducted. Table 1: Extraction method for SpeedExtractor E-916 Parameter Value Temperature 75 C Pressure 0 bar Solvent Cyclohexane 50 % Cells Vials Cycles 3 Heat-up Hold Discharge Flush with solvent Flush with gas Total extraction time Ethyl acetate 50 % 20 ml 60 ml 1 min 5 min 3 min 1 min 3 min 48 min After the extraction transfer the extracts quantitatively into 50 ml volumetric flasks and fill up to the mark with cyclohexane 50 % and ethyl acetate 50 % 5.4 Clean-up with GPC The clean-up by GPC was carried out by the Kantonales Labor Zurich If the extracts contain water transfer the extracts into 50 ml falcon tubes and centrifuge the solutions for 1 min at 100 rpm (Vaudaux-Eppendorf 5804 R). Alternatively the extracts can also be dried using sodium sulphate. Use the upper solution for the GPC clean-up. Use a syringe filter to add about 7 ml of the extract into a GPC vial Inject 2.5 ml of the filtrated solution into the GPC Carry out the GPC clean-up using the parameter of Table 2 Table 2: Clean-up by GPC Type of GPC GPC column Solvent Cyclohexane 50 % Flow Column temperature Dump time Fraction time Rinse time Gilson 233XL on-line columns switching incl. dilutor 402 with pump 307 and pump heat SC Bio-beads S-X mesh, length: 40 cm, volume: 71 ml Ethyl acetate 50 % 3 ml / min Ambient 16 min 14 min min After GPC clean-up concentrate the extracts to 2-3 ml using a rotary evaporator (220 mbar, 40 C) Application Note 064/2011 Version A, Copyright 2011 BÜCHI Labortechnik AG 5/

6 Dry the extract completely in nitrogen stream 5.5 Quantification with GC-MS/MS The quantification of the pesticides was carried out with a Varian 3800 GC with MS-1200 QQQ by the Kantonales Labor Zurich For the GC-MS/MS measurement dissolve the dried extract in 1 ml n-hexane Table 3: Quantification by GC-MS/MS Type of GC-MS/MS Injector modus PTV temperature program Varian 3800 with MS-1200 QQQ PTV Injection volume 5 µl PTV Split program Column Carrier gas Oven program Temp. [ C] Rate [ C/min] Hold [min] Total [min] Time [min] Split State Split Ratio 1 Initial On Off Off 3 7 On On 25 db-5ms m x 0.25 mm x 0.25 µm, Agilent Helium at 5.7 psi Temp. [ C] Rate [ C/min] Hold [min] Total [min] Table 4: MS-Method Substance RT [min] q1 q3 Collision energy [ev] Gamma-HCH Chlorpyrifos ,4 -Dichlorobenzophenone Application Note 064/2011 Version A, Copyright 2011 BÜCHI Labortechnik AG 6/

7 Quinalphos Profenofos Ethion Triazophos ,4 -DDT Dicofol Phosalone Cypermethrin Calculation dilution factor v m sample ( Extract ) v v ( GPC ) = (1) ( GCMS / MS ) Dilution factor : to calculate the pesticide content [g/ml] m sample v (GPC) v (Extract) v (GC-MS/MS) : sample weight [g] : volume for GPC clean-up [ml] : volume of extraction [ml] : volume for GC-MS/MS analysis [ml] 7 Results The extractions of the chilli and paprika samples were carried out in fourfold. The result of the blank measurement was in the signal to noise range and not taken into consideration. As a positive control (QC test) a spiked sample was extracted and analysed. The results received fulfilled the acceptance criteria of the Kantonales Labor Zurich. The results of the extraction with the SpeedExtractor E-916 are equivalent to the results received by extraction with the ASE 200. The determination of the pesticide content carried out with the SpeedExtractor E-916 showed lower relative standard deviations (rsd) than the extractions conducted with the ASE 200. Application Note 064/2011 Version A, Copyright 2011 BÜCHI Labortechnik AG 7/

8 7.1 Paprika Table 5: Results of pesticides extraction in a paprika sample carried out with the SpeedExtractor E-916 in comparison to extractions done with the Dionex ASE 200, n = 4 Sample: Paprika Extraction with SpeedExtractor E-916 Extraction with ASE 200 Mean value Mean value gamma-hch Chlorpyrifos ,4 -Dichlorobenzophenone Quinalphos Profenofos Ethion Triazophos ,4 -DDT Dicofol Phosalone Cypermethrin Paprika Cypermethrin Phosalone Dicofol 4,4-DDT Triazophos Ethion Profenofos Quinalphos 4,4-Dichlorobenzophenone Chlorpyrifos gamma-hch µg/kg SpeedExtractor E-916 ASE 200 Figure 2: Sample Paprika The following statistic tests were conducted with the results of the GC-MS/MS measurements: Outlier test (Grubs 95%) The determination of the extracts of the paprika sample carried out with the ASE 200 by the Kantonales Labor Zurich contained a statistic outlier for the pesticide 4,4 -DDT. Application Note 064/2011 Version A, Copyright 2011 BÜCHI Labortechnik AG 8/

9 The determination of the extracts of the paprika sample carried out with the SpeedExtractor E-916 contained a statistic outlier for the pesticides Ethion and Cypermethrin. The outliers were not taken into account. 7.2 Chilli Normal distribution (Pearson / David 95%) The extractions of the paprika sample carried out with the SpeedExtractor E-916 as well as with the ASE 200 were distributed normally. Table 6: Results of pesticides extraction in a chilli sample carried out with the SpeedExtractor E-916 in comparison to extractions carried out with the Dionex ASE 200, n = 4 Sample: Chilli Extraction with SpeedExtractor E-916 Extraction with ASE 200 Mean value Mean value gamma-hch Chlorpyrifos ,4 - Dichlorobenzophenone Quinalphos Profenofos Ethion Triazophos ,4 -DDT Dicofol Phosalone Cypermethrin Application Note 064/2011 Version A, Copyright 2011 BÜCHI Labortechnik AG 9/

10 Chilli Cypermethrin Phosalone Dicofol 4,4-DDT Triazophos Ethion Profenofos Quinalphos 4,4-Dichlorobenzophenone Chlorpyrifos gamma-hch µg/kg SpeedExtractor E-916 ASE 200 Figure 3: Sample Chilli The following statistic tests were conducted with the results of the GC-MS/MS measurements: Outlier test (Grubs 95%) There was no statistic outlier for the chilli sample Normal distribution (Pearson / David 95%) The determination of the pesticide Dicofol in the chilli sample extracted with the ASE 200 by the Kantonales Labor is not distributed normally. Nevertheless, mean value and rsd were calculated as for a normal distribution. 8 Conclusion The method presented in this application note demonstrates a fast and reliable way to extract pesticides from spice samples using the SpeedExtractor E-916. The results of the extraction with the SpeedExtractor E-916 are equivalent to results received by extraction with the ASE 200. The determination of the pesticide content carried out with the SpeedExtractor E-916 showed lower relative standard deviations (rsd) than extractions conducted with the ASE Acknowledgements We greatly acknowledge Mr. Claudio Crüzer and Dr. Andreas Schürmann of the Kantonales Labor Zurich for their measurements and support in developing this application note. References [1] US Environmental (July 24, 2007), What is a pesticide? EPA Gov. retrieved on September, [2] [3] Operation manual SpeedExtractor E-916 BÜCHI Labortechnik AG CH-9230 Flawil 1/Switzerland T F Quality in your hands Application Note 064/2011 Version A, Copyright 2011 BÜCHI Labortechnik AG /