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Practical approaches to

the analyses for pesticide residues
in essential oils

A report for the Rural Industries Research and Development Corporation by Sandra M. Garland Prof. Robert C. Menary NW Davies and Garth S. Oliver

July 2004 RIRDC Publication No 04/109 RIRDC Project No UT-36A

© 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

Published in July 2004 Printed on environmentally friendly paper by Canprint

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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

Categorisation of Pesticides ............................................................................................. 8
Chlorinated chemicals................................................................................................................. 8 Experiments undertaken for equipment assessment.................................................................. 10 Organophosphates ..................................................................................................................... 11 Urea Chemicals ......................................................................................................................... 14 Carbamate Derivatives .............................................................................................................. 16 Dithiocarbamates ...................................................................................................................... 17 Pesticides with Acidic Moieties ................................................................................................ 20 Quaternary Nitrogen Herbicides ............................................................................................... 22

Analytical Equipment..................................................................................................... 25
Gas Chromatography ................................................................................................................ 25 Liquid Chromatography ............................................................................................................ 57 Assessment of the application of ICP OES to the screening of essential oils........................... 84

Preliminary clean-up methodology ............................................................................... 87
Liquid / liquid extraction of pesticides from solvent extracted oils .......................................... 87 Preliminary Development of Solid Phase Extraction of Pesticides from Essential Oils........... 88 The application of ion exchange chromatography to the clean-up of acidic pesticides with detection by GC ECD................................................................................................................ 95

Literature Cited ............................................................................................................ 100 Appendix........................................................................................................................ 103

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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. iv .

or rather the limited applicability of standard pre-concentration steps. are often very similar to those observed for components of essential oils. UT-13A Publication No 98/123. The greater majority of commercially produced. has specific problems associated. 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. an aqueous phase is the predominant matrix from which pesticides are absorbed. For the great majority of pesticides. UT23A Publication No 04/023 and UT36A Publication No 04/104. the structure and chemical properties of the active ingredient confer physical properties. The chemical properties of the active ingredients of many pesticides. solubility and elution characteristics which can be used as a predictive indicator for their behaviour in clean-up methodology involving chromatography. such as polarity. solid phase extraction columns are designed to trap low levels of pesticide residues from large quantities of water. The components which present as the most likely to co-extract with essential oils are. having similar behaviour in liquid partition and chromatographic methodologies. Even in the analysis of vegetables. such as polarity and retention behaviour in chromatography media. presents as the greatest limitation. by the nature of their extractability. 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. This manual is designed to provide an overview of the applicability of the analytical technology generally available. Clean-up. however. Methodologies are designed based on the chemical type of the active ingredient. to the detection of analytes. 1 . 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. The detection of analytes in essential oils.Introduction Many papers have been published detailing methods for the analysis of pesticide residues in matrices such as water and vegetables. the most difficult to separate from the matrix and the most likely to interfere with the analysis.

Active ingredient acephate asulam bromacil & diuron carbaryl carbendazim chloropropham chlorpyrifos clopyralid dicamba difenoconazole dimethenamid dimethoate diuron ethofumesate fluazifop fluroxypyr glyphosate haloxyfop isoxaben linuron mancozeb MCPA mecoprop monocrotophos norflurazon oryzalin oxyflurofen paraquat & diquat pendimethalin pirimicarb procymidone prometryn propazine propiconazole sethoxydim simazine sulfosulfuron tebuconazole terbacil trichlopyr Key : Commercial preparation Orthene Asulox Krovar Carbaryl Bavistan Allicide Lorsban Lontrel Dicamba Score Frontier Rogor Diuron Tramat Fusilade Starane Roundup Verdict Flexidor Linuron Dithane MCPA mecoprop Nuvacron Solicam Snapshot Goal Gramoxone Stomp Pirimor Sumisclex Geasaguard Gesamil Tilt Sertin Gesatop Maverick Folicur Sinbar Garlon I .herbicide F .Pesticides commonly used in the essential oil industry.fungicide Function I H H I F H I H H F H I H H H H H H H H F H H I H H H H H I F H H F H H H F H H 2 .insecticide H .

d. DCM DETA DMED ECD ESI ESP FID FPD GC HPLC HR ICP OES LC MRL MS MSD MSDs MSMS PDA.Abbreviations ai amu APCI BAP C.s.P. RVE. R. 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 3 . r.E.

The solvents are normally removed from the extracts by rotary vacuum evaporation (RVE) at low temperatures (usually below 60°C). non-polar components are removed to produce a less viscous. With essential oil yields from plant material often in the vicinity of 3 to 5% by weight. Although the plant components targeted by this production method are usually aromatic volatile chemicals. are those obtained by steam distillation. Solvent extracts Concretes Many concretes are produced by the extraction of flowers. as opposed to steam distilled oils. concentrate pesticide residues. leaves or buds using low polarity organic solvents such as hexane and petroleum ether. In most of the concretes produced in Australia. ketones and diterpenoids. can in effect.Chemical Properties of Essential Oils Two of the most widely used methods for the production of essential oils include solvent extraction and steam distillation. assuming 100% recovery of the residues. patchouli oil. with or without maceration and agitation. or essential oils. Water content is often quite high. whereby waxes and other high molecular weight. The distillation is conducted at atmospheric pressure or at elevated pressures for products such as sandalwood oil. contamination of. The level of pesticide residues present in plant material is extracted into a significantly smaller volume of oil. one aspect is common for all pesticide residues which do co-extract. fresh or frozen material is used. For highly polar pesticides this increases the likelihood that much of residues present will remain in the vegetative 4 . 1 mgkg-1 in harvested crops has the potential to result in 20 a to 33 mgkg-1 level of contamination in the oil. Steam distilled oils. vetiver oil and several other oils composed mainly of sesquiterpenoid alcohols. Whatever the method of essential oil production. ethanol or acetone. such that essential oil production. more concentrated product. All methods of essential oil production have implications for the likelihood of pesticide residue contamination and confer associated chemical characteristics to be considered when clean-up and analytical techniques are applied. In addition. Absolutes are prepared by cold filtration of chilled ethanol solutions of concretes. high amount of waxes and non-volatiles are co-extracted. as much as 50% of the weight of plant material. the co-extraction of any pesticide contaminant present in the plant material is also more likely occur in the production of concretes. Concretes are produced by the steeping of raw plant material in organic solvents such as petroleum ether. for example.

However. steam distillation may present almost as a process for contaminant removal. In all. However. have few contaminants that are non-volatile. (Gould. This has implications. 1970. Starr et al. Whatever the process. however. having similar polarities and solubilities to many of the desirable essential oil components such as hydrocarbons and oxygenated hydrocarbons.. even non-volatile contaminants may be carried over in the distillation process when water droplets or particulate matter. Water is continuously washing through the oil collected. is only relevant to lipophilic chemicals. For water soluble pesticides. not only for the degree of clean-up required. Ballee et al. by the nature of their production. 5 . Distilled Oils Distilled oils. However. 1960. are washed through to the distillate. contamination of reverse phase LC columns presents as a minor consideration. as solvent extracts often contain non-volatile components not amenable to gas chromatography (GC) or liquid chromatography (LC). Highly water soluble pesticides. the likelihood of co-extraction of moderately polar pesticide residue remains. a great number of commonly used pesticides are readily soluble in organic solvents. contamination of distilled oils with volatile. co-distilled contaminants. The steam generated in the distillation process condenses to liquid and is recycled through a boiler in a closed system. Concretes are also the most chemically complex of the oils produced using the various production methods. pesticide residues are commonly detected in distilled oil. 1999). however. The removal of contaminants which precipitate out with the waxy components during the production of absolutes. 1963. splashed to the head of the distillation vessel. Absolutes Many of the considerations presented for concretes also apply to absolutes. analysis by GC may be conducted with no clean-up and little risk of accelerated GC column deterioration. moderately polar pesticide residues is a common occurrence.. but also implies that nonvolatile residues should not be present in the oil. 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. With sufficiently low detector sensitivity and specificity. As such.. However. Garland et al. clean-up techniques are almost essential. should not coextract with the oil components. such as the quaternary ammonium salts. Similarly. extracting much of any water soluble.material.

Table 1 lists some of the chemicals that are found in some of the major essential oils in Australian production.
Parsley (distilled) α-pinene β-pinene sabinene myrcene α-phellandrene limonene β-phellandrene p-cymene α-terpinolene p-menthatriene α,p-dimethylstyrene carotol tetramethoxy allyl benzene elemicin myristcin apiole Fennel (distilled) α-pinene myrcene α-phellandrene limonene β-phellandrene 1,8-cineole cis-β-ocimene fenchone estragole trans-anethole Peppermint (distilled) α-pinene β-pinene sabinene myrcene limonene 1,8-cineole cis-β-ocimene γ-terpinene 3-octanol sabinene hydrate menthone menthofuran isomenthone β-bourbonene linalool menthyl acetate neomenthol caryophyllene terpinen-4-ol menthol pulegone isomenthol germacrene D piperitone Boronia (solvent extract) α-pinene camphene β-pinene sabinene δ-3-carene limonene β-phellandrene terpinolene ethyl octanoate 2,6-dimethyl- 3,7 –octadiene-2,6- diol dihydro β-ionone dodecanol β-ionone sesquicineole dodecyl acetate methyl jasmonate methyl epi jasmonate heptadec-8-ene

Table 1. Chemical composition of essential oils

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Practical considerations for laboratory procedures in pesticide analyses
A simple guide to beginners to the field of pesticides analysis is listed. It is not intended to be a comprehensive guide to all sources of information and suggestions for specific procedures are to be instigated in addition to standard protocols common to good laboratory practices.

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: Merck index; pesticide handbooks; material safety data sheets; - available in coordinated databases marketed in comprehensive CD forms - provided by chemical companies such as Sigma Aldrich - available through several web sites, including http://ace.orst.edu/info/extoxnet/ standard literature data base search engines

Internationally standardised analytical methods usually specify detection limits to at least 1 mgkg-1 and can extend to the µgkg-1 level. In the preparation of samples and standards, the potential for contamination is very high. Procedures within an analytical laboratory must adhere to strict guidelines to prevent contamination and achieve reproducibility. Listed below are a few recommended precautions: All glassware should be washed with appropriate cleansers such as alkaline Extran® with neutralising washes of Galley® then repeated washing with distilled water. Heating glassware at 440° is also an option though vessels may become brittle. Solvents should be commercially sourced and designated pesticide-grade or redistilled in decontaminated glass stills. At the commencement of each day of analysis, all surfaces should be wiped clean before chemicals and standard solutions from storage areas are introduced into the working area. Disposable bench liners are useful for reducing contamination risk. Be aware of the surface which have come into contact with pesticides and pesticide solutions. When

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syringes are used to dispense standard solutions, place on a dedicated tissue between applications and discard the tissue at the end of each procedure. Prepare all samples to be screened for pesticide residues prior to any procedures which involve pesticide standard solutions such as those used to spike samples to establish standard curves and estimate recoveries. If practicable, complete the sample preparation and seal the vials from which the final aliquot is to be sub-sampled prior to the introduction of standard solutions to the working area. Dedicate syringes to preparations of standard curves and the spiking of samples with pesticide solutions. Have a separate set for the work-up of samples to be screened. Fortification of solvents and samples should proceed from the more dilute to the highest concentration, to minimise risk of contamination. Operators should observe all safety guidelines to comply with material safety data sheets (MSDs) protocols.

Categorisation of Pesticides
In the development of any pesticide, a particular mode of action, such as cholinesterase inhibition, inhibition of cell division or excessive stimulation of weed growth, is usually conferred by the inclusion of functional groups into the structure of the chemical. Distinct classes of pesticides are formed based on the chemical nature of these functional moieties. The chemical type of a pesticide also has implications in terms of their potential to contaminate essential oils. The functional groups unique to each class of pesticides, then, may confer the properties which affect:

translocation through plants and soils, important in terms of incorporation into plant material and potential for root uptake by current and sequential crops;

• •

stability and residual time, which relates to their potential to still be present at harvest; extractability, related to the solubilities and volatility during essential oil production.

In addition, the chemical properties of pesticides have implications in the design of analytical methodology specific to their separation and detection.

Chlorinated chemicals
The organochlorine pesticides are an extensive groups of chemicals which include chlorotriazoles, which function as systemic fungicides, and chlorotriazines, which are broadspectrum residual herbicides, used for pre and post emergence weed control. Within the essential

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However the simple liquid / liquid partition step described on page 91 is only a clean-up step with no corresponding preconcentration of target analyte. Many organochlorines are very stable. The inclusion of such as preliminary clean-up can significantly reduce the loading in to GC injectors. which despite some poor recoveries. However.The effectiveness of standard clean-up methodology is limited for the reasons already outlined. such that many of the triazines elute with excellent 9 . which in addition with their translocation and absorption properties. however. provides for a pre-concentration of target analytes and allows for the introduction of larger sample volumes into instrumentation. many organochlorines used within the essential oil industry have high efficacy with little to no residue at harvest time. makes the development of a cost-effective multi-residue screen using standard SPE techniques difficult. the life of the guard column and columns can be significantly extended and the need for washing of the columns with extended runs can be reduced. distilled oils. None the less. such as polarity and volatility. The aromatic qualities of the sesquiterpenes and oxygenated sesquiterpenes have ensured that industrial extraction protocols are designed to maximise their yield. as many have polarities similar to essential oil components.The majority of organochlorines are sufficiently volatile and thermally stable to be amenable to gas chromatography. The halogenation does. with the concomitant effective extraction of many organochlorines. modern solid phase extraction (SPE) products may allow for the development of an effective pre-concentration step for any particular organochlorine residue in essential oils. Clean-up . For LC systems. Liquid / liquid partition can remove many of the waxes and non-polar components of concretes. With sufficient dedicated resources. to those of sesquiterpenes and oxygenated sesquiterpenes common to many essential oils. Analysis . are prone to bioaccumulation. The interaction with the liquid phase of the capillary columns is often specific to the analyte. however. particularly those employing columns with non-polar phases such as C18. The similarity in properties of many of these types of pesticides to the components within essential oils. Preliminary development of an SPE methodology is described on page 92. In terms of analytical methodology the organochlorines are often amenable to GC and LC.oil industry these chemicals are at the highest risk of presenting as contaminants in products due to their systemic nature and the similarity of their chemical properties. and to a lesser extent. provide for the specificity germane to detection by Electron Capture Detectors (ECD) and distinctive isotope patterns in mass spectral analysis (MS). they are often the most difficult to pre-concentrate or separate from the matrix.

in general..01 to 1 mgkg-1 (ionisation and response specific for each pesticide) page 56 page 45 (highly specific and quantitative. well-defined peaks. however. From the roots. limiting its leaching potential (Weed Sci. In high pH soils. 1994). Am. As with gas chromatography. the standard phenyl substituted silicones are effective in the separation of many organochlorines. makes it less mobile. amenable to reverse phase high pressure liquid chromatography (RP HLPC) with elution profiles and chromatographic properties specific to each analyte determined by trial and error. leaves. 1994). however. 74 Properties typical of a commonly used organochlorine as exemplified by simazine Simazine is a selective triazine herbicide used to control broad-leaved weeds and annual grasses. Plants absorb simazine mainly through the roots. Its low water solubility.chromatographic characteristics. Soc.. and growing shoots of the plant (Kidd et al. Am.. on the other hand. may present with poor peak shape and significant tailing. organochlorines are. 1994). It acts to inhibit photosynthesis (Kidd et al. 1991. 1991. it is translocated upward to the stems. Am. Weed Sci. presenting sharp. 10 . Experiments undertaken for equipment assessment GC ECD Detection limits established 0. The triazoles. In general. will quickly distinguish which analytes have poor chromatographic properties and some experimentation with different liquid phases should be investigated.1 to 5 mgkg-1 (retention time insufficient for unequivocal peak identification) GC HRMS Detection limits established 0. Soc. Soc. It is moderately persistent with an average field half-life of 60 days (Wauchope et al. with little or no foliar penetration. 1992). Weed Sci. residual activity may remain for a year after application (2 to 4 kgha-1). specialised equipment required) LC MSMS pages 72. especially as the column ages trial and error of each..01 to 1 mgkg-1 Detection limits established 0.

000810 mPa @ 20°C Partition Coefficient: 1. phosphorous detector (NPD).5-triazine-2. LC and the related methods of detection. chloroform. 1991) Vapour Pressure: 0. The presence of a phosphorus atom is the singular feature which delineates them from the greater body of pesticides.Physical Properties: Chemical Name: 6-chloro-N2. In most other respects the suitability of GC. delineating organophosphates into a distinct class is useful only in the context of their chemical stability ie. The polarities of many of the organophosphates. generally have lower persistence and bioaccumulation compared with organochlorines. Examples dealt with specifically in this study are monocrotophos.N-diethyl-1.3. as for the organochlorines. Toxicity.4-diamine Molecular Weight: 201.5 mgL-1 @ 20°C. are similar to those of the oxygenated monoterpenes and sesquiterpenes. likelihood of residual time. acephate and chlorpyrifos. extractability in normal operations of essential oil production and lability in analytical processes.3.9600 Adsorption Coefficient: 130 (Wauchope et al. which confers a degree of polarity as evidenced by their high solubility in water and polar organic solvents such as acetone. and diethyl ether Melting Point: 225-227°C (Kidd et al. As a functional moiety able to be exploited for analytical methodology. All are soluble in water and the organic solvents used in essential oil production. but are regarded as highly toxic. has the same potential and disadvantages as any other of the pesticide classes.4-diamine N C2 H 5 NH N Cl N NHC 2 H 5 simazine 6-chloro-N.5-triazine-2. This group of pesticides which act as potent cholinesterase inhibitors. Specifically then. soluble in methanol.70 Solubility: water . ionisation 11 .N4-diethyl-1. 1992) Organophosphates The organophosphate pesticides used predominantly in the essential oil industry are low molecular weight chemicals (< 350) often with low ratios of carbon to oxygen and sometimes chlorine atoms. the phosphorus atom only confers the specificity compatible with flame photometric detection (FPD) in the phosphorus mode or a nitrogen.

12 . However. Trial and error will quickly distinguish which analytes have poor chromatographic properties and some experimentation with different liquid phases should be investigated. many of these analytes have poor chromatographic properties. Although modern SPE products may allow for the development of an effective pre-concentration step for any particular organophosphates. Page 92 details a preliminary work-up for a clean-up and pre-concentration step using SPE. on page 91. dedicated screen for the class.The effectiveness of standard clean-up methodology is limited as the polarities of organophosphates are similar to those of essential oil components. Clean-up . Some organophosphates. In addition. with peak tailing especially pronounced as the column ages. including some organophosphates. expense and robustness. when applied to the most widely used methyl silicone.The majority of organophosphates are sufficiently volatile to be amenable to gas chromatography. range of application. However a simple liquid /liquid partition step is described for a range of pesticides in solvent extracted concretes. the co-elution of these pesticides with essential oil components makes the development of a cost-effective clean-up technique for multiple pesticides in a single screen difficult. with considerations given to their increased propensity to degradation. the total number of organophosphates used widely in the essential oil industry is insufficient to warrant a separate. Organophosphates have been found to be amenable to RP HPLC with elution profiles and chromatographic properties specific to each analyte determined by trial and error. The method described results in a dilution of the pesticides and oil components into a larger volume of solvent but reduces the loading of nonpolar components into GC injectors and onto LC columns in subsequent analysis. non-polar liquid phases used in capillary GC. Analytical methodology should be adopted on the basis of. Analysis . such as monocrotophos.potential and polarity specific for each organophosphate must be assessed. are degraded in low molecular weight alcohols and this susceptibility should be taken into account in the design of analytical methodology.

Experiments undertaken for equipment assessment GC ECD (organophosphates which are also halogenated) Detection limits established 0.1 to 5 mgkg-1 (retention time insufficient for unequivocal peak identification) GC NPD GC HRMS Preliminary assessment Detection limits established 0.01 to 1 mgkg
-1

page 45

page 47 page 56

(highly specific and quantitative, specialised equipment required) LC MSMS Detection limits established 0.01 to 1 mgkg-1 page 70, 74

Properties typical of commonly used organophosphates as exemplified by acephate and monocrotophos. Acephate is an organophosphate foliar spray insecticide used for control of a wide range of biting and sucking insects. The residual systemic activity is around 10-15 days at the recommended application rate (Thomson, 1992). Physical Properties:

O CH 3 CNH

O P

OCH3

SCH 3 acephate acetylphosphoroamidothioic acid O,S-dimethyl ester
Appearance: Colourless to white solid (Montgomery, 1993), Molecular Weight: 183.17 Solubility: water 650, acetone 151, ethanol < 50, ethyl acetate 35, benzene 16, hexane 0.1 (all in g/100 ml at 20°C) (Worthing, 1987) Melting Point: 93 °C (Kidd et al., 1991) Sufficiently volatile for GC Monocrotophos is a systemic insecticide and acaricide which has a low environmental persistence. It has a half-life of 1.3 to 3.4 days on plant foliage (Chambers et al., 1992). Physical Properties:
O (CH3O)2 P O CH3 H CONHCH3 monocrotophos Dimethyl 1-methyl-3-(methylamino)-3-oxo-1-propenyl phosphate

Monocrotophos is a reddish brown crystalline solid with a mild odour. M. W.: 223.2

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Water Solubility: Soluble in water, acetone and alcohol (Meister, 1992) Melting Point: 54-55 °C Partition Coefficient: -0.22

Urea Chemicals
Phenyl-substituted ureas are used extensively in agriculture as selective herbicides, mainly for pre-and post- emergence and they act by inhibiting photosynthesis. Commonly used substituted ureas are linuron and diuron, which have low residual action and persistence. Both are soluble in the organic solvents used in the essential oil industry and sufficiently labile to codistil with oils in steam distillation.

Clean-up - The solubility of the substituted ureas in water is a parameter which may be exploited in clean-up methodologies. The polarities and chemical distinction of the ureas are also features which may be exploited using SPE and other chromatographic products. The total number of this class of pesticide used in the essential oil industry is limited, however, such that the development of a specific extraction protocol would not be cost effective. However, the inclusion of the urea based chemicals in a protocol which may effectively pre-concentrate other water soluble pesticides, such as the herbicides with acidic moieties including dicamba and fluazifop acid, may be cost effective. Several experiments have been conducted for the acidic moiety pesticides (page 77) and may have direct application to the substituted ureas.

Analysis - The application of GC to phenylurea pesticides is difficult because these compounds are thermally unstable and rapidly degrade to isocyanates and amines (Buchert et al. 1975, Deleu, R. et al. 1979, Mattern, G. C. et al. 1989). Thermal reactions in the detector and on the columns result in a lack of reproducibility and incomplete thermal degradation preclude the monitoring of the degradation products for a quantitative screen. However, the breakdown products have been monitored by GC ECD and high resolution mass spectrometry (HRMS) and residues have been detected in oils produced from field trials using this technology. Linuron and diuron degrade to the same thermal breakdown product and so are indistinguishable using this analysis method. Derivatisation to compounds which are more thermally stable would provide for a more reliable screen. The application of RP HPLC is also problematic. Under the LC conditions trialed using acetonitrile/phosphate buffer, both urea herbicides were degraded. Detection by ion trap MS/MS was also limited. Linuron was ionised by atmospheric pressure chemical ionisation (APCI), but

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the response was poor. Trials are continuing to optimise the mobile phase, specifically the pH, as urea derivatives are sensitive to slight variations in this parameter.

Experiments undertaken for equipment assessment GC ECD GC HRMS Preliminary experimentation Detection limits established 1 mgkg
-1

page 45 page 56

(not specific as ion produced is identical for linuron and diuron) LC MSMS Detection limits not established page 67

(poor chromatographic properties & poor ionisation in +ive mode APCI)

Properties typical of commonly used, urea based herbicide as exemplified by linuron Linuron is a substituted urea, pre- and post-emergence herbicide used to control annual and perennial broadleaf and grassy weeds. It works by inhibiting photosynthesis in target weed plants. Linuron is slightly to moderately soluble in water, and is not readily broken down in water (U.S. Nat. Lib. Med., 1995). It is more readily absorbed by roots from soil application, than by leaves from foliar application (Weed Sci. Soc. Am. 1994). The rate at which it is absorbed, translocated, and subsequently broken down (or metabolised) differs with various plant species (Weed Sci. Soc. Am. 1994).

Physical Properties:

Cl

NHCONCH 3 OCH 3 Cl

linuron 3-(3',4'-dichlorophenyl)-1-methoxy-1-methylurea)

Appearance: Linuron is an odourless, white crystalline solid (Kidd et al, 1991). Chemical Name: 3-(3,4-dichlorophenyl)-1-methoxy-1-methylurea Molecular Weight: 249.11

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1991) Vapour Pressure: 2 mPa @ 24°C Partition Coefficient: 3. The breakdown product of carbaryl. The carbamates used in the essential oil industry include carbendazim and carbaryl which are of intermediate polarity such that it is again difficult to remove co-extracting essential oil components in analytical methodologies. naphthalene.0043 (Kidd et al. Although the likelihood of the co-extraction of different carbamates will vary with varying representatives of this chemical class. Derivatisation to thermally stable products presents as the only option if GC is the preferred analytical methodology. Analysis .The solubilities of carbamates are quite varied. Experiments undertaken for equipment assessment LC MSMS reverse phasae HPLC and with ionisation in +ive APCI page 69 16 . 1992) Carbamate Derivatives The carbamates are N-substituted esters of carbamic acid and act as cholinesterase inhibitors that confer insecticidal activity. 1991) Melting Point: 93-94°C (Kidd et al.Water Solubility: 81 mgL-1 @ 25°C. Soluble in aliphatic hydrocarbons and acetone and moderately soluble in ethanol (Kidd et al. LC is more suited to the analysis of this chemical type. the potential to co-extract with concretes and absolutes is high. Their effects are generally less intense than the organophosphates and they have low persistence in the environment. Solubilities of the carbamates can vary quite dramatically. carbendazim and carbaryl. 1991) Adsorption Coefficient: 400 (Wauchope et al. This is particularly true for the carbamate pesticides commonly encountered in the Australian industry. slightly soluble. but low levels of this latter chemical may be found to be endogenous to essential oils. Clean-up .The majority of N-substituted carbamates are thermally unstable and therefore not amenable to GC. has been monitored. such that it is difficult to develop one clean-up procedure which is equally effective for all types.

1988) Dithiocarbamates Dithiocarbamates are broad spectrum pesticides used widely in the essential oil industry to control fungal diseases such as rust. is non-volatile and labile. It has a short residual life of less than 2 weeks. light. and acids. Dithiocarbamates are heat labile and degrade to a number of products including ethylenethiourea (ETU). acetone. Prot.S. cyclohexanone.5% zinc. Agency. It would seem unlikely that residues would contaminate 17 .3 mPa @ 25°C Partition Coefficient: Not Available Adsorption Coefficient: 300 (U. carcinogenic. It is not stable under alkaline conditions. The dithiocarbamate fungicide most commonly used in the essential oil industry is Dithane. Agency.Properties typical of commonly used carbamate as exemplified by carbaryl. Prot. Soluble in dimethylformamide. (U. Degradation of carbaryl in crops occurs by hydrolysis within the plants. containing 20% manganese and 2. ETU is suspected to have goitrogenic. They are non-systemic. Environ.S. Environ. it is insoluble in most organic solvents and water. O CH3 N H carbaryl 1-naphthyl-N-methylcarbamate Carbaryl is a wide-spectrum carbamate insecticide and an acaricide. which is soluble in water and readily absorbed and metabolised in plants. 1973. 1988) Melting Point: 142°C Vapour Pressure: <5. mutagenic and tetragenic properties (Graham. 1972). whose active ingredient is mancozeb. contact fungicides which remain on the surface of the plants until degraded or washed off with rain or abrasion. As a polymeric salt of ethylenebisdithiocarbamic acid. Physical Properties: Carbaryl is stable to heat.25 Water Solubility: 40 mgL-1 @ 30°C. Chemical Name: 1-naphthyl-N-methylcarbamate CAS Number: 63-25-2 Molecular Weight: 199.

Levels of sulfur compounds in blackcurrants are as high as 50 mgkg-1 in some clonal material (Garland et al. Research has been conducted whereby EDTA / NaOH solutions are used to extract residues of the metal complexes of the fungicides which form sodium salts of the N. Many of these components are associated with quality oils and extraction protocols are adapted to maximise yields.The lack of solubility of mancozeb and related dithiocarbamates in water and organic solvents. of course. 1986). Detection of ethylene diamine. The standard method of analysis is the headspace analysis of carbon disulfide produced by digestion of residues in acidified stannous chloride. found background levels of carbon disulfide of the order of 5 mgkg-1 (page 86). known to be free of dithiocarbamate pesticides. However. does not preclude the possibility of ETU contamination and neither does it remove the need to confirm the absence of residues of the parent dithiocarbamate.The most widely used method of carbon disulfide production using acidified stannous chloride is not sufficiently specific for the detection of residues of thiocarbamates and background levels of endogenous sulfur compounds preclude this method for qualitative and quantitative analysis. whether solvent extracted or distilled. Two methods for the quantification of these salts include 18 . Ethylenethiourea.. Analysis . in addition to precluding their co-extraction in the production of essential oil. also presents as a chemical property which may be exploited in potential clean-up techniques. instead. It could be assumed that this methodology would remove the bulk of the components of essential oils. Research. which acts as a chelating agent to solubilise the metal complexes into partly neutralised sodium hydroxide solutions (pages 87. with analysis by GC and detection by FPD (Cullen. 89). Headspace analysis of the acidified stannous chloride digest of peppermint oil. This.. assuming the interface of the oil and aqueous extraction solution is sufficient to effect reproducible recoveries. 1969). Clean-up . 2002). as for many of the organochlorines and organophosphates. sulfur chemicals are endogenous to many essential oils. is soluble in the solvents used to produce essential oils and many of the problems associated with these chemicals also apply to the clean-up of ETU from essential oils. a side product of this reaction was not successful (page 86). such as 4-methoxy-2-methyl-2-mercaptobutane in blackcurrant oil (Rigaud et al. considering the high and frequent applications implemented in commercial crops. Keppel. has focussed on the extraction of residues of thiocarbamates with EDTA. 1964. In the analysis of essential oils this presents as an excellent clean-up protocol. Nethylenebis(dithiocarbamate).essential oil.

Experiments undertaken for equipment assessment GC FPD Carbon disulfide produced by acidified stannous chloride (not specific) LC MSMS Preliminary investigation of EDTA extraction and methyl iodide derivatisation of N. nut and field crops against a wide spectrum of fungal diseases and is used in the essential oil industry to control rust. ETU may persist for longer. which had limited success (page 87). Environ. 1988). vegetable. on the order of 5 to 10 weeks (Wauchope. Prot. which had been chelated in partly neutralised NaOH / EDTA solution (page 89). 1991) Melting Point: Decomposes without melting @ 192 ˚C Vapour Pressure: Negligible @ 20 ˚C Adsorption Coefficient: >2000 19 . 1992). Mancozeb rapidly and spontaneously degrades to ETU in the presence of water and oxygen (U.derivatisation using methyl iodide and analysed by LC MSMS. with a reported field half-life of 1 to 7 days (Wauchope.S..SCNCH 2 CH2 NCS H H x x:y 10:1 Zn y Chemical Name: manganese ethylenebis(dithiocarbamate) (polymeric) Molecular Weight: 266. Mancozeb is of low soil persistence. et al. ICP OES was successfully applied to measure the manganese from mancozeb. et al. Agency. 1992). N-ethylenebis(dithiocarbamate) ICP OES page 107 page 46 Elevated levels of manganese used as a marker for mancozeb contamination Page 111 Properties typical of commonly used dithiocarbamate as exemplified by mancozeb Mancozeb is used to protect many fruit. Physical Properties: S S 22+ -Mn.. and the application of Inductively Coupled Plasma Optical Emission Spectrophotometer (ICP-OES).31 Water Solubility: 6 mgL-1 -Practically insoluble in most organic solvents (Kidd et al.

In an alkaline solution. such that the polarity of resultant ion moderates any non-polar characteristics of the remaining organic molecular framework of the residues. The excellent retention characteristics under GC and LC. are water soluble. They are directly amenable to GC and elute in the same time frame as many of the oxygenated sesquiterpenes. However. Translocation of the acidic pesticides then takes place in the roots of treated plants. The acidic forms of this class of pesticide. Clean-up . The acid forms are highly water soluble and less likely to be extracted during the production of essential oils than the parent esters. Ion exchange chromatography also presents as a promising method of pre-concentration of aqueous extracts of essential oils (page 100). However. combined with the specificity afforded by ECD of the chlorinated moieties of this chemical class. which decompose to the acidic form under alkaline or acidic conditions. making separation from such matrices difficult. the exploitation of the water solubility of acidic moieties to clean-up oil residues from oil matrices will not be compatible with many of these derivatisation processes. The breakdown of most alkanoic acids is rapid. which are soluble in organic solvents. Analysis . unless the 20 . Aqueous extractions of essential oils for the pre-concentration and removal of interfering matrix components present as the most promising approach (page 75). Phenoxy based herbicides are detected in essential oils using HRMS without clean-up (page 58) and preliminary experiments with GC ECD of the ester derivatives will allow for detection to 1 mgkg-1 (page 45 and page 99). however.The parent esters of pesticides with acidic moieties follow the same considerations as previously discussed for the organochlorines. Some of the pesticides are applied in chemical formulations as esters. The polarity and solubility characteristics closely mimic those of the oxygenated monoterpenes and sesquiterpenes which constitute many essential oils. are often sufficient to allow for the analysis of oils without residue pre-concentration. This facilitates extraction across the interface between an organic oil phase and an aqueous solution. such as methylation or trimethylsilylation can convert acidic pesticides to esters compatible with GC. which confers a physical parameter on which clean-up protocols may be designed.Pesticides with Acidic Moieties This class of broad leaf weed killers include a large range of carboxylic acid herbicides.Relevant aspects of the analysis of the ester forms of many of these chlorinated herbicides are similar to those already discussed. Acidic moieties are not amenable to GC. many derivatisation steps. and by high resolution MS and ion trap MS. the carboxylic acid moiety of the herbicides is de-protonated.

It can be applied to the leaves or to the soil to control annual and perennial broadleaf weeds. The most promising line of research.1 mgkg (acidic moieties require derivatisation) . Anion exchange discs were used to trap deprotonated acid based pesticides. and it is readily translocated to other plant parts. by acidifying the alkaline extracting solution..1 . The discs were dried then the acids derivatised and eluted with methyl iodide (page 99). dicamba accumulates in the tips of mature leaves (Weed Sci. Am.0. It is moderately persistent in soil having a typical half-life of 1 to 4 weeks (Wauchope et al. however. LC MSMS Excellent chromatography good ionisation in -ive APCI -1 page 45 page 99 page 58 page 78 Properties typical of commonly used acidic herbicide as exemplified by dicamba Dicamba is a benzoic acid herbicide. Dicamba is rapidly taken up by the leaves and roots of plants. would facilitate the re-extraction of the herbicides back into an organic solvent such as dichloromethane. Limited success in terms of recovery has been achieved in preliminary experiments investigating such an extraction protocol.5 mgkg-1 (not sufficiently specific) Preliminary method development using ion exchange discs GC HRMS Detection limits established for parent esters .. It some plant species. is the extraction of acidic herbicides into an aqueous solution with analysis by LC interfaced with APCI in the negative mode and detection using ion trap MSMS (page 77) Experiments undertaken for equipment assessment GC ECD Detection limits established for parent esters .residues are re-extracted from the aqueous phase. and tends to be faster when soil is slightly acidic. a re-protonation of the acidic herbicide residues. The rate of biodegradation increases with temperature and increasing soil moisture. Alternatively. 1992). 1994) 21 . Soc.

Very few essential 22 . They are quick acting.6-dichloro-2-methoxybenzoic acid) Chemical Name: 3. Paraquat and diquat degrade slowly when exposed to sunlight but are otherwise quite resistant to microbial degradation and can persist indefinitely when bound to soil particles. The dichloride salts are stable to heat in acidic or neutral solutions but are hydrolysed by alkaline solutions. the chemical properties which preclude their extraction in oil production are also properties which may be used to extract them from essential oils should contamination occur. would act to continuously extract water soluble chemicals. As for all potential contaminants. The processes associated with the production of steam distilled oils.As with most of the pesticides which are unlikely to contaminate essential oils. where the oil is collected from a stream of condensed water and volatiles. Paraquat and diquat are very soluble in water. (Kidd et al. 1991) Melting Point: 114-116°C Vapour Pressure: 4.5376 Adsorption Coefficient: 2 (salt) Quaternary Nitrogen Herbicides The pesticides of this class used most frequently in Australia are paraquat and diquat. non-selective.Physical properties COOH Cl OCH3 Cl dicamba (3. contact poisons which are also translocated through the plant. Clean-up . however. ethanol.5 mPa @ 25°C Partition Coefficient: -0. it is necessary to conclusively certify that no detectable pesticide residues are present in oils. Soluble in acetone.. toluene and Xylene. They are used for broadleaf weed control. The likelihood of residues in solvent extracted essential oils is therefore very low. have very low solubility in the lower alcohols and are insoluble in hydrocarbons. dichloromethane.04 Water Solubility: 6500 mg/L @ 25°C.6-dichloro-2-methoxybenzoic acid Molecular Weight: 221.

Ultraviolet light. that destroys green plant tissue on contact (and by translocation within the plant).. A highly sensitive and specific methodology using direct injection of water extracted samples into an electrospray ionisation (ESI) module configerated with an MS ion trap has been developed. paraquat and diquat should easily move into an aqueous solution from an organic phase. It is a quick-acting.. providing there is a sufficient area of phase interface. and soil microorganisms can degrade paraquat to products which are less toxic than the parent compound. The reported half-life for paraquat in one study ranged from 16 months (aerobic laboratory conditions) to 13 years (field study) (Rao et al.4'-bipyridinium dichloride (Kidd et al. 1992). Experiments undertaken for equipment assessment LC MSMS Not compatible to LC owing to high absorption on surfaces (detection limit to 0. Analysis .1'-dimethyl-4. as ion pair reagents preclude the use of some ionisation sources in LC MS interfaces.oil components extract into water. sunlight. 1980). This presents limitations as to compatible detectors.The quaternary ammonium herbicides are not amenable to GC. LC is limited usually requiring the inclusion of ion-pair reagents or the analytes must be derivatised. The dichloride salt is sparingly soluble in lower alcohols (Kidd at al..20 Water Solubility: 700 gL-1 @ 20°C. with a reported field half-life of greater than 1000 days (Wauchope et al. It is a highly toxic compound and is highly persistent in the soil environment. whereas. Physical Properties: 2+ H3C N paraquat N CH3 2Cl - Chemical Name: 1.01 mgkg-1 ) . page 85 Properties typical of commonly used quaternary nitrogen pesticide as exemplified by paraquat Paraquat is a quaternary nitrogen herbicide widely used for broadleaf weed control. 1991) Molecular Weight: 257. 1991) Melting Point: Decomposes @ 300°C Vapour Pressure: Negligible @ room temperature (paraquat dichloride) 23 . non-selective compound.

000 (estimated) (Wauchope et al.Partition Coefficient: 4.000. 1992) 24 ..4683 Adsorption Coefficient: 1.

Assuming a pesticide residue contamination level of 1 mgkg-1. amenability to automation and robustness has ensured the adoption of this technology by most analytical laboratories.02 ng of the target analyte will be present in that same 1 µL injection. The relatively low cost. as little as 0.1 ng of any one component. The volatile fraction of solvent extracted concretes or absolutes can be 20 to 80% of the total extract whilst steam distilled oils are almost 100% volatile. Most non-specific detectors such as flame ionisation detection (FID) cannot detect much below 0. then.6 µg in a 30:1 split). 25 . In samples which have not been subject to clean-up procedures. providing the detector has sufficient sensitivity and specificity. GC must be coupled to sensitive and specific detectors. The loading capacity for a standard dimethyl polysiloxane (methyl silicone). This would imply that many oils could be loaded directly onto a GC column without clean-up. non-polar phase GC column is in the vicinity 20 µg of oil in a 1 µL split injection (0.Analytical Equipment Gas Chromatography The separation of volatile oil components using temperature and pressure gradients through capillary columns in gas chromatography has become one of the widely applied technologies in analytical chemistry. The loading limit on capillary columns depends on the stationary phase and its thickness.

high detection limits & non-specific flame photometric detection .general screening limited to detection ~1 to 5 mgkg-1 without clean-up high resolution mass spectrometry .Plate 1. Gas Chromatograph Detectors assessed in this manual include: flame ionisation .halogenated chemicals NPD detector – chemicals containing nitrogen and phosphorus mass selective detection . low detection limits with minimum clean-up 26 .high specificity to sulfur and phosphorus electron capture detection .highly specific. with confirmational ions usually available for each analyte.

Pre-columns can also prolong the viability of GC columns and periodical baking of the column through a slow temperature gradient. peak tailing and low recovery of analytes. the loading of excessive amounts of essential oil components can lead to column damage. For solvent extracted concretes or absolutes (such as boronia and blackcurrant) large amounts of non-volatile components. This will show an enhanced peak for retention time confirmation. replaced or cleaned and re-silanised before re-use. programmable injectors (SPI). including waxes and tannins. There are many types of GC injectors that afford a measure of control to reduce the loading of components onto the column. The sample may also be re-chromatographed. resulting in poor resolution. Retention times can be expressed as values relative to retention time of a standard reference compound. However. Retention characteristics of target analytes under standardised GC conditions are often insufficient for an unequivocal identification as these can vary with column conditions. However. can rejuvenate poor performing columns which have been subject to excessive loading. confirming retention times. Alternatively separation may be achieved using GC columns with different immobile phases or the target analyte may be amenable to derivatisation. may not volatilise in the GC injection chamber or may not elute from the column. with a long hold at the maximum. 27 . Many non-volatile components are condensed onto the silanised glass wool within the injector liner of the GC and this can be removed after a number of injections. Selection of a chemical with similar structural and physical properties to the target analyte will ensure that slight changes in experimental conditions will change the retention characteristics of the reference compound to the same degree as those effected on the target analyte. Standard curves.The limitations of gas chromatography in the analysis of essential oils which have not undergone clean-up and pre-concentration relates to the need to load large amounts of oil components into the systems to detect low levels of pesticide residues. if matrix components co-elute with the target analyte and saturate the detector signal. should be run immediately before and after a sample is tested for pesticide contamination. the parameters which can be optimised are limited to temperature gradients and pressure. after being fortified with the target compound. A section of the column can also be removed to improve performance. such as septum equipped.

Despite reproducible and standardised methods the retention characteristics are often insufficient for unequivocal peak assignment. 28 . Methods for confirming peak identity include. - application of clean-up techniques to remove interfering peaks. reference to mass spectral data. It is useful to pre-determine the retention indices of the components of the relevant essential oils. benchtop MSMS and HR MS. The introduction of a new pesticide into a GC screen can then be easily assessed by first determining the retention indices for the new analyte and relating them to the established indices for the essential oil components. When considering the suitability of GC ECD to the analysis of a new target analyte. contaminated with halogenated pesticide residues. derivatisation and application of different detectors specific to different functional groups on the target compound. Gas chromatographic retention indices (Kováts' indices) relate the retention time of a particular analyte to the retention time of a series of CnH(2n+2) hydrocarbons. The retention characteristics of the target analyte relative to the major components of essential oils are therefore of critical relevance. The following sections detail the experiments undertaken in the assessment of a range of GC compatible detectors. co-elution of a major oil component would mask the specificity of the ECD through saturation of the detector with a component 105 times more concentrated than the halogenated pesticide. however. the determination of the retention characteristics relative to essential oil components can provide an indication as to whether the analyte response will be masked by co-eluting extractives. NPD. As discussed previously. Obviously. Assessment of GC ECD in the Analysis of Pesticide Residues in Essential Oils.0001%. Both essential oils and solvent extracts are predominantly hydrocarbons and oxygenated hydrocarbons. The bulk of the matrix of an essential oil. This will allow an assessment as to whether the pesticide residue will elute in a time window separated from other major essential oil components. should not register by ECD. - adjustment of experimental conditions to improve peak resolution. comparing relative retention times on capillary columns having different liquid phases. compared to pesticide contamination levels which are often around 0. including ECD. FPD. any one component of an essential oil can constitute in excess of 10% of the oil. such as halogenation. Electron Capture Detectors (ECD) are specific to halogenated chemicals.

at 60°C.: Oven Temp: Injection Temp: Detector: Hewlett Plackard 5890 gas chromatograph Hewlett Plackard Flame Ionisation Detector Processing Software . Kovát's indices were established for the major components of essential oils and for the halogenated pesticides commonly used in the industry. method validations were conducted for eight halogenated pesticides in fennel.HP Chem 1 µL. A 1 mgmL-1 solution of a range of CnH(2n+2) hydrocarbons was prepared and analysed in the same column. split automatic injections 30 m HP 5MS. This indicated that co-elutions of essential oil peaks with the target analyte would be minimal. it was shown that the majority of the components of distilled oils have Kovát's indices below 1500. None the less. Retention Indices of Components of Essential Oils Aim : To establish the Kovát's indices of essential oils on a HP 5MS column under the GC conditions to be used in standard GC ECD pesticide analysis using GC FID Experimental : GC FID has the capacity to detect a 1µL split injection of a 1 mgmL-1 solution. Analytical parameters Instrumentation Injection: Column: Carrier gas: Head Press. and the adequate retention and response properties of some of the halogenated pesticides tested. 260°C FID 280°C 29 . Experiments Undertaken in the Process of Method Development Example 1. then programmed at 20°C/min to 290°C for 10 min.To assess the application of GC ECD to the detection of pesticide residues in the matrix of essential oils. whilst the majority of pesticides have Kovát's indices above this figure. as those to be used in the proposed GC ECD analysis. it was evident that the retention times alone were insufficient to unequivocally identify peaks. and under identical conditions of pressure and temperature. parsley and peppermint distilled oils and boronia extracts. Despite the high degree of specificity of the GC ECD. 0. In the experiments detailed in the following pages. GC ECD can only be used as a screen to determine that no gross contamination of essential oils has occurred. The high number of components of boronia extract with retention indices greater than 1500 effected the masking of pesticide residue peaks.22 mm id. 0.25 µm film thickness Instrument grade nitrogen 10 psi 1 min.

.Results: Figure 1. 2.GC FID of distilled fennel oil 30 . 3 and 4 are the GC FID chromatograms for injection of 20 mgkg-1 solutions of parsley. fennel and peppermint oils and boronia oils.GC FID of distilled parsley oil Figure 2. Figure 1 .

GC FID of solvent extracted boronia oil 31 . Figure 4 .GC FID of distilled peppermint oil.Figure 3 .

834 22. Time (mins) 14.817 24.t'R(N) I ab = 100N + 100n -------------------t'R(N+n) . Retention times of CnHn+2 hydrocarbons The retention times of the major components and the calculated Kováts' indices are listed in Table 3.582 20.381 18. t'R(A) (ref 1.t'R(N) Where I is the retention index on phase a at temperature b and t'R(N) and t'R(N + n) are the adjusted retention times of n . The Kovát's indices are calculated using the formula t'R(A) .126 17.794 21. 32 .paraffin hydrocarbons of carbon numbers N and (N+ n) that are respectively smaller and larger than the adjusted retention times of the unknown.Table 2 records the retention times of mixed 1 mgmL-1 solutions of hydrocarbon standards ranging from C8H18 to C36H74.804 16.435 Kovát's indices 1500 1600 1700 1800 2000 2100 2200 2400 2800 3400 Table 2.697 35.682 28.) Standard Peak nC15H32 nC16H34 nC17H36 nC18H38 nC20H42 nC21H44 nC22H46 nC24H50 nC28H58 nC34H70 Ret.

Figures 5 to 8 record the GC ECD traces of injection of 20 mgkg-1 solutions of essential oils.11 Kovát’s indices 674 743 778 827 913 962 1535 1626 2061 Kovát’s indices 821 1032 1079 1152 1221 1412 1492 Fennel oil α-pinene α-phellandrene limonene β-phellandrene fenchone estragole trans-anethole ret.96 10.07 10. ret.50 8.71 11.07 15.26 15. time 7. time 6.17 ret.49 12.47 15.Parsley oil α-pinene β-pinene myrcene β-phellandrene α-terpinolene menthatriene tetramethoxyallyl benzene myristicin apiole Peppermint oil 1. time 6. time 6. Figure 5.40 16.71 12.57 16.30 7.53 21.02 7.86 9.34 Kovát’s indices 665 733 808 1488 1520 1614 1704 2233-3266 Table 3.13 7.37 23 . chem.45 7.83 15.94 9.52 Kovát’s indices 667 773 812 812 925 1079 1249 Boronia extract α-pinene β-pinene terpinolene β-ionone dodecyl acetate methyl epijasmonate heptadec-8-ene waxes & high M.86 7. GC ECD of distilled parsley oil 33 .03 8.41 17.8-cineole menthone menthol pulegone isomenthol germacrene D piperitone ret.40 7.22 14.21 10.33 7.W. Kováts indices for major components of essential oils and extracts.

GC ECD of distilled fennel oil 34 .Figure 6. GC ECD of distilled peppermint oil Figure 7.

has interfering components eluting through to 35 minutes. The loading capacity of standard non-polar capillary columns such as HP1 or HP5MS. 35 . GC ECD would need to be able to detect 0. in 20 mg of oil constitutes a 1 mgkg-1 solution. GC ECD of solvent extracted boronia oil Conclusion: Evident in Figures 5 to 7 is that the components of the distilled essential oils which effect an ECD response.02 ng of analyte in a 1 µL injection. The solvent extracted boronia extract shown in Figure 8. however. Experiments Undertaken in the Process of Method Development Example 2. This concentration should be sufficiently high for easy detection so as to determine the retention time of each analyte and provide some indication as to the likely response under GC ECD conditions. For each halogenated pesticide a concentration of ~2 µg of analyte in 1 mL of solution is equivalent to 100 mgkg-1 in a 20 mg sample of oil. Without clean-up techniques.Figure 8. A sample containing 0. have Kovát's Indices less than 1500.02 µg of an active ingredient of a pesticide. is in the vicinity of a 1 µL split injection of a 20 mgmL-1 solution of an essential oil. Retention Indices of Pesticides in Essential Oils Aim: To establish the Kovát's indices for pesticides on a HP 5MS capillary column Experimental: An acceptable limit of detection for a basic screen would approach 1 mgkg-1.

derivatised Dicamba . split automatic injections 30 m HP 5MS. at 60°C. GC amenable pesticide were injected in a gas chromatograph with detection by ECD under the following conditions.25 µm film thickness Instrument grade nitrogen 10 psi 1 min.derivatised mecoprop .derivatised Table 4. Distilled oils (20 . 0. then programmed at 20°C/min to 290°C for 10 min. Analytical parameters Instrumentation Hewlett Packard 5890 gas chromatograph Hewlett Packard Electron Capture Detector Processing Software . Results: ECD was found to be successful in the detection of terbacil. 36 . Vials were spiked with 10 µL of an acetone solution containing a 0.derivatised trichlopyr .derivatised clopyralid . haloxyfop ester. bromacil. Sub-samples (20 .derivatised MCPA . propiconazole.Table 4 lists the pesticides commonly used in the essential oil industry which contain at least one halogen in their molecular structure. tebuconazole and difenaconazole.HP Chem 1 µL. Boronia extracts were warmed and mixed thoroughly to ensure an even distribution of all oil components.30 mg) were weighed into 2 mL GC vials. Figure 9 shows the chromatograms obtained by GC ECD.30 mg) were weighed into GC vials.derivatised fluroxypyr .22 mm id.2 mgmL-1 mix of each pesticide standard. Halogenated. Halogenated pesticides used within the essential oil industry Acetone solutions (2 µgmL-1) of each halogenated. 260°C ECD 260°C Injection: Column: Carrier Gas: Head Press.: Oven Temp: Injection Temp: Detector: The effects of the matrix on the detection of halogenated pesticides were then assessed. 0. GC amenable pesticides tebuconazole propiconazole linuron diuron simazine oxyflurofen chloroprofam bromacil terbacil procymidone difenoconazole ethofumesate dimethenamid chlorpyrifos norflurazon haloxyfop esters fluazifop esters glyphosate .

Figure 9. 37 .951 22.596 18.099 19. peppermint. time 20. and fennel oils and boronia extract.146 24. Kováts indices calculated for the pesticides shown in Figure 9.00 32.620 Kovát's indices 2013 2007 1773 1871 2270 2416 2153 2380 2421 3136 Table 5.388 24.652 20. Figures 10 to 13 shows the GC ECD chromatograms for spiked parsley. GC ECD traces of 2 µgmL-1 solutions of halogenated pesticides pesticide linuron ethofumesate simazine terbacil fluazifop ester fluroxypyr ester procymidone propiconazole tebuconazole difenaconazole ret.563 25.147 23.

GC ECD chromatogram of parsley oil fortified with 100 mgkg-1 mixed pesticide standard Figure 11. GC ECD chromatogram of peppermint oil fortified with 100 mgkg-1 mixed pesticide standard 38 .Figure 10.

GC ECD chromatogram of fennel oil fortified with 100 mgkg-1 mixed pesticide standard Figure 13. GC ECD chromatogram of boronia extract fortified with 100 mgkg-1 mixed pesticide standard 39 .Figure 12.

s are listed in Table 6. Samples were analysed using the same parameters as listed on page 40. bromacil. Method Validation for Detection of Pesticides by GC ECD in Parsley. terbacil. is limited. haloxyfop and fluazifop esters. Peppermint and Fennel Oils and Boronia Extracts.01 to 10 mgkg-1. and the repeatability as expressed by the r. Experiments Undertaken in the Process of Method Development Example 3. Repeat injections at each concentration of fortified oil were analysed to determine repeatability. linearity as expressed by the r2 value of a linear regression.1 mgkg-1.d. propiconazole. without clean-up techniques.Discussion: The application of GC ECD to the detection of halogenated pesticides in essential oils. Detection limits are unlikely to be less than 0. Method validation has been undertaken for the peaks which showed good response and chromatographic properties. 40 . a preliminary method validation experiment was designed to include simazine. Experimental: Four 20 mg aliquots of each oil were spiked with standard solution mixes to produce a range of concentrations of 0. The large number of interfering peaks from co-eluting extract components makes the unequivocal identification of unknown peaks impossible. Results: The detection limits.s. Aim: From the results presented in Figures 10 to13. tebuconazole and difenaconazole.

Tests were undertaken with acephate.999 0.02 18.19E-09 5.3 7.000 1.92 20. with 41 . with partitioning of carbon disulfide into n-octane was trialed (adapted from methodology published by Woodrow 1995).02 1 0.92 32. This well established technique for the detection of dithiocarbamate residues was applied to the analysis of essential oils.02 Table 6.01 0. due to thermal degradation and poor interaction with the liquid phase of the GC columns.5 1 1 1 50 5 1 1 5 1 5 5 1 22.998 0.Pesticide ret.5 0.000 0. The gaseous product can undergo head-space sampling with analysis by GC FPD in the sulfur mode. Assessment of GC FPD GC FPD was not found to be particularly suitable for the analysis of pesticide residues in essential oils and extracts without considerable clean-up procedures. ECD will be effective to establish that no gross contamination is present in oils or extracts in the absence of a co-eluting peak. Poor chromatography.mint parsley bor. such as high resolution mass spectrometry.2 4. Detection limits and repeatability for the analysis of halogenated pesticides in essential oils and extracts by GC ECD Discussion: Analysis of pesticide residues in essential oils and extracts by application of GC ECD are not specific enough to allow for an unequivocal identification of any contaminant.12E 1 -2.5 1 0. As a screen. In the situation where a peak is recorded with the correct elution characteristics.01 0.5 0.s.31 26. The detection of mancozeb in peppermint oil using acidified stannous chloride digestion.92E-09 -1 1 2.39E-06 6.15E-05 1.50E-05 8.02 0.7 12. curve coefficients x 2 x curve fit 2 r detection limits solvent µgmL -1 fennel pep.56E 0 -1 1 0. though with several adaptations to the sampling method for the gaseous end product.5 5 0. % simazine terbacil bromacil haloxyfop ester fluazifop ester propiconazole tebuconazole difenaconazole 18.d. and which is enhanced when the sample is fortified with the target analyte. Digestion of dithiocarbamate pesticides with acidified stannous chloride produces carbon disulfide.26E-06 5.5 1 5 0.29E-09 9.34E-05 1.55 22.995 0.7 5. ECD is a screen for contamination and will not provide a positive identification for any pesticide contaminant. time std.05 23. precludes this detection method in specific screens for the analysis of residues in essential oils.02 0. Recoveries were as low as 10%.70 9. methedimos and monocrotophos. r. a second means of contaminant identification would be required.85E-05 2.00 21.997 0.2 13.3 2.

However. the methodology for the analysis of peppermint leaves is included in the appendix 1. The quantification of the amount of ethylenediamine produced was trialed. The presence of endogenous sulfur chemicals precludes this methodology for the analysis of mancozeb in most essential oils. The NPD collector contains a small alumina cylinder coated with a rubidium salt (active element) which is heated electrically. Assessment of GC NPD in the Analysis of Pesticide Residues in Essential Oils.detection limits of around 10 mgkg-1.6%. The ionised organic molecules are collected and the resulting current is measured. None the less experiments were undertaken to determine the potential of GC NPD to detect pesticides in essential oils. of 10. gave background levels as high as 5 mgkg-1. The specificity afforded by the efficient ionisation of organic molecules containing nitrogen or phosphorus may allow for the NPD to selectively respond to nitrogen and phosphorous containing pesticides. Nonetheless. Much of the points noted under the assessment of GC ECD apply for GC NPD with the probability of excessive amounts of the components quenching the signal. peppermint oil samples that had not been treated with mancozeb gave positive results for the production of carbon disulphide using the digestion methods described. The NPD is based on the ionisation of nitrogen and phosphorus in a thermionic source within a jet flow of hydrogen and air. Residual stannous chloride or hydrochloric acid may have promoted the depletion of the derivatising agent. Many essential oils have endogenous sulfur components and analysis of carbon disulfide by acid digestion of samples.d. A by-product of acidified stannous chloride digestion of dithiocarbamates is ethylenediamine. The response for carbon disulfide by GC FPD was non-linear and less sensitive than that recorded for phosphorus compounds using the 524 µm filter. Detection of mancozeb at concentration levels equivalent to 20 mgkg1 gave a r. Detection of the carbon disulfide using headspace analysis with detection by FPD has been trialed. preferentially detecting them from amongst the C H and O containing components of essential oils. not previously treated with dithiocarbamate chemicals. However. Derivatisation of ethylenediamine with BSTFA to convert the product to a GC amenable analyte was successful. Mancozeb was digested in the matrix of peppermint leaves and oils and compared to solvent only digestions.s. 42 . detection of ethylenediamine produced from the digestion of mancozeb standard was unsuccessful.

22 mm id. 1 µL. Varian NPD.25 µm film thickness Instrument grade helium 10 psi 60°C (1 min. The Detection of Pesticides by GC NPD in Peppermint Oil. simazine. The retention times of the proposed analytes were established by injecting 1 mgkg-1 solutions of each pesticide into a GC with detection by FID.) -10°C/min-280°C (15 min. Aim: To undertaken an assessment of the response of GC NPD to pesticides in the matrix of peppermint oil.Experiments Undertaken in the Process of Method Development Example 4.1 mV -4. Experimental: The pesticides included in this experiment were monocrotophos. 0. cyanazine. propazine. tebuconazole and difenaconazole. propazine. 20 and 2 ngmL-1 in acetone were prepared for direct comparison to equivalent solutions but also containing 20 µLmL-1 of peppermint oil. 0. simazine. Figures 15 and 16 show the GC NPD traces of pesticide fortification levels of 1 mgkg-1 in solvent only and in peppermint oil respectively. The column and GC parameters used to establish the retention times were replicated when transferring analysis to the GC NPD. cyanazine. propiconazole. simazine. propiconazole.0 mV Injection: Column: Carrier gas: Head Press: Oven Temp: Injection Temp: Detector: Filament voltage Voltage offset Results: Figure 14 show the chromatogram of a 10 µgmL-1 acetone solution of monocrotophos. Processing Software – Varian Starr Workstation. tebuconazole and difenaconazole was analysed by GC NPD using the conditions listed. 10 and 1 mgkg-1 pesticides concentrations in peppermint oil. tebuconazole and difenaconazole. cyanazine. 43 . This translated to 20. Analytical parameters: Instrumentation: Varian 3300 gas chromatograph. Solutions of 200. Retention times were used to identify peaks. propazine.) 260°C NPD 300°C 3. An injection (1 µL) of a 20 µgmL-1 mixed solution of monocrotophos. propiconazole. splitless manual injections 30 m HP5MS.

44 . Pesticides (1 mgkg-1) in solvent analysed by GC NPD. Figure 15.Figure 14. Pesticides (10 µgmL-1) in acetone analysed by GC NPD.

simazine propazine cyanazine propiconazole propiconazole tebuconazole ret. Relative response of nitrogen and phosphorous containing pesticides fortified at a level equivalent to 1 mgkg-1 relative to oil in solvent and peppermint oil.429 18.327 25.487 25.445 18.Figure 16. The oil has limited effect on the retention time but suppresses the response of the NPD by over 80 % for all the pesticides. The relative response of pesticides in oil compared to pesticides in solvent expressed as a percentage is also listed. time mins 18. 45 .864 peppermint oil 1186 2139 729 190 344 436 % response pepp.335 25. Pesticides (1 mgkg-1) in peppermint oil analysed by GC NPD The areas obtained for each fortification levels of pesticides in acetone are compared to the response of equivalent concentrations of each pesticide in peppermint oil in Table 7. the selectivity of the NPD is encouraging and the analyte peaks stand well above the background signal.319 25.883 solvent only 8505 12091 3444 13760 2238 2335 ret. However.506 25.344 25.645 21.630 21. The earlier eluting isomer of propiconazole is suppressed by 99%.oil to solvent only 14 18 21 1 15 19 Table 7. despite the quenching of the response. time mins 18.

with encouraging results. The equipment accessed by this project was a Varian Starr 3400 CX GC coupled to a Varian Saturn 4D MS/MS. only ions enter directly into the ion trap. The introduction of a 1 µL split injection may contain as much as 20 µg of essential oil components. Unlike other mass spectrometry systems. Although several of the pesticides relevant to the essential oil industry were trialed in solvent solutions.Assessment of Benchtop GC MSMS Despite the high expectations based on the sensitivity and selectivity of the application of benchtop gas chromatography daughter / daughter mass spectrometry. With a split ratio of 1 : 50 the injector will deliver 0. Ionised components of the background matrix are often in sufficient abundance to fill the ion trap prior to the ionisation of the target analyte. it was not considered practical to continue method development within the matrix of essential oils without comprehensive clean-up methodologies. 46 . severe limitations relating to the configuration of the system. In addition essential oil components would quickly accumulate within the ion trap. precluded its application in the analysis of pesticide residues in essential oils without considerable clean-up techniques. The Saturn MS/MS. collects a specific number of ions. In some mass spectrometers.4 µg onto the column. The mass spectrometer is required to be shut down for several days for the trap to be cleaned. the effluent from the gas chromatograph elutes into ion trap before ionisation. under the control of the 'Autogain'. The entire eluant then enters the trap such that the isolation of specific target parent ions is difficult.

thiols. a high level of expertise is required to maintain and operate such an instrument. However. HRMS has the capacity to measure the m/z ratio to such an accuracy as to be able to determine the molecular mass of fragments to 4 and 5 decimal places. Benchtop GC MSMS Assessment of GCHRMS in the Analysis of Pesticide Residues in Essential Oils. with the difficulties inherent in the analysis of pesticide residues within essential oils GC HRMS affords a capacity for specific and sensitive detection for all GC amenable analytes irrespective of the presence or absence of halogens. 47 . This provides a high degree of specificity and when used in the selected ion monitoring mode (SIM) can detect target analytes even when they are in a much lower abundance amongst co-eluting matrix components. amines or phosphates. In addition.Plate 2. Very few analytical laboratories have access to this sophisticated and expensive equipment.

dimethanamid. norflurazon and diuron by GC TIC. As a case example. High Resolution Mass Spectrometer The Application of GC HRMS in the Analysis of GC Amenable Pesticides. Gas Chromatograph. corresponding to the fragmentation 48 . Figure 18 shows the MS of sethoxydim. oryzalin. simazine. The retention time is recorded and the ions most suitable for monitoring are selected on the basis of their abundance and diagnostic potential. sethoxydim. dichlorpyrifos. bromacil. Ions with masses similar to those originating from co-eluting components of essential oils can be selected. The ions selected for monitoring were 191 and 219. then the first full scan run can include a range of pesticides for the determination of retention time and ions suitable for monitoring. which elutes at 10:53 minutes on the GC temperature ramp used.Plate 3. Each analyte is run by GC MS in full scan mode. If the relative retention and basic MS of analytes are known. Figure 17 shows a sample run of acephate. as the degree of mass discrimination afforded by high resolution allows distinction of the ions separated by as little as 10 ppm in mass or 0.05 atomic mass units (amu) at m/z 500. The MS and retention characteristics of the active ingredient of each pesticide are first assessed.

TIC of pesticides by GC HRMS Figure 18.0946 and 219. Figure 17.1259. MS of sethoxydim 49 .ions C11H13NO2 and C13H17NO2. The masses for the ions to be monitored by high resolution SIM were calculated to be 191.

norflurazon. simazine. octadecane was observed to separate. preventing quantitative determination. 50 . The actual retention times are omitted as this parameter is subject to variations depending on column type. temperature and pressure gradients etc. bromacil.Having determined the retention times and suitable mass fragments for monitoring.. Should this analyte be present in oils. When samples were prepared in polar solvents. the high level at which the standard is spiked should overwhelm any background contamination and not significantly affect quantification. It chromatographs well on capillary GC and it is not commonly used in the essential oil industry. Table 8 lists the analytes. However. Figure 18 shows a typical GC HRMS run established to monitor diuron. chlorpyrifos and sethoxydim. In HRMS the response of ions monitored will change with instrument tuning which must be optimised each day of use. In early method development octadecane was selected and the ion monitored was 254. Endosulfan was selected as an internal standard as the chemical structure and properties are similar to that of other pesticides. Method development has been undertaken for 19 pesticides that can be directly analysed by GC HRMS. dimethenamid.2973. The ion selected for monitoring of the internals standard was 194. the ions monitored and general comments with regard to their chromatographic behaviour. the mass spectroscopist establishes an instrument run whereby the HRMS is programmed to switch between ions to be monitored within time windows spanning the retention time relevant to each particular analyte included in the screen. An internal standard is necessary. as a result of extraction protocols etc.9534. acephate. The pesticides are listed in order of elution under the conditions trialed. the solubility of this hydrocarbon presented some limitations.

Figure 18. GC HRMS chromatogram of 9 pesticides 51 .

0714 191.1344 261.1259 Confirmation ion 186.9534 230.1 0.1361 201.1 0.05 0.Active ingredient linuron diuron acephate monocrotophos terbacil chloropropham int.0262 252.05 0.1 0.0035 393. Propiconazole.02 0.1 0. Vials were fortified with the standard solutions as listed in Table 52 .ions monitored and chromatographic characteristics Experiments Undertaken in the Process of Method Development Example 5.0875 194.05 0.0984 204.05 0. Experimental: Standards were purchased from Sigma Aldrich and approximately 25 mg of each were weighed into 25 mL volumetric flasks and made up to volume with acetone.05 1 -1 226. into 25 mL of acetone.05 0. Simazine. Solutions of 10µgmL-1 and 1 µgmL-1 were prepared by diluting 250 µL and 25 µL of the 1 mgmL-1 solutions.9584 186.1 0.0386 219.05 0.1 1 1 1 0.0546 good med. prometryn simazine ethofumesate int.05 0. Method Validation of the Analysis of Tebuconazole.9555 188.0781 286. poor poor good good good Det.0527 Chrom.0291 250.0406 252.1376 198.1126 186.9202 252.0946 good good good good good good diastereomers some tailing good some tailing Table 8. properties degrad.02 0.9172 300.0557 254.05 0.5 0.1 0.02 0. limit peppermint mgkg 1 1 1 0.0160 161.05 0. Terbacil.01 0. dimethenamid pendimethalin bromacil chlorpyrifos oxyflurofen fluroxypyr propiconazole tebuconazole norflurazon sethoxydim Trade name Linuron Krovar Orthene Nuvacron Sinbar Allicide octadecane Gesaguard Gesatop Tramat endosulfan Frontier Stomp Krovar Lorsban Goal Fusilade Tilt Folicur Solicam Sertin Ion mass 188.9555 136. prod. limit boronia mgkg 0.02 0.05 0.05 1 -1 Det.2973 241. Analysis of 19 pesticides by GC HRMS .05 0.02 0.0743 303. std.0742 259.05 0.0377 281. Bromacil and Oxyflurofen in Boronia Extract by GCHRMS Aim: To validate the methods developed for the detection of 6 pesticides in the matrix of boronia extract using GC HRMS. std. good good 232.05 0. degrad.0117 213. prod.05 0.9584 97. respectively.0164 127. Standard curves were prepared by weighing 22 x ~20 mg of boronia concrete into GC vials.0398 282.9613 196.

02 0 - 1 no.9. Recoveries were determined by establishing a standard curve in boronia solutions which had been subject to the same partition clean-up step described above. min. 1. Hexane (200 µL) was added to each. propiconazole.1 min. then at 1 psi.d. Spiking protocol for the establishment of standard curves and repeatability experiments Methanol (0. between 7:50 and 8:12 minutes. The bases of the vials were gently warmed by briefly placing them on the base of an oven where required. The GC injection temperature was 260°C and the oven temperature programmed from 60°C to 290°C at 20°C min. 0.25 µm film thickness). the lids were placed firmly on top.-1 to 35 psi. and the mixtures gently swirled to dissolve the concrete.-1. monitored between 8:12 to 9:00 minutes. 0. The GC was equipped with a BPX5 fused silica capillary column (25 m.0743. without sealing. Splitless injections of 1µL of sample were analysed using a carrier gas flow program of 30 psimin-1 from 25 to 40 psi.. ion 252. tebuconazole. Ion monitored for terbacil was 161.1µgmL -1 endosulfan 5 5 5 5 5 5 5 20 20 10 4 0 Table 9. The lower layer was filtered through cotton wool into 200 µL glass GC vial inserts for analysis. then at 30 psi. Analytical parameters: Samples were analysed on a HP 5890 Gas Chromatograph directly coupled to a Kratos Concept ISQ Mass Spectrometer.0117 between 5:00 and 7:50 mins.05. held for 0. and 10 mgkg-1 in oils. to allow for 6 replicate samples at 3 concentrations to establish repeatability. Concentrations (mgmL ) 20 10 1 0. but spiked after that process and using the same fortification levels as listed in Table 9. After a settling period of ~ 5 minutes the lids were removed and 4 drops of distilled water were added to each vial.-1 to 25 psi. Endosulfan (5 µg) was added and the vials sealed for analysis.0398. which were then sealed and shaken vigorously to ensure complete mixing. endosulfan. For oxyflurofen. 53 . Ion 194. Endosulfan (5 µg) was added to each vial as an internal standard.. 1 6 6 1 6 1 1 10µgmL 40 20 -1 1µgmL -1 0.8 mL) was added to each vial. ion 250. min.1 0. In addition a standard curve was prepared by fortifying methanol / water solutions with standard pesticide solutions to concentrations equivalent to 0.22 mm i.9534 was monitored for internal standard.05 0.

899 r.050 0.9613 and simazine.3 kV. limit (mgkg-1) 0. Electron ionisation was undertaken at a source temperature of 210°C and an electron energy of 70 eV. Results: Table 10 records the detection limits and the relevant statistical data for the detection of the 6 analytes by GC HRMS.9856 from perfluorokerosene was used as the lock mass for all analytes and the internal standard.s increased as the concentration at which they were obtained decreased.998 0.146 0.999 1. Detection limits. The poor peak shape of tebuconazole. A dwell time of 300ms/ion and 50 ppm voltage sweep were employed for all ions.000 (10% valley definition) and the ion m/z 242.995 0.046 0. 54 . The Application of GC HRMS in the Analysis of Pesticide Residues with Acidic Moieties.s. When the esters come in contact with the soil the ester is cleaved to form the acid. The parent ester is often present in more than one form and may include methyl.05 0. The fluazifop and fluroxypyr esters have been incorporated into GC HRMS and ECD methodologies. however. Discussion: The chromatographic properties presented the primary limitation to low detection levels. require derivatisation to render them amenable to analysis by GC.445 r2 0.05 0.05 0. ethyl or ethoxy ethyl derivatives. The acidic forms of this group of pesticides.995 0.S. recoveries and r.1 0.1 x coefficient 0. ion 201.D. ether solutions of diazomethane are convenient and effective.0210. analyte tebuconazole propiconazole oxyflurofen bromacil terbacil simazine det. resulted in the interference from background noise at low levels such that the detection limit was 10 fold higher than that obtained for propiconazole.054 0.5 0.000 0.0871.043 0. Of the many methylating reagents available. Resolution of 10. for example.s. The commercially produced formulations available for growers usually contain the ester form of the carboxylic acid specific to this group of pesticides.d. ion 204. The reaction of the herbicide trichlopyr and diazomethane is shown. bromacil. The ester form of the active ingredient can often be included in the general screen for GC amenable pesticides.d.s for pesticides analysed by GC HRMS. (at 1 mgkg-1) 17 9 13 12 14 9 recovery (at 1 mgkg-1) 95 85 72 82 107 90 Table 10.ion 259. with an accelerating voltage of 5. R.

with and without derivatisation. MCPA. trichlopyr. The retention times and the ions to be monitored are accurately calculated and a suitable screen is programmed within which the mass spectrometer switches between the ions to be monitored in each time window. Figure 19 shows the chromatogram for the analysis of haloxyfop. dicamba and clopyralid.Cl N OCH2 COOH CH2 N2 Cl N OCH2 COOCH3 Cl trichlopyr Cl ether/10mins Cl trichlopyr methyl ester Cl [(3. with endosulfan included as an internal standard. The amount detected in the underivatised sample can then be subtracted from the amount detected in the methylated sample. In this circumstance the sample should be analysed twice. If the parent ester of the carboxylic acid is a simple methyl ester. Following the inclusion of a derivatisation step method development is the same as that undertaken for the other GC amenable pesticides. A TIC is obtained. This will give the amount of acid contaminating any one sample. the derivatisation of the acid with diazomethane will produce an analyte identical to that which originally present in the commercial formulation. 55 . Often cleavage from the ester to the acid form in the field situation is incomplete and derivatised acid residues are indistinguishable from residues of the ester.6-trichloro-2-pyridinyl)oxy]acetic acid The preparation of the diazomethane solution is presented in appendix 3. either be GC with a mass selective detector (MSD) or on the GC HRMS.4.

mcpa. ions monitored and detection limits for haloxyfop. 56 . GC HR MS of herbicides with acidic moiety Table 11 shows the analytes.Figure 19. dicamba and clopyralid. trichlopyr.

providing numeous options of solvent combinations within one chromatographic run. However. The concentration of the target analyte may then be measured. HPLC systems can be interfaced with a range of detection systems.1 -1 Table 11. such that the collision of the ions effect a controlled breakdown of the parent ion to form daughter ions.0352 209. the effluent from the LC system is nebulised and can be ionised by a range of methods as described below.0397 202. polarity of both mobile and stationary phases and gradient profile of the liquid phase. this manual only includes the assessment of detection using ion trap MS. into an ion trap where the electromagnetic field oscillates to reject ions without the correct m/z ratio. The charged constituents of the aerosol are then transferred to the mass separation system by the action of a high vacuum. The ionisation methods interfacing liquid chromatography to ion mass detectors assessed are: Thermospray (TSP).1 0.5 0.9666 173.The LC effluent is sprayed in the vicinity of a small ion sampling orifice. The second level of fragmentation.Proton transfer reactions may be stimulated by using a filament or discharge electrode. termed filament assisted TSP or discharge assisted TSP respectively.Active ingredient haloxyfop trichlopyr dicamba mcpa clopyralid Trade name Verdict Garlon Dicamba MCPA Lontrel Ion 316. • Electrospray (ESP).1 0.9280 214. however. Assessment of HPLC in the Analysis of Pesticide Residues in Essential Oils In ion trap MS. good good good good good Det.1 0. High pressure liquid chromatography (HPLC) systems can be configerated with multiple pumps. GC HRMS of derivatised residues of pesticides with acidic moieties Liquid Chromatography Liquid chromatography presents as a less deleterious separation process for thermally labile chemicals. known as MSMS. The stream of ionised molecules passes through a system by which many of the non-ionised particles are eliminated.9513 Chrom. Ionisation is effected by applying a high voltage to the needle through which the spray exits. 57 . These include flow rate. has application for non-volatile components and has more flexibility in terms of adjustable parameters. limit mgkg 0. is normally very specific to the target ion and the level of interfering ions is greatly reduced. co-eluting ions may confuse the background and so daughter ions can be formed from the target analyte by increasing the energy of the selected ions in the ion trap.

. The primary ions may be solvent molecules ionised by a corona discharge or desolvated buffer ions. Waters 2690 HLC and Ion Trap Mass Spectrometer Retention of Essential Oil Components on HPLC Columns Clean-up methods were not to be considered within the initial framework of the analytical methodology. Without clean-up techniques the threshold of detection is limited by the amount of oil which can be safely loaded onto the HPLC system without the loss of column functionality. Ultraviolet (UV) detection was applied to determine the total elution profile of essential oils and extracts introduced into the HPLC and to ensure that 58 . The retention characteristics of essential oils on a standard reverse phase (RP) HPLC C18 column were therefore assessed. Plate 4. itself.Ionisation occurs by molecular collisions. The potential of oil components to be retained within the column under a given mobile phase protocol may lead to a shorter working life of the column and the infusion of oil components into the MS system.• Atmospheric pressure chemical ionisation (APCI). has the potential to permanently contaminate the ion trap. A Total Ion Current (TIC) by MS of the effluent from a LC column will only detect chemicals which have been ionised.

3. The flow from the column was diverted to a Waters photo diode array detector (PDA). 59 . Analytical parameters: LC .9 x 150 mm column was used to establish a mobile phase with gradient of 50:50 acetonitrile / 0. Peppermint and Fennel Distilled Oils on RP HPLC Columns. Experiments Undertaken in the Process of Method Development Example 6. Aim: To assess the retention characteristics of essential oils on RP HPLC columns using acetonitrile / ammonium acetate buffer mobile phase Experimental: Four 200 µL (~200 mg) aliquots of each of peppermint. Results: Figures 20 to 22 show the LC UV traces of the essential oils analysed under the conditions listed. the flow from the HLPC was diverted to a collection vessel and the sample analysed by GC MS. Injections of 20 µL were introduced into the HPLC system detailed below.1M ammonium acetate buffer ramped to 90:10 over 15 minutes.A Waters 2690 separation module (Alliance) system equipped with a NOVAPAK C18. In addition.components do not remain on the column. fennel and parsley oil were dispensed with an Eppendorf into LC vials and 800 µL of analG acetone was added. Behaviour of the Components of Parsley.

20 0.00 6.80 AU 0.00 Minutes 10.00 0.00 22.00 Minutes 14.40 1.00 10.60 0. HPLC UV trace of distilled parsley oil (220 nm) 60 .00 14.50 3.00 0.00 6.20 2.00 2.00 8.00 4.50 2.00 2.00 AU 1.00 Figure 20.00 18.00 Figure 21.20 1.00 -0.00 12.00 4.40 0.00 12.00 16.3.00 8.50 0.00 20.50 1. HPLC UV trace of distilled fennel oil (270 nm) 1.

or for the separation of pesticide residues from essential oil components. 3. the flexibility afforded by the interchangeable nature of the stationary phase of the column. HPLC UV trace of distilled peppermint oil (270 nm) The fractions collected from the HPLC were subject to GC MS analysis to confirm the elution of the majority of essential oil components from the LC system.00 12.00 14.9 x 150 mm column was selected with acetonitrile / ammonium acetate buffer as the mobile phase. Optimisation of these parameters for each analyte would be a difficult undertaking.00 0. the solvents.50 3.00 2.50 0. the solvent gradients and the flow rates allows for adaptations to accommodate specific analytes. In the assessment of the HPLC equipment a reverse phase NOVAPAK C18. HLPC MSMS Method Development In HPLC.00 AU 1.50 1. This system may not necessarily be optimal for each analyte.00 8.00 Minutes 10. but this commonly used system served to give workable results from which more specific methods may be developed.3.00 4.00 Figure 22.00 6.50 2. Discussion: The main components of distilled essential oils do not remain on the RP column under the parameters tested.00 2. 61 .

All the chemicals were trialed using APCI in the negative and positive mode. 62 . Bromacil and terbacil were successfully ionised in the negative mode using ESI but combined with the poor sensitivity or lack of ionisation for the great majority of analytes trialed.The first steps in method development are the determination of the mass spectrum of the analyte and the establishment of tune file optimising the collision energy and isolation width specific for each parent and daughter ion for each ionisation source. ESI linuron oxyflurofen propiconazole prometryn tebuconazole sethoxydim simazine endosulfan bromacil terbacil haloxyfop ester fluazifop ester fluroxypyr ester NR . Table 13 shows the general parameters established. Hence MS/MS could not be used to increase the specificity and lower background noise. For analytes such as tebuconazole the molecular mass ion was detected but no useful daughter ions were produced.8 mLmin-1. Mass ions and collision energies detected by fragmentation of pesticide parent molecules by ESI Preliminary results listed in Table 12 indicate that ESI has limited application. No response was recorded for some of the most commonly used pesticides in the essential oil industry such as linuron. oxyflurofen and bromacil.no response parent ion NR NR 342 242 308 328 202 NR NR 215 (-ive) 434 384 369 159 316 328 255 15 25 19 27 6 6 5 6 159 200 NR 282 124 17 18 5 4 21 20 6 5 daughter ion collision energy isolation width (amu) no response no response notes Table 12. ESI appears to have limited application for a wide ranging screen. Table 12 records the parameters established for a selection of pesticides analysed by ESI and APCI in the positive and negative modes. To undertake the optimisation a 10 µgmL-1 solution is infused directly into the MS using a worm driven syringe at a rate of 0.

APCI is the preferred ionisation method for several reasons. making overnight runs less costly. simazine.201 282. though poor. compared to ESI.163 200.161. Chromatography Once the ions to be monitored by mass spectrometry were established the retention characteristics of the analytes were assessed using the acetonitrile / ammonium acetate buffer elution profile on the RP column. bromacil. sethoxydim.330 202.283 124 203.205 161. APCI uses less nitrogen gas compared to ESI.243. In addition. Mass ions and collision energies detected by fragmentation of pesticide parent molecules by APCI Results indicated that APCI had limited application for the detection of linuron. In addition.218 NR NR NR daughter ions 182 316 159.310 328. propiconazole and tebuconazole were poor.APCI Linuron Oxyflurofen Propiconazole Prometryn Tebuconazole Sethoxydim Simazine Endosulfan Bromacil Terbacil Haloxyfop ester Fluazifop ester Fluroxypyr ester NR . simazine and terbacil eluted in a very narrow time frame under the mobile phase conditions tested. tebuconazole and propiconazole and showed a response. to linuron and oxyflurofen. However. oxyflurofen and the esters of the acid moiety pesticides. which is pressure sensitive.329. excellent results were obtained for prometryn. Also.344 242.163 collision energy 19 21 21 isolation width (amu) 6 5 6 notes poor response poor response 19 19 5 5 Table 13. poor results were obtained for tebuconazole.244 308.343.204 NR 261.no response parent ions 251 363 342. Again.259(-ive) 215. The chromatographic characteristics of the triazole pesticides.217. • APCI does not have the high back pressure associated with ionisation by ESI such that APCI can be run in conjunction with UV-VIS without risk of damaging the UV cell. bromacil and terbacil. APCI was slightly more sensitive for the commonly used pesticides. with no daughter ions generated. Terbacil had a higher 63 .

Method Development for the Analysis of 14 Pesticides by HPLC MSMS Using APCI in the Positive Mode. An example of method development is shown below. pirimicarb. dicamba. Fine tuning of the phase gradient. Experiments Undertaken in the Process of Method Development Example 7. collision energy (CE) and isolation width were determined for each compound. however. mcpa. Overall. pendimethalin. Experimental: As previously described. 64 . adjustments of the pH to provide better resolution of the haloxyfop esters and triazine pesticides and trials of alternative mobile phases are all aspects which warrant further experimentation. the similar elution characteristics of simazine. The MS trap has the capability to switch from the positive mode to the negative several times within a single run. dimethoate. The preliminary method developments described form the foundation on which comprehensive method development can continue. [M + H]+ ion. carbaryl. standards of all the target analytes were prepared to concentrations of 10 µgmL-1 in acetone and each were introduced into the MS via the continuous flow worm drive with the mobile phase of 50/50 0. procymidone.response in the negative mode by APCI. precluded the monitoring of these three pesticides in the same screen as there was insufficient separation to allow for switching between the ionisation modes. ethofumesate. which is ionised in the positive mode. cyanazine. difenoconazole. daughter ion.8 mLmin-1. Target compounds which had good ionisation potential in positive mode APCI were then analysed by HPLC MS using the mobile phase previously established. monocrotophos and propazine. However. Aim: To undertake method development for acephate. The process of establishing the ionisation properties and optimising tuning files. collision energies and isolation widths was undertaken for a range of chemicals to be included in a general screen.1M ammonium acetate buffer / acetonitrile flowing from the HPLC at 0. The ionisation potential. glyphosate. the mobile phase has general application to most of the pesticides trialed.

sethoxydim. Negative ionisation mode for APCI gives improved characteristics for dicamba. Acephate.341 259. pirimicarb.163 141.E.232 Table 14.284(-) 199. tebuconazole.216 175. Parameters established for the analysis of pesticides by APCI in the positive mode. Int. Analytes that ionised in the negative APCI mode were incorporated into a separate screen which included bromacil and the pesticides with an acidic moiety. std. 239.98 188. 221 230 406.143 141. pirimicarb. peppermint. parsley and fennel. which requires positive ionisation.143 193. The method validation was conducted for three oils. The thirteen pesticides included in this general screen were monocrotophos. cyanazine. Bromacil and terbacil were not included as both require negative ionisation and elute within the same retention time window as simazine.Results: Table 14 lists the optimised parameters for the analytes to be included in the screen.=poor possible ESI or -ive APCI strong signal strong signal but lost in LC weak signal-alternative LC poor signal +ive & -ive strong signal & LC peak 229 230.283 282.195 212.213 161. daughter ion weak. carbaryl. MCPA and mecoprop but this ionisation mode was compromised by the acetate buffer.207 182.190 band width 3 4 5 8 4 6 4 3 4 6 5 4 5 5 C. propiconazole. 183 201 240 220 229 405 286 169 238 281 283 200 parent ions 184 202. ethofumesate and pendimethalin all require further work for enhanced ionisation and/or improved elution profiles. difenoconazole and the esters of fluroxypyr.203 241.214 224. prometryn. dimethoate. propazine and difenaconazole into the standard screen already established.339. fluazifop and haloxyfop.145 214.201(-) 213.240 282.177 199 337.225 daughter ions 143 143. analyte acephate carbaryl cyanazine dicamba dimethoate difenaconazole ethofumesate glyphosate pirimicarb pendimethalin procymidone MCPA mecoprop monocrotophos propazine M. simazine.243 219.408 287 no ion. 65 .W. propazine.double peak 3 daughters similar inten.167. 13 20 18 17 20 22 20 22 20 23 18 18 22 18 notes buffer adduct=loss signal good ionisation but lost in LC pot.241. Discussion: The results listed above formed the basis for the inclusion of monocrotophos. procymidone. try -ive good ionisation but lost in LC good signal .

50 2.1.6. 238 229 327 241 307 341 406 centre of parent m/z window [M+H] 224 202 242 239 231 328 242 n/a 344 408 isolation collision product tentative identification [M+H-CH3NH2] [M+H-HCN] [M+H-C3H6] [M+H-C3H6] [M+H] + + + + + + + retention time (mins) 1. Oils were fortified with solutions of the mix of the 13 pesticides to the equivalent of 0. fennel and parsley oil. A Finnigan MAT LCQ was used to monitor the ions listed in Table 15 within the relevant time windows.7. 0.50 6.1M ammonium acetate buffer ramped to 90:10 over 15 minutes.310 159. 3.12 + monocrotophos simazine cyanazine pirimicarb propazine sethoxydim prometryn tebuconazole propiconazole difenaconazole width (amu) energy (CE) ions monitored % 5 22 193 4 5 3 5 5 5 n/a 6 6 18 18 22 18 17 20 n/a 21 22 124 214. 66 .66 7.0 and 10 mgkg-1.W.45 3. analyte M.58.190 282 200 308.339 [M+H-C2H5ClN] [M+H-C3H7N] + [M+H-C2H6O] + 3.15 5.9 x 150 mm column was used to establish a mobile phase with gradient of 50:50 acetonitrile / 0. All fortified oils were spiked with 20 µL of 100 µgmL-1 solutions of cyanazine as an internal standard.62. which were then diluted to 100. 1.11 4. Experimental: Four 200 µL (~200 mg) aliquots of each of peppermint.56 2.90 5. The oils were analysed by LC using the following conditions.216 182 188. 10 and 1 µg/mL standard solutions.5. Stock solutions (1 mgmL-1) of the 10 pesticides were prepared in volumetric flasks in acetone.Experiments Undertaken in the Process of Method Development Example 8. Analytical Parameters: A Waters 2690 separation module (Alliance) system equipped with a NOVAPAK C18.81 [C7H4Cl2+H] [M+H-C2H3N3] Table 15. of major isotope 223 201 240. fennel and parsley oil were dispensed with an Eppendorf pipette into GC vials and 800 µL of acetone was added. Method Validation for the Analysis of 10 Pesticides in Distilled Oils Aim: To validate the method developed for the analysis of 10 pesticides in peppermint.161 337. Parameters and conditions for the monitoring of ions monitored by MS.

01 .18 MA: 30994994 NL: 1.50 309.50 .0 2.50] RT: 2.0-200.5 F: + c SRM ms2 344.5 4.00] NL: 2.5-182.74 .00 [ 122.217.5-124.5 F: + c SRM ms2 408.00 [ 280.96E6 m/z= 336.5.42E6 m/z= 181.49 AA: 4137884 monocrotophos NL: 7.189.68 MA: 2375398 tilt RT: 7.10.201.62 MA: 32135613 difenoconozole 4 5 6 Time (min) 7 8 9 10 Figure 23.194.0 3.5 F: + c SRM ms2 239.00 . 67 .5-282. RT: 0.11 MA: 23349203 propazine 1. HLC MS trace for the analysis of 10 pesticides.54 MA: 8354032 folicur RT: 6.5 2.17 RT: 1.09 MA: 10313193 sethoxydim RT: 5.0-218.5-188.00 [ 186.5 F: + c SRM ms2 224.125.0 4.47E5 m/z= 192.50 .00 .19E6 prometryn m/z= 199.00 [ 181.50 .5 5.5-159.339.00] 100 50 0 100 50 0 100 50 0 100 50 0 100 50 0 2 3 RT: 5.50] NL: 8.Results: Figure 23 shows the HPLC MS trace for the analysis of pesticides listed in Table 15.90E6 m/z= 187.00 [ 212.50 .5-193.64E5 m/z= 307.5-308.50] 100 50 0 100 50 0 100 50 0 100 50 0 100 50 0 0.27 RT: 4.196.50 .52E5 m/z= 212.5 Time (min) 3.0 F: + c SRM ms2 242.50] NL: 4.50 .45 MA: 1986999 pirimicarb RT: 4.8 F: + c SRM ms2 242.14E5 m/z= 123.13E6 m/z= 281.160.00 [ 165.5 F: + c SRM ms2 231.50] RT: 3.0 RT: 2.5 F: + c SRM ms2 202.5-337.0 RT: 1.5 F: + c SIM ms [ 306.283.00 [ 198.56 MA: 2522163 simazine NL: 4.50 .5 1.00 [ 335.13 AA: 9862826 NL: 1.00 [ 157.50] NL: 1.50] NL: 3.03E5 m/z= 158.50] NL: 2.5 F: + c SRM ms2 328.

558 7.374 2.000 1.865 0. limit µgkg 20 20 5 10 25 10 200 50 10 -1 x coefficient 1.s.248 r2 1.995 4.737 2. Method validation statistics for the analysis of pesticides in fennel distilled oil.998 0.s. the coefficients for the standard curves.69 7.000 1.000 1.999 1.998 0.000 0.d. = (standard deviation / mean) x 100.547 1.12 4. (at 1 mgkg ) 3 6 7 4 4 6 7 5 3 -1 Table 16. (at 1 mgkg ) 9 4 8 6 9 6 13 19 6 -1 Table 17.000 1. 17 and 18 present the detection limit achieved.s calculated for the responses recorded at the 1 mgkg-1 detection level in fennel.493 6.926 0.999 0.s.125 0.000 0.d.000 0.904 0.000 1. (at 1 mgkg ) 7 11 4 3 5 7 16 11 4 -1 Table 18.45 2.645 2. Fennel analyte monocrotophos simazine pirimicarb propazine sethoxydim prometryn tebuconazole propiconazole difenoconazole det.000 r.993 r2 0.5 200 100 10 -1 x coefficient 0.000 1. Peppermint analyte monocrotophos simazine pirimicarb propazine sethoxydim prometryn tebuconazole propiconazole difenoconazole det.000 r.d. r.869 1. Method validation statistics for the analysis of pesticides in parsley distilled oil.997 1.134 r2 1.000 1.553 7.999 1.858 2.000 1.000 r.029 0.000 1.585 2.5 50 0.000 1. 68 .335 5. limit µgkg 20 10 10 5 25 10 100 100 10 -1 x coefficient 0.s.509 7.d.827 1.54 5.000 0. parsley and peppermint respectively.000 1.000 1.d.932 0.Tables 16.999 1.996 1. the 'r' square value and the r. Parsley analyte monocrotophos simazine pirimicarb propazine sethoxydim prometryn tebuconazole propiconazole difenoconazole det. Method validation statistics for the analysis of pesticides in peppermint distilled oil. limit µgkg 50 20 20 0.s.

the values increase as the detection limit is approached.s of repeat injections of fortified fennel. The parsley oil selected for background matrix for the method validation has background levels of prometryn such that the standard curve did not pass through zero.01 mgkg-1.s.S.5 mgkg-1. parsley and peppermint distilled oils fortified with the thirteen pesticides. The type of oil analysed had minimal effect on the response function as expressed by slope of the standard curve. 10 mgkg Fennel monocrotophos simazine pirimicarb propazine sethoxydim prometryn tebuconazole propiconazole difenoconazole Parsley monocrotophos simazine pirimicarb propazine sethoxydim prometryn tebuconazole propiconazole difenoconazole Peppermint monocrotophos simazine pirimicarb propazine sethoxydim prometryn tebuconazole propiconazole difenoconazole 4 4 4 2 6 4 6 5 3 7 11 4 3 5 6 16 11 4 9 20 5 34 10 5 15 26 5 7 14 38 14 8 22 8 6 4 4 4 6 2 1 4 4 9 4 8 6 9 6 13 19 6 9 8 2 3 6 5 19 21 5 11 12 15 21 14 20 7 5 8 7 7 6 7 4 7 6 3 6 7 4 4 6 7 5 3 11 15 9 4 13 5 12 13 8 15 48 28 4 14 17 19 -1 1 mgkg -1 0.5 mgkg -1 0. Predictably.Table 19 below records the r. which can only be detected to levels of 0.s for repeat injections of fennel.1mgkg -1 Table 19. R. parsley and peppermint distilled oils. Overall method development experiments have found contamination of most parsley oils with this 69 . These levels are in the order of 0.3 mgkg-1. excluding propiconazole and tebuconazole.D.d. Discussion: HPLC MSMS provides an effective screen for pesticides in essential oils for ten analytes with detection limits to 0.

was used to establish a mobile phase with gradient of 50:50 acetonitrile / 0. These were used to prepare mixed solutions of 100. Samples for recovery determinations were analysed under the same conditions as the standard solutions. 5 µm guard cartridge.. with auxillary gas at 20 psi.1M ammonium acetate buffer ramped to 90:10 over 15 minutes. 3. 13 x 100 mm disposable test tubes) to produce a calibration curve of 0.A Waters 2690 separation module (Alliance) system equipped with a NOVAPAK C18. A simple partition step was included in the extraction process to remove non-polar components of the waxy extract. The parent m/z. Capillary temperature was 150°C with a voltage of 16 V. The corona current was 5 µA. For determinations of recoveries. 10 and 50 mgkg-1 relative to oil weight. 1.5 g of boronia concrete (weighed into 9 mL. collision energies and product ions monitored are listed in Table 15. The mixtures were again vortexed for 3 x 20 second bursts then centrifuged for 5 minutes at 3000 rpm to effect a partition. 70 .9 x 150 mm column preceded by a Alltech Econosphere C18. 1mL of methanol/water (5:1) were spiked at concentrations equivalent to those prepared for the standard curve. Experimental: Stock solutions of 1 mgmL-1 of each of the 10 pesticides were prepared in acetone using volumetric flasks. sheath gas (nitrogen) at a pressure of 60 psi. Cyanazine (25 µL of a 100 µgmL-1 solution) was added as an internal standard. 250 µL of acetone was added to ensure the polarity was similar to the samples in boronia matrix. The boronia concretes were dissolved in 1 mL of hexane. Analytical parameters: LC . Standard acetone solutions of 10 µgmL-1 and 100 µgmL-1 were used to fortify 0. The samples were analysed by LC MS/MS using the listed conditions. The samples were vortexed and 1 mL of methanol/water (5:1) was added. Method Validation for the Analysis of 10 Pesticides in Boronia concrete Aim: A separate screen for detection in boronia extract of all the analytes listed in example 8 was developed. Location of an oil. not contaminated with prometryn. Experiments Undertaken in the Process of Method Development Example 9.pesticide. A Finnigan MAT LCQ APCI source had a vaporiser temperature 450°C. The aqueous layer was sub-sampled using a disposable pipette and filtered through cotton wool into a 200 µL GC vial insert. will allow for a repeat of this validation experiment. The total volume of acetone in each tube was brought to 250 µL. 10 and 1 µgmL-1 by volumetric serial dilution.25.

on the other hand are very low. The recoveries for propiconazole. probably as a result of co-eluting components in the boronia extract. propazine and sethoxydim are also less than 50%.5 35 ± 1 72.7% of cyanazine is offset by the excellent reproducibility.5 1 mean ± SE 178 ± 11 34 ± 2 115 ± 2 47. Table 21 presents the detection limits achieved.6 ± 0. the relative contribution of the co-eluting contaminant increases.6 ± 0. The signal for ion m/z 193 is enhanced in the matrix of boronia.8 105 ± 2 24. Recoveries for simazine.5 ± 0.4 ± 0. 71 . with an r.s. The low recovery of 41. however.4 ± 0.3 66 ± 1 105 ± 3 17. Percentage recoveries for target analytes in clean-up step for pesticides in boronia The high recoveries recorded for monocrotophos in boronia preclude the application of this screen in quantitative experiments.25 mean ± SE 214 ± 11 25 ± 3 109 ± 4 49 ± 1 17 ± 2 81 ± 2 88 ± 7 nil 66 ± 2 Table 20. of 7% at 1 mgkg-1 still allows for the application of this analytical method for the detection of gross contamination of propiconazole in boronia concrete. the repeatability of that low recovery.d.8 21. mg/kg analyte monocrotophos simazine pirimicarb propazine sethoxydim prometryn tebuconazole propiconazole difenaconazole n 4 4 4 4 4 4 4 4 4 50 mean ± SE 123 ± 4 38.5 74 ± 0.4 ± 0.3 ± 0.7 ± 0. the 'r2' values and the relative standard deviations for the detection of all nine pesticides investigated in boronia concrete.4 72 ± 1 10 mean ± SE 155 ± 7 34 ± 1 107 ± 2 43. However.2 ± 0. the coefficients for the standard curves. the consistency in the percentage of analyte recovered across the range of fortification levels allows for the application of this analytical method to the screening of these three analytes in boronia extract.5 101 ± 1 38. Table 20 lists the recoveries for the target analytes across 4 concentration levels.5 20. As the level of monocrotophos approaches the ppb level.6 ± 0.9 74 ± 2 119 ± 3 15 ± 1 75 ± 2 0.Results & Discussion: The clean-up step used in the analyses of boronia required an assessment of the suitability of cyanazine as an internal standard.

000 0.287 0.997 1. The process for method development for the detection of this class of pesticide followed the same protocols described in the preceding section. fluroxypyr acids and their parent esters.298 0. fluazifop.000 0. Table 22 records the ions monitored and the relevant retention times. Tuning files were established optimising the fragmentation and ionisation of haloxyfop. effectively splitting the relevant peak. 72 .038 0.717 0.01 0. fit (r ) 0.993 1. Parameters were established by direct infusion of 1 µgmL-1 acetone solutions into the MS.115 0. This variation in response was partly due to the haloxyfop ester being present in the standards as both the ethyl and methyl esters. reg.393 0. The application of HPLC MSMS to the Analysis of Pesticides with Acidic Moieties.Boronia concrete det.01 0. The elution profile for the esters and the acids under the acetonitrile / ammonium acetate buffer RP chromatography showed acceptable resolution but the responses for haloxyfop and fluroxypyr esters were poor with haloxyfop registering a response thirty times lower than that of fluazifop.25 mg/kg 10 22 5 6 17 2 16 n/a 8 Table 21. Fluazifop possessed good ionisation characteristics using ESI.2 0.990 0.15 0. limit analyte monocrotophos simazine pirimicarb propazine sethoxydim prometryn tebuconazole propiconazole difenaconazole mg/kg 0.1 0.998 1.02 coefficient 0.02 0.140 0.000 2 %rsd 50 mg/kg 2 3 4 3 3 3 3 6 6 10 mg/kg 2 3 3 3 4 2 2 10 6 1 mg/kg 8 11 6 4 7 3 9 18 1 0.030 1.1 0. Method validation for pesticides boronia oils by LC MSMS using ionisation with APCI in the positive mode.2 0.000 0.994 1.108 x lin.

258 MS 384 (+) 326 (-) 434/436 (+) 360/362 (-) 361 367. 73 .290 288. Poor chromatography and response necessitated an improved mobile phase and the effect of pH on elution characteristics was considered the most critical parameter. Recoveries and reproducabilities were improved with these modifications.1 10 isol. precluded the simultaneous analysis of the parent esters with the carboxylic acid forms.analyte fluazifop-butyl fluazifop acid haloxyfop ethyl haloxyfop acid fluroxypyr methyl heptyl fluroxypyr acid MW 383 327 433.435 361.369. the acid moiety of this class of pesticide is de-protonated facilitating the extraction of the analytes from the essential oil into an aqueous layer. MS parameters for the detection of the parent ester and acid forms of fluazifop. In addition. which was adjusted to a pH of 10 with NaOH. The inclusion of an aqueous extraction.01M NaOH. which was acidified and back extracted with dichloromethane (DCM) had limited success.370 255.257 (-) MSMS 328 254 316.7 6 amu 18% 6 amu 17% Table 22.235 ret.363 366. 5 amu 19% 3 amu 17% 6 amu 25% 4 amu 26% 12. The 0.01M NaHCO3. The pesticides within this class are weak acids. however. LC UV analysis of the DCM confirmed that very little essential oil residue was co-extracted through the process but recoveries were poor. time 11. window & C.E.318 288.255. Preliminary experiments whereby distilled oil samples were extracted with 0.368.290 255. haloxyfop and fluroxypyr.01M NaOH was replaced with 0. the acidification and subsequent back extraction with DCM was removed as it was considered that only a limited amount of essential oil components should be soluble in an aqueous extract which was analysed directly. To prevent excessive dilution of the pesticide extracts the ratio of essential oil to NaHCO3 solution was lowered to 4 : 1. The acidic nature of the active ingredient of this class of pesticide presented a moiety which may be exploited in an extraction protocol. Extraction Protocol for Pesticides with Acidic Moieties Under basic conditions. The development of a screen for the pesticides with an acidic moiety proceeded with the inclusion of an extraction step whilst the esters were to be included in the general screen using APCI in the positive mode.371 (+) 253.256.257 233.

Experiments Undertaken in the Process of Method Development Example 8.4.4.16 2.290 [M-H-H2O] [M-H-C2H2O2] [M-H-C2H2O2] [M-H-C2H2O2] [M-H-C3H4O2] [M-H-C2H4O2] Table 23. The agents 2. Method Validation for the Analysis of Acidic Pesticides by HPLC MSMS.13 4. Aliquots (20 x 5 mL) of each of peppermint and fennel oils were dispensed into 50 mL test tubes.4-D.50 4.163 141.4-D and 2. and for the acidic pesticides dicamba.The inclusion of an internal standard negates the errors introduced by inaccurate subsampling and dilutions.5-T mecoprop haloxyfop width (amu) energy (CE) ions monitored % n/a n/a 219. analyte M.199 141. MCPA and mecoprop.W. 2.4-D MCPA 2. MS parameters for the monitoring of 2. Extracting solvent (1 mL of 0. The parameters established in the preliminary investigations were used in the validations for the detection of pesticides with acidic moieties were conducted.51 5. of major isotope 220 254 220 200 254 214 361 centre of parent m/z window [M+H] n/a 254 221 200 255 214 361 isolation collision product tentative identification [M-H] + + + + + + + retention time (mins) 2. Experimental: Commercially produced oils were combined to produce a blended oil with uniform characteristics.4-D.5-T dicamba.5-T were proposed as suitable internal standards and both were introduced in the MS using direct infusion to determine the ionisation and fragmentation parameters. Table 23 records the parameters established for 2.5-T.143 195. Validations were conducted for peppermint and fennel distilled oils only. MCPA and mecoprop.235 161. The emulsions were 74 . 2.86 6. The densities of parsley oil were quite variable such that multi-layers were present when non-polar/aqueous partitions were established during the extraction process.221 6 6 5 8 4 4 17 20 18 20 18 26 233.197.4.4.02 8. Aim: To conduct method validation for the extraction and analysis for residues of pesticides with acidic moieties in fennel and peppermint distilled oils.01M NaHCO3 adjusted to pH 10 with NaOH) was added and the mixtures were vortexed for 4 x 15 second bursts.89 dicamba fluroxypyr 2.143 288.

7 F: .00] NL: 2.7+142.c SRM ms2 361.00 [ 287.91 NL: 1.291.13 2.9 x 150 mm column was used to establish a mobile phase with gradient of 50:50 acetonitrile / 0.7 F: .7-195. The mixtures were centrifuged at 2000 rpm for 5 minutes and 250 µL of the aqueous layers were quantitatively transferred to a GC vial inserts.7+288.3-221.4D 4.7-197.81 100 2.144.00 .76E5 2. the slopes of the linear regression describing the standard curves established.1M ammonium acetate buffer (adjusted to 3.201. A Finnigan MAT LCQ was used to monitor the ions listed in Table 22 and 23 within the relevant time windows.00] 0 100 0 100 5.02 mecoprop m/z= 140. Analytical Parameters: A Waters 2690 separation module (Alliance) system equipped with a NOVAPAK C18.5+234.5-233.7-161.7-141.c SRM ms2 255.7-143.00 .44 4.7 F: .7+196.236.8 with acetic acid) ramped to 90:10 over 15 minutes.73E6 m/z= 232.c SRM ms2 200.7 F: .164.c SRM ms2 254.51 MCPA m/z= 160.57E6 m/z= 140.7+162.00] NL: 1. Hexane (1 mL) was added to each and the mixtures were vortexed for 2 x 15 second bursts to remove residual oil contaminants.c SIM ms [ 218.50 fluroxypyr 2.7-288.c SRM ms2 214.56E6 m/z= 218.02E6 0 100 0 100 4.3 F: .5-T) was added to each and the samples analysed using the conditions listed.00] 0 1 2 3 4 5 6 Time (min) 7 8 9 10 11 Figure 25. 3. 'r2' value pertaining to the fitted curves and the reproducibilities as 75 .4.00 . RT: 0.86 NL: 5.284.5 F: .3-219.11.3+220.15E6 m/z= 287.00 .4.00 .89 haloxyfop NL: 3. Table 24 records the detection limits.7-289.7+198.7-143.00 [ 193.5-235.01 .00] NL: 1.53 2.transferred to 13 x 100 mm borosiliate glass tubes and centrifuged at 2500 rpm for 15 minutes.c SRM ms2 221.00 . The aqueous layers were transferred to a 10 x 75 mm borosilicate glass tubes using pasteur pipettes.00] NL: 2.16 dicamba 2.00 [ 232.00] 0 100 8. Internal standard (10 µl of a 50 µgmL-1 solution of 2.7-163.5-T m/z= 194. Results: Figure 25 shows the trace for the analysis of acidic pesticides by HPLC MS.00 [ 140.85E6 0 100 6. HPLC MS trace of analyse of acidic pesticides.00 .7-199.7-141.7 F: .00 [ 140.00 [ 160.7+142.144.

limit mgkg 0. 10 mgkg Fennel dicamba fluroxypyr MCPA mecoprop haloxyfop Peppermint dicamba fluroxypyr MCPA mecoprop haloxyfop 5 2 5 4 8 13 15 18 20 14 42 10 6 18 13 9 31 7 51 9 8 9 11 12 2 7 4 6 6 16 8 6 8 5 22 42 50 -1 1 mgkg -1 0.S.01 0.995 0.20E-06 2.998 0.s.D. mcpa.86E-06 3.99E-06 3.28E-06 2. Detection limits correlation co-efficients and standard curve statistics for the detection of pesticides with acidic moieties in fennel and peppermint distilled oil. Table 25 records the r.01 3.described by the r. Discussion: The aqueous extraction and detection by HPLC MSMS in the negative mode of pesticides with acidic moieties has excellent detection limits.s for the analysis of the acidic forms of dicamba.5 mgkg -1 0.1 0.996 0. R. 76 .995 0.01 0. analyte Fennel dicamba fluroxypyr MCPA mecoprop haloxyfop Peppermint dicamba fluroxypyr MCPA mecoprop haloxyfop 0.01 0.63E-06 0.25E-06 3.1 0.d.s for each pesticide at each fortification level in fennel and peppermint oil.28E-06 3. is reproducible and gives a linear relationship between analyte concentration and peak area.999 13 15 18 20 14 det.997 2 7 4 6 6 Table 24.78E-06 6. mecoprop and haloxyfop acid.999 0.01 0.s for the detection of pesticides with acidic moieties in fennel and peppermint distilled oil.1mgkg -1 Table 25.s.992 0.999 0.999 0.89E-06 1.01 -1 x coefficient 4.01 0. fluroxypyr.d.24E-06 r 2 %rsd (at 1 mgkg ) -1 0.1 0.

Acetone / water solutions (50:50) of paraquat and diquat (1 mgmL-1) were diluted to 10 µgmL-1 and introduced into the MS using a continuous flow worm driven syringe at a flow rate of 0. MS parameters for the detection of diquat and paraquat. however. The power of mass spectrometry allows for the monitoring of ions specific to the target analyte despite the collection of large amounts of background ions.The Application of Direct MSMS to the Analysis of Quaternary Ammonium Salts The quaternary ammonium salts form a distinctive class of pesticides.W.5 17. the problems encountered are very specific to this chemical type. An alternative approach to the analysis of quaternary ammonium salts was required. Initial trials of ESI showed excellent ionisation response. 77 . A water extraction of essential oils would be expected to effectively remove paraquat and diquat from an oil extract with very little of the oil components co-extracting. A post-column injection would still require the continuous flow of mobile phase from the LC system through the injection loop of the mass spectrometer. The associated loss of analyte within the LC system would be avoided. The application of liquid chromatography to the separation of paraquat and diquat presented difficulties. This would offset the loss of automation that direct injection into the loop of the mass spectrometer would represent.1% paraquat C12H14N3 2+ 186 186 + 171 1. An aqueous extract of paraquat and diquat from essential oils may contain so few co-extracted oil components that direct injection of samples into the injection loop of the mass spectrometer may be conceivable. The method development for the two main products used in the Australian essential industry. Paraquat and diquat are both very soluble in water and virtually insoluble in hydrocarbons. however the approach would also have the added advantage of a quick analysis turn around time. paraquat and diquat. analyte diquat molecular formula C12H12N2 2+ M.1% Table 26. initially follow the same processes as the preceding methodologies.8 mLmin-1. 184 parent ion (+ive) 183 + daughter ion 157 isolation width 4 collision energy 17. The effects of low solubility in organic solvents and the predisposition of both analytes to absorb on to most surfaces was manifest by their complete loss when analysed by the standard HPLC protocols. Parameters established are listed in Table 26.

with decreases extremely pronounced for diquat as illustrated in Figure 26. The replacement of acetonitrile with methanol alleviated this problem.91 20.0-1 73.73 AA: 1 33825 28 30 32 34 36 38 40 42 44 46 paraquat NL: 1 ..62 26.40 A A : 585527 RT: 42.33 42.1 6 1 3.72 4.1 7 Figure 26.04 A A : 61 8054 RT: 36.35 7.68 50 40 30 20 1 0 0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 20 22 24 26 Time (min) 28 30 32 34 36 38 40 42 44 46 2.00] RT: 0.29E5 TIC F: + p SRM ms2 1 83.07 1 6.61 4.48 1 0.47.25 25.46 37.60 A A : 67351 6 RT: 30.74 A A : 71 2836 A A : 734053 A A: 668402 NL: 1 .80 46.96 1 .48 23.57 40.00 26.04 20.48 1 3.42 A A: 839050 RT: 24. Kaniansky et al. RT: 0.86 23.00 .23 35.07 26.27 21 .00 [ 1 56. Table 27 presents the areas for repeat injections of the fennel oil extracted in glassware pre-conditioned with 5% of a 0.30 1 3. In addition to the loss of analyte by absorption other problems encountered were shorting across the ionisation source which was possibly related to the acetonitrile / ammonium acetate buffer.60 23.89 A A : 557462 RT: 38.00] RT: 27.5 mgmL-1 water solution of paraquat and diquat showed a rapid decrease in response over 5 minutes.56 29.96 23.61 35.1 6 8.38 2.55 27.75 1 00 90 80 Relative Abundance 70 60 1 0.01.75 1 5.1 59. The adsorption of quaternary ammonium salts onto surfaces has been encountered by other research groups.72 30.27 42.00 [ 1 70. DETA was found to preferentially accumulate on the surfaces of sample containers.78 A A : 71 3276 RT: 41 .69 1 9.22E5 m/z= 1 56.79 37.35 A A : 583405 RT: 5.1M DETA solution compared to extractions in glassware not conditioned.47 4.22 32.48 A A : 61 2331 RT: 33. competitively binding on the adsorption sites.77 32.89 46.89 40.92 1 7.1 0 34.76 1 00 90 80 Relative Abundance 70 60 50 40 30 20 1 0 0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 20 22 24 26 Time (min) RT: 2.56 A A : 740456 diquat RT: 21 . 78 . Repeated 20µL loop injection of a 0.47.01. These were eliminated by spiking samples with diethylenetriamine (DETA).1 73.1 RT: 1 4.66 A A : 7951 52 RT: 9. (1994) reported the introduction of serious analytical errors in the analysis of paraquat and diquat at low concentrations by adsorption losses of the analytes in sample storage containers.98 AA : 61 71 1 8 RT: 1 . Repeat injections of paraquat and diquat solutions.1 9 29.85 1 RT: 1 8 8.05 1 1 3.The adsorption problems also had consequences for the reproducibility of direct manual injections into the mass spectrometer.29 1 7.00 .90 8.49 A A: 622433 RT: 44.69 43.1 5 1 6.36 40.0 F: + p SRM ms2 1 86.

4 fold increase in diquat response at the 0.01 mean std. Effect of the inclusion of DETA treated glassware in the area response for the analysis of paraquat and diquat. 79 .8 0. R.S. Dev. R.D.1 mgkg-1 fortification level.9 mean std.984 Table 27. Dev.S. The inclusion of a DETA solution pre-treatment of all glassware has effected: • • a 3.998 432032 56317 13 0.D. a 2.DETA 186 170-173 + DETA 23234 15028 17824 32109 35127 32640 20810 0.5 213177 36064 17 1.3 186155 206904 273962 236205 183563 231997 173454 7671 3273 43 149364 112825 106703 107986 97298 88943 110520 20859 19 124055 22701 18 2.DETA 5860 12206 4145 6302 9840 156-159 + DETA (peak area) . R.3 fold increase in paraquat response at 0. + deta / -deta linear regression r2 0.1 25253 7986 32 3.4 374669 505016 474457 420107 385911 763011 490703 438389 407946 525012 162305 31 52322 10907 21 144892 126952 146982 99221 102230 54473 44854 43708 48236 70337 mean std.paraquat parent ion daughter range mgkg -1 diquat 183 (peak area) .01 mg/kg-1 fortification level. + deta / -deta 0.D.986 0. Dev.999 0. + deta / -deta 0.S.

including standard stock solutions. 100 mgkg-1 samples of paraquat and diquat in fennel oil stored for two weeks in un-treated glassware and glassware pre-treated with DETA solutions were compared to freshly prepared standard extracts. minimising of contact with all metallic surfaces in the flow path. an increase in the linearity of the standard curve at low concentrations as expressed by the improved 'r2' values for both paraquat and diquat. Areas for the peaks of the respective treatments are presented in Table 28.d.). Effect on the area response of the inclusion of glassware pre-conditioned with DETA on the stability of paraquat and diquat in storage. Results in Table 28 confirm that despite the improved response and reproducibility afforded by the use of glassware pre-treated with DETA. 0. lowered from 43 to 32%.1 mgkg -1 paraquat 186 170-173 + DETA 2 weeks < 12 hours 8866 213177 (peak area) . changing of mobile phase to 50 : 50 100 µM DETA / methanol. In addition to this experiment. depletion of both paraquat and diquat in solutions necessitates the preparation of standards and samples on the day of analysis.DETA bdl 110520 diquat 183 156-159 + DETA 16903 124055 (peak area) .DETA bdl 52322 parent ion daughter range Table 28. 80 . the inclusion of DETA in all solutions. including the replacement of the metal injection loop with teflon. extraction of all oil samples with DETA solutions.• • an increase in the repeatability of the analysis for paraquat as expressed by the relative standard deviation (r.s. Reproducibility in the analyses of paraquat and diquat was further improved by including the following measures: • • • • • pre-conditioning of all glassware to be used in the extraction procedures.

9 x 150 mm column was used to establish an isocratic mobile phase of 50 : 50 DETA : methanol mobile phase. The fortifications were repeated four times at each concentration to determine repeatability of the method.Experiments Undertaken in the Process of Method Development Example 10.s. diquat (mgkg ) 0.d.0 0.5 1.1M DETA and dried.5 and 1. Analytical parameters: A Waters 2690 separation module (Alliance) system equipped with a NOVAPAK C18.5 5 Response (mean area) 0 9528314 14776066 18640057 Response (mean area) 0 4494099 14490788 17807392 r. 14 12 10 Table 29.5 2. The oils were extracted with 2 mL of 0.1M DETA solution and vortexed for 3 x 20 second bursts. Repeatability of the detection of paraquat and diquat using HPLC MSMS 81 .s. 3.0 -1 -1 µg 0 0. Aim: To determine the repeatability of the detection of paraquat and diquat using HPLC MSMS Experimental: Fennel oil (16 x 5 mL) were weighed into 10 mL glass tubes. The solutions were washed with 0. Freshly prepared 10 µgmL-1 and 50 µgmL-1 solutions of paraquat and diquat were used to spike each of 5 mL of fennel oils to produce fortification levels of 0. previously washed in a 5% solution of 0. A Finnigan MAT LCQ was used to monitor the parent ion 186 for diquat with the daughter ion in range 170 to 173 and the parent ion 186 for paraquat with the daughter ion range of 156 to 159.5 2. 14 6 8 r.d. The tubes were centrifuged at 2500 rpm for 15 minutes and the aqueous layers were transferred into 10 x 74 mm 'Kimbal' tubes. Each injection was repeated six times for each of the four repeats of each fortification level and the areas averaged. Results: Table 29 records the results obtained.5 5 µg 0 0.1.1 0.1 0.0 0. Method Validation for the Detection of Paraquat and Diquat.75 mLs of DCM. Manual injections to fill a 25 µL teflon loop were interposed into the LC mobile phase post-column and introduced directly into the MSMS.0 mgkg-1.5 1.0 paraquat (mgkg ) 0. 0.

1 mgkg-1 level. The widely accepted method for the analysis of dithiocarbamate pesticides. is not suitable for the analysis of essential oils. Problems affecting linearity of the response at low levels are evident but the method has been shown to be robust and efficient. been overcome.971 and 1. DMED was first synthesised using the method described by Gustafsson and Thompson (1981). to produce dimethyl ethylenebis(dithiocarbamate) (DMED). The parent m/z ion 82 . The product was then dissolved in acetone and introduced into the MS via the worm drive syringe effecting direct infusion into the MS at a flow of 0. Sulfur chemicals are endogenous to many essential oil crops and will produce carbon disulfide when subjected to acidic digestion.Discussion: The results in Table 29 show a non-linear relationship between analyte concentration and peak area for the analysis of paraquat and diquat by HPLC MSMS. tetrabutylammonium hydrogen sulfate (TBAS). At low concentrations adsorption problems seem more pronounced for paraquat. N'ethylenebis(dithiocarbamate). such that the response for this analyte is half that seen for diquat at the 0. S EDTA S SNa SNa TBAS CHCl3 -hexane (3:1) aq.5) CH2 CH2 NH C NH C CH2 CH2 NH C NH C SCH 3 SCH 3 mancozeb NaOH S S When mancozeb is extracted in an NaOH / EDTA solution the salt produced is disodium N. This method was adapted with the intention of using detection by HPLC MS/MS. The Application of LC MSMS to the Analysis of Mancozeb This methodology is still in developmental stages. Gustafsson and Thompson (1981) developed a HPLC UV method for the determination of dithiocarbamates using a EDTA / NaOH extraction followed by methylation with methyl iodide. The problems encountered for the detection of paraquat and diquat at levels of 1 mgkg-1 have.8 mLmin-1. A curve fit describing the shape of the curve with a second order polynomial gave a 'r2' value of 0. the details of progress to date may be useful as an indication as to the applicability of LC MS/MS to the analysis of dithiocarbamates.(pH 6.5-8. to the greater extent. which is transferred across the aqueous organic interface into a methyl iodide hexane / chloroform solution with the phase transfer reagent. however. that is headspace GC FPD analysis of carbon disulfide produced by the acidified stannous chloride digestion of dithiocarbamates (page 46).000 for paraquat and diquat respectively.

which are interfering in the analytical procedure. It was found that the precursors and/or the target product were thermally labile and that without heat.8 which when subject to a collision energy of 17% produced the daughter ion of m/z 192. However. 83 . no DMED was detected. Analyses by LC MS/MS gave positive results for the mancozeb derivatives extracted from fortified NaOH / EDTA solutions down to the 0. when introduced into the HPLC MS. Emporite™ Anion-SR exchange discs were to be used to selectively bind the salt produced by NaOH / EDTA solvation of mancozeb.8. The discs were then dried at 60°C and the bound salt methylated with methyl iodide in acetonitrile. However. In preliminary experiments the target analyte was not detected in any of the extracted samples. TBAS contaminated the entire system. resulted in a marked increase in DMED response. Adapting the method of Wells et al. such that any signal from the target analyte was overwhelmed. Instead attention was diverted to the monitoring of manganese levels in oils to be used as an indication of mancozeb contamination as detailed in the following pages. It was also concluded from further tests that increasing the concentration of the phase transfer reagent. despite showing some promise as a highly specific analytical technique.1M ammonium acetate buffer mobile phase with a gradient of 90 : 10 over 15 minutes. This was overcome by the washing the organic layer with water. (2000). this methodology was not taken to conclusion. Mancozeb was dissolved in an EDTA / NaOH solution and TBAS was used to transfer the solvated product into the chloroform / hexane layer containing methyl iodide. However. TBAS. derivatisation could be accomplished.was 240. Further experimentation found that pre-washing the oil with NaOH / EDTA improved the yield of DMED showing that the components of the oil. This would indicate that the essential oil components co-extracted with the mancozeb may be competitively binding with methyl iodide The separation of the ethylenebis(dithiocarbamate) salt from the aqueous phase was attempted using specialised empore discs to allow for direct methylation without the need for phase transfer reagent.2 minutes. are soluble in NaOH / EDTA and once removed allow for a successful extraction and derivatisation of mancozeb. was detrimental but that increasing the concentration of the derivatising agent. The analyte eluted at 5.25 mgkg-1 level. when essential oil was spiked with mancozeb and extracted with NaOH / EDTA solutions and transferred into the organic layer for derivatisation with MeI. methyl iodide. The analyte was then introduced into the HPLC to be chromatographed on a NOVAPAC C18 column with a 50 : 50 methanol : 0.

Parsley reacted violently with the acid and combusted instantly resulting in the loss of the sample. Fennel reacted moderately with the acid but formed amorphous lumps that were difficult to digest and spontaneously combusted toward the end of the digestion. Sample volumes are then reduced to 1-2 mL on a heating block under a stream of air. increased mancozeb fortification levels coinciding with higher levels of manganese detected. cooled and made up to 25 mL in volumetric flasks using distilled water then filtered. This light is recorded by one or more optical spectrometers and when calibrated against standards. Samples are heated to 100°C for 2 hrs then heated to 240°C and refluxed for 4 hrs.Assessment of the application of ICP OES to the screening of essential oils ICP OES is used for elemental analysis. Despite this. It was concluded that in addition to perchloric acid digests being dangerous and unlikely to meet OH&S standards. are introduced into a plasma formed by ionised gas in an oscillating magnetic field that reaches extremely high temperatures. However. which normally are present as contaminants at very low concentrations. would not produce enough of any one element to register above background noise in a crude extract. peppermint. Obviously most pesticides. however. fennel and boronia oils. No ICP analysis was possible. usually dissolved in aqueous solutions. The manganese : zinc ratio recorded was not that expected 84 . metals such as manganese.25M EDTA / 0. Elevated levels of these elements in samples treated with the pesticide may be indicative of mancozeb contamination. reacted only moderately. solutions had to be diluted 1 : 20 as the carbon content of co-extracted oil components was too high. Peppermint. Mancozeb contains manganese and zinc in a ration of 10 : 1. Digestions are traditionally performed using nitric acid:perchloric acid (5:1). Due to the difficulty in digesting essential oils and the limited quantity of oil available for analysis it was concluded part of the method proposed by Gustafsson and Thompson (1987) could be adopted. Initial trials were instigated to determine base levels of endogenous manganese / zinc ratios and trial the effectiveness of standard digestion procedures. This methodology was applied to the digestion of essential oils. Digestions using H2SO4:H2O2 were trialed in samples of parsley. are trace elements in most vegetative materials and are only present at very low levels.45M NaOH was used to complex mancozeb residues allowing free manganese ions to pass into the aqueous phase. Sulphuric acid charred the oils such that a black solid mass formed in the bottom of the tube. Samples. Atoms entering the plasma emit light (photons) with each element producing a characteristic wavelength. A solution of 0. on the other hand. essential oils do not react consistently under the conditions trialed. This method was found to be successful. provides a quantitative analysis.

USA) were analysed regularly as quality control samples (accurate to <10% for all elements considered). These were extracted with 2 x 5 mL of a 1 in 50 dilution of 0. Boronia extract (0. ICPMO143-5 and ICPMO165-5. optimisation and calibration were performed daily. Mn and Zn were monitored using an 'IRIS" Inductively Coupled Plasma Optical Emmission Spectrometer (ICP-OES. 85 . High carbon content was overcome by using a partly neutralised solution of 5 mM EDTA / 9 mM NaOH.25. Standard mixtures were prepared in 5% (v/v) HNO3. Ma.0 and 10. Results: Table 30 records the results obtained. fennel and parsley were spiked with mancozeb (suspended in acetone) at levels of 0. University of Tasmania.6. Experiments were conducted to test the parameters of the method developed in parsley. Instrument tuning.from the breakdown of mancozeb.0 mgkg-1. Fortifications of boronia extracts were conducted as for that undertaken for distilled oils. Higher than expected levels of zinc may have been due to contamination of glassware/reagents etc. Thermo Jarrell Ash (Franklin. New Haven. Each calibration protocol typically consisted of a blank and 3-5 standards covering the concentration range 0 to 200 mgL-1. Aim: To establish detection limits and linearity of response for the analyses of mancozeb in essential oils using ICP OES Experimental: Samples (5 mL) of peppermint. for metals analysis. CT.1 0. peppermint and fennel distilled oils and in boronia concrete. 1. with quantitation by means of external calibration. while the rinse time between samples was also ~60 seconds. USA)). Commercial reference preparations (AccuStandard Inc.45M NaOH and adjusted to pH 9.5 g) was dissolved in 5 mL of hexane prior to the sub-sampling of 5 mL such that only 2. The EDTA extracts were combined and washed with 3 mL of hexane to remove any oil contamination.5 grams of boronia matrix was extracted. Method undertaken for the monitoring of manganese by ICP OES to screen for mancozeb in fortified essential oils.25M EDTA / 0. Processes undertaken in the process of method development Example 11. Sample uptake time to the ICP-OES was ~60 seconds. Analytical parameters: Samples were submitted to the Central Science Laboratory. Solutions were vortexed for 3 x 20 seconds and centrifuged for 10 minutes at 2500 rpm.

00 0.53 zinc 0. This experiment provides the basis for a sensitive. Contamination is more likely in solvent extracted concretes than in distilled oils.04 -0.00 -0.01 0. economical screen.1 mgkg-1 whilst a level of 0.00 0.02 0. The results indicate that the detection limit for mancozeb in distilled oils approaches 0. The boronia results recorded a higher manganese blank.01 0.01 0.05 0 -0.07 0.00 0.07 0.01 Table 30. 86 .02 0.25 mgkg-1 is achievable in the matrix of boronia extracts.01 0.79 0 0.04 0. The extraction and analyses using adaptations of this basic methodology from the surface of peppermint leaves is described in appendix 2.00 0.02 0. This may be due to endogenous manganese or possible mancozeb contamination.00 0.01 0.06 0.03 -0.85 0 0.Sample Name Parsley blank -1 Pars_100µgkg -1 pars_250 µgkg -1 pars_1000 µgkg -1 pars_10000µgkg Peppermint blank -1 pep_100 µgkg -1 pep_250 µgkg -1 pep_1000 µgkg -1 pep_10000 µgkg Fennel blank -1 fen_100 µgkg -1 fen_250 µgkg -1 fen_1000 µgkg -1 fen_10000 µgkg Boronia blank -1 Bor_100 µgkg -1 Bor_250 µgkg -1 Bor_1000 µgkg -1 Bor_10000 µgkg Manganese 0 0. Manganese levels in dilute 5mM EDTA / 9mM NaOH extracts of essential oils and boronia extracts fortified with mancozeb.00 0.03 0 0.01 0 0.79 0 0.03 -0.01 0.00 0.

Cyanazine (0. 87 . To allow for an estimation of recoveries fortified oil extracts were to be compared to aliquots of 1mL of methanol:water (5:1) which had been fortified at concentrations equivalent to those prepared for the standard curve. 13 x 100 mm disposable test tubes) to produce fortification levels of 0. The aqueous layer was sub-sampled using a disposable pipette and filtered through cotton wool into a 200 µL GC vial insert. Many of the non-polar compounds removed are non-volatile so the inclusion of a liquid/liquid extraction can also reduce the loading into the injector of GC based analytical methodologies. The mixtures were again vortexed for 3 x 20 second bursts then centrifuged for 5 minutes at 3000 rpm to effect a partition. 10 and 50 mgkg-1. Samples. were analysed under the same conditions as the standard solutions. The boronia concretes were dissolved in 1 mL of hexane. The samples were analysed by LC MS/MS using the listed conditions. Acetone (250 µL) was added to each to ensure the polarity was similar to the samples in boronia matrix.5 g of boronia concrete (weighed into 9 mL. The samples were vortexed and 1 mL of methanol : water (5:1) was added. The total volume of acetone in each tube was brought to 250 µL. prepared to determine recoveries. A simple partition clean-up step can be effective in removing non-polar components. 1.025 mL of a 100 µgmL-1 solution) was added to each as an internal standard. Experimental: Standard acetone solutions of 10 µgmL-1 and 100 µgmL-1 of the pesticides were used to fortify 0. Experiments Undertaken in the Process of Method Development Example 12 The application of liquid / liquid partition in the preliminary clean-up of pesticides in solvent extracted oils.Preliminary clean-up methodology Liquid / liquid extraction of pesticides from solvent extracted oils Direct analysis of solvent extracted concretes such as boronia and blackcurrant is limited as the complex mixtures usually include low polarity hydrocarbons and waxes which contaminate and reduce sensitivity in most analytical machines.25. relative to oil weight. This allows for the introduction of the samples onto LC systems with reduced risk of blocking the guard columns or non-polar phase LC columns. Aim: To determine the effectiveness and recoveries of pesticides extracted from boronia concrete using liquid / liquid partition.

3 66 ± 1 105 ± 3 17. propazine and sethoxydim are also less than 50%. Successful solvent / media combinations were then scaled up for application in SPE. Preliminary Development of Solid Phase Extraction of Pesticides from Essential Oils.5 74 ± 0.4 ± 0. with an RSD of 7% at 1 mgkg-1 still allows for the application of this analytical method for the detection of gross contamination of propiconazole in boronia concrete. mg/kg analyte monocrotophos simazine pirimicarb propazine sethoxydim prometryn tebuconazole propiconazole difenaconazole n 4 4 4 4 4 4 4 4 4 50 mean ± SE 123 ± 4 38. probably as a result of co-eluting components in the boronia extract.5 1 mean ± SE 178 ± 11 34 ± 2 115 ± 2 47.9 74 ± 2 119 ± 3 15 ± 1 75 ± 2 0.6 ± 0. The recoveries for propiconazole. As the concentration level of monocrotophos approaches the ppb level.6 ± 0. The high recoveries recorded for monocrotophos in boronia preclude the application of this screen in quantitative experiments.4 72 ± 1 10 mean ± SE 155 ± 7 34 ± 1 107 ± 2 43.5 ± 0. on the other hand are very low. The signal for ion m/z 193 is enhanced in the matrix of boronia. However. However. 88 . the repeatability of that low recovery. the relative contribution of the co-eluting contaminant increases.6 ± 0.Results & Discussion: Table 31 lists the recoveries at each fortification level using the liquid / liquid partition method described. as the elution characteristics of the components are often very similar to the analytes we wish to separate from the oils.8 105 ± 2 24.5 20. thin layer chromatography (TLC) provides a quick effective method for selection and optimisation of solvent regimes. the consistency in the percentage of analyte recovered across the range of fortification levels allows for the application of this analytical method to the screening of these three analytes in boronia extract. however.5 101 ± 1 38. The large array of parameters which can be varied in SPE methodology make the number of possible chromatographic conditions unmanageable.3 ± 0. Within this section TLC was used to establish a basic method for the separation of a range of pesticides.4 ± 0.25 mean ± SE 214 ± 11 25 ± 3 109 ± 4 49 ± 1 17 ± 2 81 ± 2 88 ± 7 nil 66 ± 2 Table 31.8 21. % Recoveries of pesticides using liquid / liquid partition. Recoveries for simazine. Solid phase extraction (SPE) has limited application for pesticides in essential oils.4 ± 0.5 35 ± 1 72.7 ± 0.

peppermint oil and boronia extracts were spotted onto the base line with glass capillaries. Commercially available.Experiments Undertaken in the Process of Method Development Example 13 The application of Thin Layer Chromatography for Development of a Solvent Combination Suitable for the Separation of Pesticides from Essential Oil Components.V absorbances of a range of pesticides were also established. After resolution of each plate the solvent front was marked and the Rf calculated for the oil components and each pesticide where Rf = elution distance of target analyte elution distance of solvent front Results & Discussion: Table 32 records the Rf of pesticides for some example solvent combinations. Experimental: Thin layer chromatography aluminium sheets layered with silica gel 60 F254 supplied by Merck were cut into 15 x 4 cm sections. A range of solvent combinations was trialed. hexane / ether and chloroform / methanol in varying ratios. Aim: The behaviour of oil components and pesticides were first trialed using TLC. peppermint oil and boronia extracts trialed under a range of solvent combinations. Solvent combinations trialed included hexane / acetone. The retention characteristics and U. Pencil lines were inscribed on the silica above the level of the chromatography chamber sumps and dilute aliquots of fennel oil. activated silica TLC plates were used to test the separation of parsley oil. 89 . parsley oil.

6 not tested not tested not tested not tested hexane/ether 50/50 0.2 0. The analytes are then eluted from the column with acetone. simazine.1 0.0 0. SPE Method Validation Aim: In the experiment described previously it had been established that the target pesticides remained on silica columns when hexane / ether (90 / 10) is used as a mobile phase.5 0. The movement of oil components and the relative immobility of pesticides presented the hexane / ether solvent combination as the most favourable.hexane/acetone pesticide difenaconazole linuron bromacil terbacil simazine procymidone ethofumesate chloropropham fluroxypyr ester sethoxydim mecoprop pendimethalin pirimicarb 50/50 0.0 Table 32.5 0.8 0. cyanazine.2 0. Prepacked "Bond Elut" TM MEGA BE-SI 6 mL columns. Experiments Undertaken in the Process of Method Development Example 14. For all oils tested hexane / diethyl ether (90 / 10) effected separation of oil components.2 0.8 0. 1 and 10 µg of monocrotophos. Acetonitrile (200 µL) 90 . Experimental: 1 mL of peppermint oil and boronia concrete were spiked with 0.0 1.9 0. 0.0 0.1 0. 0.4 0.1 not tested not tested not tested not tested hexane/ether 90/10 0. tebuconazole and difenaconazole to give fortification levels of 0.2 0. propazine.0 0. The acetone fraction was collected into 9 mL Kimbal disposable test tubes and dried under nitrogen on a heating block.7 0. propiconazole.1.7 0.9 0.0 1.0 0. were purchased from Varian.9 0.9 not tested not tested not tested not tested chloroform/ methanol 50/50 0.0 0.0 0.4 0. pirimicarb.01.0 0.2 0. Fortified oil (100 µL) was applied to the pre-conditioned columns and 4 x 1 mL of the hexane / ether was used to remove oil components from the column.0 0. A validation of the application of this silica based SPE to the clean-up of essential oils was undertaken.1 0. 1 and 10 mgkg-1. 5 µL of a 1 mgmL-1 solution of cyanazine was added to all as an internal standard. Acetone (4 mL) was used to elute the retained pesticides.0 0.8 0.01.6 1. Columns were pre-conditioned with 2 mL of hexane / ether (9:1).0 0.8 1. containing 1 g of silica.0 0.2 0. Chromotagraphic characteristics of selected pesticides resolved on TLC under differing solvent regimes.1.

GC column: carrier gas injection volume: injector temp: detector temp: initial temp: head pressure : Hewlett Packard 6890 Hewlett Packard 5MS 30m. The volatiles. This process was repeated with boronia oil to obtain a measure of the volatile material removed by SPE from the polar fraction containing the target pesticides. 10°Cmin-1 to 270°C (7 mins) 10psi.was added and the solution transferred to 200 µL insert in a 1 mL LC vial.236 mg of the internal standard. This experiment was also conducted in quadruplicates. Also included in this experiment was an assessment of the amount of volatiles eluted in each fraction relative to the total amount in oil not subject to SPE. octadecane. This was undertaken to obtain a measure of signal suppression due to oil components alone and overall recovery of pesticides from the acetone fraction. The fractions collected of ~4 mL of hexane / ether (9:1) and ~4 mL of acetone were each spiked with 4. The responses recorded for fortified solvents and peppermint oils applied to SPE were compared to samples fortified with equivalent concentrations of pesticides but which had not passed through columns. To obtain a measure of total volatiles in the oils used in the SPE validation experiment 25 µL of peppermint oil (22 mg) was dissolved in 1 mL of acetone and spiked with 1. The extractions were repeated 4 times for each concentration.927 mg of octadecane and subject to analyses by GC flame ionisation detection (FID). The percent volatiles recovered in each SPE fraction were determined by GC FID under the following conditions. and analysed directly. Analytical parameter: Samples were analysed by LC MS/MS using the conditions detailed on page 70. were expressed as pecentage recoveries relative to the amount of oil analysed by assuming a 1 : 1 response ratio to the known quantity of internal standard added.32µm instrument grade nitrogen 1µL (split) 250°C 280°C (FID) 50°C (3 min). defined as all peaks eluting before the octadecane under the conditions described. To assess the effect of oil on recoveries 100 µL of solvent spiked with the equivalent level of pesticides as that prepared in oil were also passed through silica columns. 91 . Results & Discussion: Table 33 records the %volatiles recovered in each SPE fraction relative to the amount determined in the neat oil. A further 100 µL of the peppermint oil was applied to the SPE column as described. i.d 0.

8 3.6 22. Recoveries of volatile components form peppermint and boronia oil in SPE fractions.2 0. which is a solvent based extraction using low polarity solvents such as hexane.3 49. has a high proportion of waxes and low polarity components which are not volatile within the GC environment. only 4.4 mg or 4.7 2. however.7 15.3 hexane/ether fraction 97.6 acetone fraction 97. are of limited relevance as boronia concrete.3 3.sample parameter wt of oil (mg) wt of volatiles calculated (mg) %volatiles % menthol sample parameter wt of oil (mg) wt of volatiles calculated (mg) %volatiles %post C18 peaks by GC FID %yield total GC-able peaks peppermint oil neat 21. In boronia extract only 22. The results presented for boronia. reveals on closer analyses that 85% of the volatiles in the acetone fraction is menthol. Of that percentage only 19% remained in the acetone fraction along with the pesticides. Excluding menthol.3% of the oil is detectable by GC FID.4 0.6 84. In some circumstances menthol can be selectively vented to waste. Peppermint oil on the other hand is produced by steam distillation and has a higher proportion of volatile components.3 boronia concrete neat 12. This result.1 25.9 Table 33.5 mg of the original 87.1 34.1 22. A total of 71.3% of the volatiles in peppermint oil are detectable by GC FID under the conditions listed.8 6.4 2. 92 . 84% of those volatiles come through in the 2 fractions collected with 58% eluting with the pesticides in the acetone fraction.6 6.4 3.5 71.8 15.1 30.4 hexane/ether fraction 87.6% of volatiles coelute with the target pesticide. which at first appears discouraging.0 acetone fraction 87.3 12.3 15.

