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Book 04-109
Book 04-109
A report for the Rural Industries Research and Development Corporation by Sandra M. Garland Prof. Robert C. Menary NW Davies and Garth S. Oliver
2004 Rural Industries Research and Development Corporation. All rights reserved. ISBN 1 74151 018 X ISSN 1440-6845 Practical approaches to the analyses for pesticide residues in essential oils Publication No. 04/109 Project No. UT-36A The views expressed and the conclusions reached in this publication are those of the author and not necessarily those of persons consulted. RIRDC shall not be responsible in any way whatsoever to any person who relies in whole or in part on the contents of this report. This publication is copyright. However, RIRDC encourages wide dissemination of its research, providing the Corporation is clearly acknowledged. For any other enquiries concerning reproduction, contact the Publications Manager on phone 02 6272 3186.
Researcher Contact Details Prof. R. C. Menary Tasmanian Institute of Agricultural Research (TIAR) University of Tasmania GPO Box 252-54 HOBART Tas 7001 Phone: Fax:( Email: (03) 6226 2723 (03) 6226 7609 R.Menary@utas.edu.au
In submitting this report, the researcher has agreed to RIRDC publishing this material in its edited form.
RIRDC Contact Details Rural Industries Research and Development Corporation Level 1, AMA House 42 Macquarie Street BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: Fax: Email: Website: 02 6272 4539 02 6272 5877 rirdc@rirdc.gov.au http://www.rirdc.gov.au
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Contents
Acknowledgments...................................................................................................................... iv
Introduction....................................................................................................................... 1
Pesticides commonly used in the essential oil industry. ............................................................. 2 Abbreviations .............................................................................................................................. 3 Chemical Properties of Essential Oils......................................................................................... 4 Practical considerations for laboratory procedures in pesticide analyses ................................... 7
Analytical Equipment..................................................................................................... 25
Gas Chromatography ................................................................................................................ 25 Liquid Chromatography ............................................................................................................ 57 Assessment of the application of ICP OES to the screening of essential oils........................... 84
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Acknowledgments
Our appreciation is extended to Dr Ashley Townsend whose expertise in ICP OES was instrumental in developing the methodology for the screening of essential oils for mancozeb contamination.
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Introduction
Many papers have been published detailing methods for the analysis of pesticide residues in matrices such as water and vegetables. The detection of analytes in essential oils, however, has specific problems associated. The chemical properties of the active ingredients of many pesticides, such as polarity and retention behaviour in chromatography media, are often very similar to those observed for components of essential oils.
Clean-up, or rather the limited applicability of standard pre-concentration steps, presents as the greatest limitation. The greater majority of commercially produced, solid phase extraction columns are designed to trap low levels of pesticide residues from large quantities of water. Even in the analysis of vegetables, an aqueous phase is the predominant matrix from which pesticides are absorbed. Essential oils are usually a complex mixture of medium polarity and non-polar extracts of plant material concentrated to as little as 5% of the source material. Development of an analytical methodology for any one contaminant can be achieved, but the pre-concentration of a number of pesticide residues within one screen is problematic.
For the great majority of pesticides, the structure and chemical properties of the active ingredient confer physical properties, such as polarity, solubility and elution characteristics which can be used as a predictive indicator for their behaviour in clean-up methodology involving chromatography. The components which present as the most likely to co-extract with essential oils are, by the nature of their extractability, the most difficult to separate from the matrix and the most likely to interfere with the analysis, having similar behaviour in liquid partition and chromatographic methodologies. This manual is designed to provide an overview of the applicability of the analytical technology generally available, to the detection of analytes. Methodologies are designed based on the chemical type of the active ingredient.
The Manual can be read in conjunction with reports on four RIRDC projects detailing the development of analytical techniques for the determination of pesticide minimum residue limits in essential oils: UT-8A, UT-13A Publication No 98/123, UT23A Publication No 04/023 and UT36A Publication No 04/104.