6 70.4 78.2 242. The retention times of sethoxydim within the LC MS/MS were not consistent and did not elute within the time window established for the target ion. that in peppermint oil.9 56.0 76. 61% of the response is lost.E.3 0. the response for sethoxydim was found to be linear with increasing analyte concentration and that 15% of that signal is suppressed when compared to the same fortification level in solvent only at 10 mgkg-1.4 77.80E+07 3.17E+07 %C.7 83. Percentage response of pesticides in peppermint oil relative to solvent only injections 93 .1 377.0 propiconazole 69. Table 35 records the signal suppression caused by the presence of peppermint oil which is calculated by the formula % response = area of signal in peppermint oil x 100 area of signal in solvent only mgkg 10 1 0.V. m/z 282.3 64. Prometryn also did not elute from the silica column but the response of this analyte when analysed in peppermint oil without SPE was 78% (S.0 simazine 68.0 0.4 66. 2 2 2 22 18 %response 100 39 24 9 7 Table 34.2 0.1 0.2 0.0 tebuconazole 56.9 propazine 62.01 Table 35.7 80.V.0 0. Note should be made. The combined effect results in only 9% overall response to cyanazine. Recoveries of cyanazine from SPE with hexane / ether mobile phase The results listed in Table 34 indicate that cyanazine is not suitable as in internal standard for the analyses of pesticides using silica columns with the mobile phase tested.66E+08 1.0 pirimicarb 53.23E+08 1. and not subjected to SPE. however.9 0. This is further exacerbated when only 24% of the analyte is recovered from the silica column. The %C.0 difenaconazole 66.9 90. of 22% is too high to allow for the application of a correction factor to relate the final result for cyanazine on silica to the result obtained for the analyte not applied to silica.2 0. cyanazine n Response in solvent without SPE Response in peppermint oil without SPE Response in solvent with SPE Response in peppermint oil with SPE Response in boronia oil with SPE 4 4 16 16 16 area 4. Results are not reported for this analyte.03E+08 3.Table 34 lists the recoveries for the internal standard.6 67.1 96. 4% n=4) relative to that recorded when analysed in solvent only. When cyanazine is analysed within the matrix of peppermint oil.1 -1 monocrotophos 69.

5 tebuconazole 74±6 76±2 93±4 propiconazole 16±0 9. simazine and propazine the response is reasonably constant across the concentration ranges tested.8 60±1 13±13 48±3 90±5 propiconazole 68. then by 4 to allow for direct comparison with the areas detected by the 10µL injection of 94 .7 37±2 29±2 41±6 simazine 46.9±0.4±0. The recoveries are reported as percentages.4±0.7 52.1 0. mgkg 10 1 0.6 Table 38.0±0. For monocrotophos.3±0. Percentage recoveries of pesticides in solvent only when applied to SPE. Tables 37 and 38 list the recoveries of pesticides from SPE in peppermnt oil and boronia extract respectively. Percentage recoveries of pesticides in boronia oil by SPE relative to pesticides recovered from SPE without oil.4. propiconazole and difenaconazole there may be a co-eluting peak which enhances the signal disproportionately at lower concentrations. In addition this had been diluted by 4:1. Alternatively the responses for propiconazole and difenaconazole may be non-linear as the detection limits are approached.0 propiconazole 47±2 61±1 47±2 45±3 difenaconazole 44±2 57.7±0. However it would appear that for pirimicarb.9 52±3 35±2 48±7 propazine 43±2 53±3 46±1 64±5 tebuconazole 45±2 58.7±0. Percentage recoveries of pesticides in peppermint oil by SPE relative to pesticides recovered from SPE without oil.6 65.5±0.01 -1 monocrotophos 74±6 60±5 62±6 simazine 59.9 56±2 34.8 difenaconazole 97±6 95±2 119±11 181.8 65.1 0.Table 36 lists the responses of the target analytes applied to SPE in 100 µL of solvent.3±0.4±0.7±0.6 68±3 73±1 pirimicarb 111±11 114±5 158±4 179.4 49±3 58±7 pirimicarb 64±1 70±1 82±2 154±12 propazine tebuconazole 57.5 47±1 51±2 Table 36.01 -1 monocrotophos 137±11 127±5 210±11 514±7 simazine 49±3 41.9 178±3 571±89 difenaconazole 64±2 59±2 65±3 67±7 Table 37.1 0.7 32±1 pirimicarb 32.3±0. The matrix effect of peppermint oil on the analyses of all the pesticides tested was marked.01 mgkg-1. as a peak was not recorded for either analyte at concentrations around 0.01 -1 monocrotophos 26. The amount of essential oils able to be injected onto an LC MS/MS system is limited as contamination of inlet systems and the ion trap itself is possible. relative to the same concentrations of analytes not subject to SPE.7 61±1 0. mgkg 10 1 0.0±0.8 propazine 74±5 64±16 192±2 785±2.7 15.3±0.0±0. mgkg 10 1 0. As such only 10 µL of the oil solution not subject to SPE was injected into the LC MS/MS. The areas recorded for each peak were multiplied by 2.

In actuality this would mean that the LC MS/MS is attempting to detect a lower level of pesticide in peppermint oil than in solvent only such that any non-linear behaviour at low levels would be exasperated. This presents the potential to bind such pesticides onto ion exchange media whilst interfering matrices. which recorded a strong positive result even in non-fortified oil. 95 . Excluding the signal suppression observed for pesticides in essential oils the recoveries of pesticides in peppermint oil by SPE using the solvent protocol are low but the standard errors recorded at each concentration level are within an acceptable range indicating that with further work the SPE method developed has potential. In the following experiments the binding of six herbicides onto ion exchange discs is undertaken with detection by gas chromatography electron capture detection (GC ECD). The application of ion exchange chromatography to the clean-up of acidic pesticides with detection by GC ECD In basic solution acidic pesticides are present as negative ions. The SPE methodology allows for the processing of larger amounts of essential oil and reduces loading of essential oil components into analytical instrumentation. This would result in a false positive being recorded for pirimicarb in essential oil screens using LC MS/MS. Standard curves must be established within the matrices of contaminant free essential oil and must undergo the same work-up as that undertaken for the samples to be tested. It would be difficult to identify an internal standard without using isotopes of the analytes to be tested but the inclusion of an internal standard post-SPE would negate the effects of poor instrument precision etc.undiluted spiked solvents. are eluted. Recoveries from fortified boronia concretes detailed in Table 38 are higher than those obtained from peppermint oil further highlighting the effect different matrices can have on SPE methodology. This is not the case for pirimicarb. The retained pesticides can then be eluted from the ion exchange media in an acidic mobile phase and submitted for analysis. such as components from essential oils.

1 mgkg-1.HP Chem 1 µL. Whilst still wet.25 µm film thickness Instrument grade nitrogen 10 psi 60°C (1min. haloxyfop acid. adjusted to pH 10 with NaOH. 5 mL of the fortified buffer solution was passed through the preconditioned discs under gravity. splitless automatic injections 30 m HP 5MS. Empore™ ion exchange discs. provided by Varian. Table 40 lists the areas obtained for the two fortification levels tested. A non-linear relationship between analyte concentration and area detected is evident. 96 . 1 mL of acetonitrile and 100 µL of methyl iodide was added and then heated at 85°C for 60 mins. Experimental: Standard solutions of 1 mgmL-1 of clopyralid. The discs were then rolled and inserted into a 1 mL GC vial. A 5 mL aliquot of NaHCO3 buffer.Experiments Undertaken in the Process of Method Development Experiment 15 The trapping and derivatisation of acidic pesticides on ion exchange discs. transferred to a 100 µL GC insert and subject to GC ECD under the following conditions.) 260°C ECD 280°C Injection: Column: Carrier gas: Head Press : Oven Temp: Injection Temp: Detector: Results & Discussion: Of the six acids included in the experiment only four were successfully methylated using the method outlined. Analytical parameters: Instrumentation Hewlett Packard 5890 gas chromatograph Hewlett Packard Electron Capture Detector Processing Software . 0.22 mm id. was fortified with the standard solutions at levels equivalent to 10 mgkg-1 and 0. Aim: The effectiveness of trapping of six commonly used acidic pesticides in basic solutions onto anion exchange discs with subsequent methylation of the fixed acids with methyl iodide was to be tested. an effect inherently associated with ECD. fluroxypyr acid.) -20°C/min-290°C (10 min. 0. were punched into 13 mm diameter discs and washed under a light vacuum with 5 mL of acetone followed by 5 mL of methanol. MCPA and mecoprop were made up in acetone. dicamba. The discs were removed from the holders and dried at 80°C for 60 minutes. The GC lid was then opened and the solvent removed under a stream of nitrogen. The samples were then reconstituted in 200 µL of acetonitrile.

clopyralid. Whilst still wet the 3 mL of the buffer solution extract was passed through the pre-conditioned discs under gravity. Experiments Undertaken in the Process of Method Development Experiment 16 The isolation and derivatisation of acidic pesticides on anion exchange discs.18min clopyralid 972898 13180 7.1mgkg Table 40.retention time analyte 10mgkg -1 -1 4.94min fluroxypyr 216267 3131 11.71min dicamba 79026 917 5. Acetonitrile (1 mL) and 100 µL of methyl iodide was added and then the vial was heated to 85°C for 60 mins. fluroxypyr acid and haloxyfop acid were made up in acetone. fluroxypyr and haloxfop were used to fortify peppermint and fennel oil with view to application of the technique described. Aim: The success of the previous experiment in methylating 4 acidic pesticides using anion exchange discs justified the application of the technique to the pesticides in the matrix of essential oils. were punched into 13 mm diameter discs and washed under a light vacuum with 5 mL of acetone followed by 5 mL of methanol.24min haloxyfop 1588199 24770 0. The samples were then reconstituted in 200 µL of acetonitrile. Empore™ ion exchange discs.5 mgkg-1. The stock solutions of dicamba. Results & Discussion: Table 41 records the ECD response for the methylated acidic pesticides extracted from peppermint and fennel oil. Experimental: Standard solutions of 1 mgmL-1 of clopyralid. provided by Varian. 97 . The GC lid was then opened and the solvent removed under a stream of nitrogen. Aliquots (5 mL) of peppermint and fennel oil were fortified at levels equivalent to 10 and 0. dicamba. transferred to a 100 µL GC insert and subject to GC ECD under the conditions previously listed. ECD response to methylated acidic pesticides derivatised on anion exchange discs. The discs were removed from the holders and dried at 80°C for 60 minutes. The discs were then rolled and inserted into a 1 mL GC vial. Samples were extracted with 3 mL of NaHCO3 adjusted to pH 10 with NaOH.

eluting at around 12. However.908min 438271 786839 14497 588610 58584 haloxyfop 13. a very large contaminant was evident.5mgkg peppermint oil 10mgkg fennel oil 0.7 mins and elevating the base line through to the next sample run.477min 1576381 1981816 33778 1462200 67826 Table 41.5mgkg fennel oil -1 -1 -1 -1 -1 clopyralid 5.dicamba sample 10mgkg solvent only 10mgkg peppermint oil 0. Chromatogram of 0.5 mgkg-1 methylated acidic pesticides in peppermint oil by GC ECD 98 . Areas as obtained by GC ECD for acidic pesticides in peppermint and fennel oil methylated on ion exchange discs.5 mgkg-1 in peppermint oil. Figure 27 records the chromatogram as obtained by GC ECD for acidic pesticides fortified at a concentration of 0.059min 711900 574182 18148 684607 fluroxypyr 8.121min 269085 85959 5871 217000 4. Figure 27. The results indicate successful derivatisation of the acids on the ion exchange media.

This may indicate that the contaminant is moved through the column by acetonitrile. if the source is an indispensable ingredient in the process. Ideally the source of the contaminant should be isolated and removed from the sample work-up. much of the contaminant was removed following the elution of the solvent peak from the next injection. followed by a 6 minute run to a GC oven temperature of around 100°C. 99 . However. however.The contaminant bleed evident in Figure 27 continued through to subsequent injections. may remove the contaminant. However. the elution of the methylated haloxyfop acid rides the shoulder of the contaminant such that quantification of this analyte using the methodology detailed would be problematic. Interspersing such a run between samples would ensure the GC was relatively clean between injections. an injection of acetonitrile.

W. Oil and Hazardous Materials/Technical Assistance Data System. Chromatogr. Variation during dormancy and the effect of freezing and postharvest incubation on the chemical composition of blackcurrant buds (Ribes nigrum L. J. 1979. J. C. & Biotech. 1975. Residues of DDT and Analogues in Midwestern Mint Hay and Oil. 63. and Effects. Assoc. 115 682 -686. E. Inc. W. Organophosphates Chemistry. 2002. 53. E. and Claye. 1960. 221 Davies. T. E. 1964 . Anal. 1990 503 1-24. Problems in the Control of Mint Insects. Dissipation of propiconazole and tebuconazole in peppermint crops (Mentha piperita (Labiatae)) and their residues in distilled oils. New York. Food Chem. 171 263 . Food Chem.10-16 Cullen. 47 294-298 Gould. NY. Entomol. Buchert.. Academic Press.. J. J. and Lokke. Chem. and Davies. J. S. A. 21. Agric.. J.. A. and Patricia Levi. C. Fate. C. Menary. Inc. L.. Off. Gas Chromatographic Retention Indices of Monoterpenes and Sesquiterpenes on Methyl Silicone and Carbowax 20M Phases. J. J. M. S.268 Garland. Sci. 526-527. 1973. 1999. Baltimore. D. and Fahey. Econ. L. Deleu. R. MD. S. R. 1658-1660. Chambers.Literature Cited Ballee. N. J. G. Hort.. 234 100 . Entomol. 77.. 1988. Menary. (4) 489-497 Garland. J. 47. J.. G. J. 1992. E. R. Econ. N. Gould. Graham. H. and Copin.. 1970.. E. Agric. Chromatography. Chemical Information Systems. Chromatogr.). M.

Willoughby. Anal.. (11).I. J. F.). M. and Shibamoto.. Chelsea. P. The Agrochemicals Handbook. G. Food Chem. Kaniansky. Toxicol. 29.1994. C. 52. Assoc. and Davidson. S. A. Royal Society of Chemistry Information Services. R. Eds. Farm Chemicals Handbook 1992. D. Rao. C. M. and James.. J. Etievant. 72 970-974 Meister. Qualitative Analysis of Flavour and Fragrance Volatiles by Gas Capillary Column Gas Chromatography. 1980. Ann Arbor Science. Contam... F. Singer. J. Henry.. M. Environmental Data.1817-1824 Keppel. 1972. 729 . des Aliments. A. S.. OH. W. M. Academic Press. Anal. J.) Sci. 1981.. H. New York. Rigaud. 1992. J. and Rosen. R. J. 6. E... Louis.. Published by Lewis Publishers. Robson. Chem. G. High-pressure liquid chromatographic determination of fungicidal dithiocarbamates.Graham. Montgomery. and Onuska... 1969. W.T. Agric. M. Cambridge. 7. and Hansen. Davidson.. Chem. Off. Estimation of pesticide retention and transformation parameters required in nonpoint source pollution models. 1980. 1993. H. R.T. Environ. G. MI. 66. Latrasse. UK. 19 Gustafsson.. J. Thompson. Meister Publishing Company. J. Third Edition. D. 1986. R. Agrochemicals Desk Reference. 213-220 101 . 1991 Mattern. un constituant majeur de l'arome du bourgeon de cassis (Ribes nigrum L. (ed. K.732 Jennings. Eds. H. D. Le methoxy-4-methyl-2 butanethiol-2. 1989. J. Off.. In Environmental Impact of Nonpoint Source Pollution. Analytical Chemistry.167 Kidd. Ivanyi. L.. Overcash. H. Assoc. Bull. R. P. 162 . On-Line Isotachophoretic Sample Pretreatment in Ultratrace Detemination of Paraquat and Diquat in Water By Capillary Zone Electrophoresis.

Starr. (1-2) 237-250 Woodrow. (ed. 1963. J. Augustijn-Beckers. Thomson Publications. R. Chromgr. P. 2000. G. Analytical Method for the Dithiocarbamate Fungicides Ziram and Mancozeb in Air.. 1992. Thomson. The Pesticide Manual: A World Compendium. . J. 1992 U.and Terriere. Agric. Bethesda. Buttler. Z. T. Book 1: Insecticides. 1524-1529 Worthing.. M. Published by The British Crop Protection Council. 43.. 1987. Agric.. C. 482-486. Fresno. Washington. R.9-9 Wauchope.S. Hornsby A. and Yu. M. 1995. IL. Seventh Edition. Weed Science Society of America. U. National Library of Medicine. 123: 1-157. D. Kiigemagi. M. Guidance for the Registration of Pesticide Products Containing Maneb as the Active Ingredient. Solid-phase extraction of acidic herbicides. 1995. L.. Herbicide Handbook. CA. M. 1994 Wells. 11. 1988. Environmental Protection Agency. J. 885. L. Toxicol. W. Eighth edition.) . Contam. and Burt..4-11 U. J. Insecticide Residues in Peppermint and their Distillation with Peppermint Oil. DC. C.S. W. Environ. Agricultural Chemicals. SCS/ARS/CES Pesticide properties database for environmental decisionmaking. Food Chem. Hazardous Substances Databank. Rev. 102 . MD. Champaign. J.. H.. J. P. T. Food Chem.

The response for carbon disulfide was non-linear and less sensitive than that recorded for phosphorus compounds using the 524 µm filter 103 .W. Analysis for Mancozeb in Peppermint Leaves by Detection of Carbon Disulfide Produced by Digestion with Acidified Stannous Chloride.Appendix 1. 2 mL of distilled water and 2 mL of acidified stannous chloride solution were added.618 g of SnCl2. Note that mancozeb is insoluble in water. = 225. but vigorous shaking of mancozeb / water mixtures produces a fine suspension from which sub-samples can be taken with some degree of accuracy.31 so 1 µmole of mancozeb produces 146 µg CS2.2H2O (M. Peppermint leaves were ground under liquid nitrogen in a stainless steel mortar and pestle. Each unit of a mancozeb molecule produce 2 molecules of carbon disulfide. The FPD detector had a 393 nm filter specific for the detection of sulphur. Standard curves were produced by fortifying leaf samples with known quantities of mancozeb. Acidified stannous chloride was prepared by dissolving 1. The gaseous CS2 trapped in the headspace vial was pressurised and vented to the injection port of a gas chromatograph. The analytical system was calibrated with solutions of known quantities of analytical grade carbon disulphide dissolved in acidified stannous chloride.65) in 100 mL of the 5M HCl. The molecular weight of a unit of the mancozeb chain is 266.. The vials were shaken vigorously and placed in an oven at 90ºC for 1 hour. 2 g of the ground leaves were weighed into 25 mL headspace vials.

5 X 20 samples of 50 leaves were collected to establish a de-sorption profile of mancozeb into 5µM EDTA / 9 µM NaOH. 15 and 30 minutes. A further selection of leaves / sample were weighed and then dried at constant temperature and humidity to determine dry weight. The desorption isotherm established (Figure 28) was used to determine the optimum time for extraction of mancozeb into the chelating reagent. In the laboratory the samples were divided into 5 groups of 4. An extra 50 leaves were collected and passed through a Paton Electronic Planimeter to give an estimate of total leaf area sub-sampled. Unfortunately the isotherm did not reach an asymptote within 30 minutes 104 . Maceration of leaves was therefore not undertaken as the co-extraction of vegetative matrix would not enhance the amount of mancozeb extracted yet would increase the load of carbon compounds. 50 leaves were removed from treated peppermint plants and placed in pre-weighed 250 mL 78 x 70 mm polycarbonate vials. Another 4 samples were immersed in 150 mL 5µM EDTA / 9 µM NaOH for periods of 5 minutes whilst the remaining 3 x 4 leaf samples were subject to passive extraction for 10. De-sorption Isotherm: Following an application of Mancozeb. As neither maceration nor agitation were to be applied during the extraction process a desorption isotherm was established to determine how long leaves should be immersed in the extracting solution to effect solvation of mancozeb. 150 mL of the 5µM EDTA / 9 µM NaOH was added ensuring all the leaves were covered. After decanting all samples were submitted for analyses by ICP OES. The leaves were collected into 250 mL. The containers were left for 1 minute. thereby precluding the application of ICP-OES to the monitoring of residual manganese from the mancozeb polymer. Analysis for Mancozeb in Peppermint Leaves by Detection of Manganese Quantified by ICP OES.2. The samples were re-weighed. Mancozeb is a non-systemic fungicide which remains on the surface of the leaf. 78 x 70 cm polycarbonate specimen containers sealed with a polypropylene lid. without agitation before the aqueous extract was decanted into plastic 40 mL vials. Using tweezers to grasp the base of the petiole.

Derivatisation .20 -30 mg of essential oil was weighed into a GC vial. Desorption isotherm of mancozeb into NaOH / EDTA 3. The mix was transferred to a separating funnel and the aqueous layer drained and washed with a further 25 mL of ether. ready for analysis. The mixture was placed on ice. 40 mL of distilled water and 50 mL of diethyl ether was added. The vials were left at room temperature for 10 minutes. 105 . N-methylN-nitrosourea (3 g) was added slowly and the mixture was stirred for 20 minutes. 0. was weighed into a 250 mL conical flask. Derivatisation of Residues of Pesticide with Acidic Moieties in Essential Oils Diazomethane Preparation .75 mL of the diazomethane in ether solution was added and the solutions were gently swirled to ensure the oil was completely in solution. 200 µL of glacial acetic acid was added to each vial to quench the remaining diazomethane.5 mancozeb mg/kg DMB 1 0.1. 16 g. The ether solutions were combined and stored at -4°C until use.Potassium hydroxide. GC vials were crimp sealed.5 0 0 10 20 30 40 minutes Figure 28.