Abbreviations ai amu APCI BAP C.E. DCM DETA DMED ECD ESI ESP FID FPD GC HPLC HR ICP OES LC MRL MS MSD MSDs MSMS PDA. R.P. r.s.d. RVE. SIM SPE SPI TBAS TIC TLC TSP active ingredient Atomic Mass Units Atmospheric pressure chemical ionisation Best Agricultural Practices collision energy Dichloromethane Diethylene triamine
Dimethyl ethylenebis(dithiocarbamate)
Electron Capture Detector Electrospray ionisation Electrospray Flame Ionisation Detector Flame Photometric Detector Gas Chromatography High Pressure Liquid Chromatography High Resolution Inductively Coupled Plasma Optical Emission Spectrophotometer Liquid chromatography Maximum Residue Limit Mass Spectrometry Mass Selective Detector Material Safety Data sheets Daughter Mass Spectra Generated by Fragmentation of a Parent Ion Photo Diode Array Detector Reverse Phase Relative Standard Deviation Rotary Vacuum Evaporation Single Ion Monitoring Solid Phase Extraction Septum Equipped Programmable Injector Tetrabutylammonium hydrogen sulfate Total Ion Chromatogram Thin Layer Chromatography Thermospray
Solvent extracts
Concretes Many concretes are produced by the extraction of flowers, leaves or buds using low polarity organic solvents such as hexane and petroleum ether. Although the plant components targeted by this production method are usually aromatic volatile chemicals, high amount of waxes and non-volatiles are co-extracted. In addition, the co-extraction of any pesticide contaminant present in the plant material is also more likely occur in the production of concretes, as opposed to steam distilled oils. In most of the concretes produced in Australia, fresh or frozen material is used. Water content is often quite high, as much as 50% of the weight of plant material. For highly polar pesticides this increases the likelihood that much of residues present will remain in the vegetative
material. Highly water soluble pesticides, such as the quaternary ammonium salts, should not coextract with the oil components. However, a great number of commonly used pesticides are readily soluble in organic solvents, having similar polarities and solubilities to many of the desirable essential oil components such as hydrocarbons and oxygenated hydrocarbons. Concretes are also the most chemically complex of the oils produced using the various production methods. As such, clean-up techniques are almost essential, as solvent extracts often contain non-volatile components not amenable to gas chromatography (GC) or liquid chromatography (LC).
Absolutes Many of the considerations presented for concretes also apply to absolutes. However, the cold ethanol extraction process usually removes many of the waxes and lipids common to concretes which would otherwise present difficulties for volatilisation in GC or contaminate columns in liquid chromatography. However, the likelihood of co-extraction of moderately polar pesticide residue remains. The removal of contaminants which precipitate out with the waxy components during the production of absolutes, is only relevant to lipophilic chemicals.
Distilled Oils
Distilled oils, by the nature of their production, have few contaminants that are non-volatile. This has implications, not only for the degree of clean-up required, but also implies that nonvolatile residues should not be present in the oil. However, even non-volatile contaminants may be carried over in the distillation process when water droplets or particulate matter, splashed to the head of the distillation vessel, are washed through to the distillate. Whatever the process, pesticide residues are commonly detected in distilled oil. (Gould, 1960; Starr et al., 1963; Ballee et al., 1970, Garland et al., 1999). With sufficiently low detector sensitivity and specificity, analysis by GC may be conducted with no clean-up and little risk of accelerated GC column deterioration. Similarly, contamination of reverse phase LC columns presents as a minor consideration. For water soluble pesticides, however, steam distillation may present almost as a process for contaminant removal. The steam generated in the distillation process condenses to liquid and is recycled through a boiler in a closed system. Water is continuously washing through the oil collected, extracting much of any water soluble, co-distilled contaminants. In all, however, contamination of distilled oils with volatile, moderately polar pesticide residues is a common occurrence.
Table 1 lists some of the chemicals that are found in some of the major essential oils in Australian production.
Parsley (distilled) -pinene -pinene sabinene myrcene -phellandrene limonene -phellandrene p-cymene -terpinolene p-menthatriene ,p-dimethylstyrene carotol tetramethoxy allyl benzene elemicin myristcin apiole Fennel (distilled) -pinene myrcene -phellandrene limonene -phellandrene 1,8-cineole cis--ocimene fenchone estragole trans-anethole Peppermint (distilled) -pinene -pinene sabinene myrcene limonene 1,8-cineole cis--ocimene -terpinene 3-octanol sabinene hydrate menthone menthofuran isomenthone -bourbonene linalool menthyl acetate neomenthol caryophyllene terpinen-4-ol menthol pulegone isomenthol germacrene D piperitone Boronia (solvent extract) -pinene camphene -pinene sabinene -3-carene limonene -phellandrene terpinolene ethyl octanoate 2,6-dimethyl- 3,7 octadiene-2,6- diol dihydro -ionone dodecanol -ionone sesquicineole dodecyl acetate methyl jasmonate methyl epi jasmonate heptadec-8-ene
Collect as much information concerning the chemical nature of the pesticides and reagents to be used in all procedures. This information provides a starting point from which to assess the methodologies most suited to the chemical type and creates an awareness of significant safety issues including toxicity and potentially hazardous materials. Sources of information include: