Determination of Pesticide Minimum Residue Limits in ...

Determination of Pesticide Minimum Residue Limits in Essential Oils

Report No 3

A report for the Rural Industries Research and Development Corporation By Professor R. C. Menary & Ms S. M. Garland

June 2004 RIRDC Publication No 04/023 RIRDC Project No UT-23A

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© 2004 Rural Industries Research and Development Corporation. All rights reserved. ISBN 0642 58733 7 ISSN 1440-6845 ‘Determination of pesticide minimum residue limits in essential oils’, Report No 3 Publication No 04/023 Project no.UT-23A 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 Professor R. C. Menary & Ms S. M. Garland School of Agricultural Science University of Tasmania GPO Box 252-54 Hobart Tasmania 7001 Australia Phone: (03) 6226 2723 Fax: (03) 6226 7609 Email: [email protected] 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: 02 6272 4819 Fax: 02 6272 5877 Email: [email protected] Website: http://www.rirdc.gov.au Published in June 2004 Printed on environmentally friendly paper by Canprint.

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FOREWORD International regulatory authorities are standardising the levels of pesticide residues present in products on the world market which are considered acceptable. The analytical methods to be used to confirm residue levels are also being standardised. To constructively participate in these processes, Australia must have a research base capable of constructively contributing to the establishment of methodologies and must be in a position to assess the levels of contamination within our own products. Methods for the analysis for pesticide residues rarely deal with their detection in the matrix of essential oils. This project is designed to develop and validate analytical methods and apply that methodology to monitor pesticide levels in oils produced from commercial harvests. This will provide an overview of the levels of pesticide residues we can expect in our produce when normal pesticide management programs are adhered to. The proposal to produce a manual which deals with the specific problems associated with detection of pesticide residues in essential oils is intended to benefit the essential oil industry throughout Australia and may prove useful to other horticultural products. This report is the third in a series of four project reports presented to RIRDC on this subject. It is accompanied by a technical manual detailing methodologies appropriate to the analysis for pesticide residues in essential oils. This project was part funded from RIRDC Core Funds which are provided by the Australian Government. Funding was also provided by Essential Oils of Tasmania and Natural Plant Extracts Cooperative Society Ltd. This report, an addition to RIRDC’s diverse range of over 1000 research publications, forms part of our Essential Oils and Plant Extracts R&D program, which aims for an Australian essential oils and plant extracts industry that has established international leadership in production, value adding and marketing. Most of our publications are available for viewing, downloading or purchasing online through our website: • downloads at www.rirdc.gov.au/fullreports/index.html • purchases at www.rirdc.gov.au/eshop Simon Hearn Managing Director Rural Industries Research and Development Corporation

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Acknowledgements Our gratitude and recognition is extended to Dr. Noel Davies (Central Science Laboratories, University of Tasmania) who provided considerable expertise in establishing procedures for chromatography mass spectrometry. The contribution to extraction methodologies and experimental work-up of Mr Garth Oliver, Research Assistant, cannot be underestimated and we gratefully acknowledge his enthusiasm and novel approaches. Financial and ‘in kind’ support was provided by Essential Oils Industry of Tasmania, (EOT).

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Abbreviations ADI Average Daily Intake AGAL Australian Government Analytical Laboratories ai active ingredient APCI Atmospheric Pressure Chemical Ionisation BAP Best Agricultural Practices CE collision energy DETA Diethylene triamine ECD Electron Capture Detector ESI Electrospray ionisation FPD Flame Photometric Detection GC Gas Chromatography HR High Resolution LC Liquid Chromatography LC MSMS Liquid Chromatography with detection monitoring the fragments of Mass Selected

ions MRL Maximum Residue Limit MS Mass Spectrometry NRA National Registration Authority R.S.D. Relative Standard Deviation SFE Supercritical Fluid Extraction SIM Single Ion Monitoring SPE Solid Phase Extraction TIC Total Ion Chromatogram

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Contents

FOREWORD.......................................................................................................................................III

ACKNOWLEDGEMENTS................................................................................................................ IV

ABBREVIATIONS .............................................................................................................................. V

CONTENTS.........................................................................................................................................VI

EXECUTIVE SUMMARY............................................................................................................... VII

1. INTRODUCTION ............................................................................................................................. 1

1.1 BACKGROUND TO THE PROJECT ........................................................................................................ 1 1.2 OBJECTIVES ....................................................................................................................................... 2 1.3 METHODOLOGY ................................................................................................................................. 2

2. EXPERIMENTAL PROTOCOLS & DETAILED RESULTS ..................................................... 3

2.1 METHOD DEVELOPMENT ................................................................................................................... 3 2.2 MONITORING OF HARVESTS ............................................................................................................ 42 2.3 PRODUCTION OF MANUAL............................................................................................................... 46

3. CONCLUSIONS.............................................................................................................................. 47

IMPLICATIONS & RECOMMENDATIONS................................................................................. 50

BIBLIOGRAPHY ............................................................................................................................... 50

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Executive Summary The main objective of this project was to continue method development for the detection of pesticide residues in essential oils, to apply those methodologies to screen oils produced by major growers in the industry and to produce a manual to consolidate and coordinate the results of the research. Method development focussed on the effectiveness of clean-up techniques, validation of existing techniques, the assessment of the application of gas chromatography (GC) with detection using electron capture detectors (ECD), flame photometric detectors (FPD) and high pressure liquid chromatography (HPLC) with ion trap mass selective (MS) detection. The capacity of disposable C18 cartridges to separate components of boronia oil was found to be limited with the majority of boronia components being eluted on the solvent front, with little to no separation achieved. The cartridges were useful, however, in establishing the likely interaction of reverse phases (RP) C18 columns with components of essential oils, using polar mobile phases . The loading of large amounts of oil onto RP HPLC columns presents the risk of permanently contaminating the bonded phases. The lack of retention of components on disposable SPE C18 cartridges, despite the highly polar mobile phase, presented a good indication that essential oils would not accumulate on HPLC RP columns. The removal of non-polar essential oil components by solvent partitioning of distilled oils was minimal, with the recovery of pesticides equivalent to that recorded for the essential oil components. However application of this technique was of advantage in the analysis of solvent extracted essential oils such as those produced from boronia and blackcurrant. ECD was found to be successful in the detection of terbacil, bromacil, haloxyfop ester, propiconazole, tebuconazole and difenaconzole. However, analysis of pesticide residues in essential oils by application of GC ECD is not sufficiently sensitive to allow for a definitive identification of any contaminant. As a screen, ECD will only be effective in establishing that, in the absence of a peak eluting with the correct retention time, no gross contamination of pesticide residues in an essential oil has occurred . In the situation where a peak is recorded with the correct elution characteristics, and which is enhanced when the sample is fortified with the target analyte, a second means of contaminant identification would be required. ECD, then, can only be used to rule out significant contamination and could not in itself be adequate for a positive identification of pesticide contamination. Benchtop GC daughter, daughter mass spectrometry (MSMS) was assessed and was not considered practical for the detection of pesticide residues within the matrix of essential oils without comprehensive clean-up methodologies. The elution of all components into the mass spectrometer would quickly lead to detector contamination. Method validation for the detection of 6 common pesticides in boronia oil using GC high resolution mass spectrometry was completed. An analytical technique for the detection of monocrotophos in essential oils was developed using LC with detection by MSMS. The methodology included an aqueous extraction step which removed many essential oil components from the sample.

Further method development of LC MSMS included the assessment of electrospray ionisation (ESI) and atmospheric pressure chemical ionisation (APCI. For the chemicals trialed, 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. Overall, there was very little difference between the sensitivity for ESI and APCI. However, APCI was slightly more sensitive for the commonly used pesticides, tebuconazole and propiconazole, and showed a response, though poor, to linuron and oxyflurofen. In addition, APCI was the preferred ionisation method for the following reasons, ♦ APCI uses less nitrogen gas compared to ESI, making overnight runs less costly;

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♦ 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 fracturing the cell, which is pressure sensitive.

Analytes that ionised in the negative APCI mode were incorporated into a separate screen which included bromacil, terbacil, and the esters of the fluazifop and haloxyfop acids. Further work using APCI in the positive mode formed the basis for the inclusion of monocrotophos, pirimicarb, propazine and difenaconazole into the standard screen already established. Acephate, carbaryl, dimethoate, ethofumesate and pendimethalin all required further work for enhanced ionisation and / or improved elution profiles. Negative ionisation mode for APCI gave improved characteristics for dicamba, procymidone, MCPA and mecoprop. The thirteen pesticides included in this general screen were monocrotophos, simazine, cyanazine, pirimicarb, propazine, sethoxydim, prometryb, tebuconazole, propiconazole, , difenoconazole and the esters of fluroxypyr, fluazifop and haloxyfop.. Bromacil and terbacil were not included as both require negative ionisation and elute within the same time window as simazine, which requires positive ionisation. Cycling the MS between the two modes was not practical. The method validation was tested against three oils, peppermint, parsley and fennel. Detection limits ranged from 0.1 to 0.5 mgkg-1 within the matrix of the essential oils, with a linear relationship established between pesticide concentration and peak height (r2 greater than 0.997) and repeatabilities, as described by the relative standard deviation (r.s.d), ranging from 3 to 19%. The type of oil analysed had minimal effect on the response function as expressed by slope of the standard curve. The pesticides which have an carboxylic acid moiety such as fluazifop, haloxyfop and fluroxypyr, present several complications in any analytical method development. The commercial preparations usually have the carboxylic acid in the ester form, which is hydrolysed to the active acidic form on contact with soil and vegetation. In addition, the esters may be present in several forms, such as the ethoxy ethyl or butyl esters. Detection using ESI was tested. Preliminary results indicate that ESI is unsuitable for haloxyfop and fluroxypyr ester. Fluazifop possessed good ionisation characteristics using ESI, with responses approximately thirty times that recorded for haxloyfop. Poor chromatography and response necessitated improved mobile phase and the effect of pH on elution characteristics was considered the most critical parameter. The inclusion of acetic acid improved peak resolution. The LC MSMS method for the detection of dicamba, fluroxypyr, MCPA, mecoprop and haloxyfop in peppermint and fennel distilled oils underwent the validation process. Detection limits ranged from 0.01 to 0.1 mgkg-1 Extraction protocols and LC MSMS methods for the detection of paraquat and diquat were developed. ESI produced excellent responses for both paraquat and diquat, after some modifications of the mobile phase. Extraction methodology using aqueous phases were developed. Extraction with carbonate buffer proved to be the most effective in terms of recovery and robustness. A total ion chromatogram of the LC run of an aqueous extract of essential oil was recorded and detection using a photodiode array detector confirmed that very little essential oil matrix was co-extracted. The low background noise indicated that samples could be introduced directly into the MS. This presented a most efficient and rapid way for analysis of paraquat and diquat, avoiding the need for specialised columns or modifiers to be included in the mobile phase to instigate ion exchange. The adsorbtion of paraquat and diquat onto glass and other surfaces was reduced by the inclusion of diethylenetriamine (DETA). DETA preferentially accumulates on the surfaces of sample containers, competitively binding to the adsorption sites. All glassware used in the paraquat diquat analysis were washed in a 5% solution of 0.1M DETA, DETA was included in all standard curve preparations, oils were extracted with aqueous DETA and the mobile phase was changed to 50:50 DETA / methanol. The stainless steel tubing on the switching valve was replaced with teflon, further improving

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reproducibility. Method validation was undertaken of the analysis of paraquat and diquat using the protocols established. The relationship between analyte concentration and peak area was not linear at low concentrations, with adsorption more pronounced for paraquat, such that the response for this analyte was half that seen for diquat and the 0.1 mgkg-1 level. The development of a method for the detection of the dithiocarbamate, mancozeb was commenced. Disodium N, N'-ethylenebis(dithiocarbamate) was synthesised as a standard for the derivatised final analytical product. An LC method, with detection using MSMS, was successfully completed. The inclusion of a phase transfer reagent, tetrabutylammonium hyrdrogen sulfate, required in the derivatisation step, contaminated the LC MSMS system, such that any signal from the target analyte was masked. Alternatives to the phase transfer reagent are now being investigated. Monitoring of harvests were undertaken for the years spanning 1998 to 2001. Screens were conducted covering a range of solvent extracted and distilled oils. Residues tested for included tebuconazole, simazine, terbacil, bromacil, sethoxydim, prometryn, oxyflurofen, pirimicarb, difenaconazole, the herbicides with acidic moieties and paraquat and diquat. Problems continued for residues of propiconazole in boronia in the 1998 / 1999 year with levels to 1 mgkg-1 still being detected. Prometryn residues were detected in a large number of samples of parsley oil. Finally the information gleaned over years of research was collated into a manual designed to allow intending analysts to determine methodologies and equipment most suited to the type of the pesticide of interest and the applicability of analytical equipment generally available.

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1. Introduction 1.1 Background to the Project Research undertaken by the Horticultural Research Group at the University of Tasmania, into pesticide residues in essential oils has been ongoing for several years and has dealt with the problems specific to the analysis of residues within the matrix of essential oils. Analytical methods for pesticides have been developed exploiting the high degree of specificity and selectivity afforded by high resolution gas chromatography mass spectrometry. Standard curves, reproducibility and detection limits were established for each. Chemicals, otherwise not amenable to gas chromatography, were derivatised and incorporated into a separate screen to cover pesticides with acidic moieties. Research has been conducted into low resolution GC mass selective detectors (MSD and GC ECD. Low resolution GC MSD achieved detection to levels of 1 mgkg-1 in boronia oil, whilst analysis using GC ECD require a clean-up step to effectively detect halogenated chemicals below 1mgkg-1. Dithane (mancozeb) residues were digested using acidified stannous chloride and the carbon disulphide generated from this reaction analysed by GC coupled to FPD in the sulphur mode. Field trials in peppermint crops were established in accordance with the guidelines published by the National Registration Authority (NRA), monitoring the dissipation of Tilt and Folicur residues in peppermint leaves and the co-distillation of these residues with hydro-distilled peppermint oils were assessed. Development of extraction protocols, analytical methods, harvest monitoring and field trials were continued and were detailed in a subsequent report. Solvent-based extractions and supercritical fluid extraction (SFE) was found to have limited application in the clean-up of essential oils In conjunction with Essential Oils of Tasmania (EOT), the contamination risk, associated with the introduction of a range of herbicides, was assessed through a series of field trials. This required analytical method development to detect residues in boronia flowers, leaf and oil. The methodology for a further nine pesticides was successful applied. Detection limits for these chemicals ranged from 0.002 mgkg-1 to 0.1 mgkg-1. In addition, methods were developed to analyse for herbicides with active ingredients (ai) whose structure contained acidic functional groups. Two methods of pesticide application were trialed. Directed sprays refer to those directed on the stems and leaves of weeds at the base of boronia trees throughout the trial plot. Cover sprays were applied over the entire canopy. For all herbicides for which significant residues were detected, it was evident that cover sprays resulted in contamination levels ten times those occurring as a result of directed spraying in some instances. Chloropropham, terbacil and simazine presented potentially serious residue problems, with translocation of the chemical from vegetative material to the flower clearly evident. Directed spray applications of diuron and dimethenamid presented only low residue levels in extracted flowers with adequate control of weeds. Oxyflurofen and the mixture of bromacil and diuron (Krovar) presented only low levels of residues when used as a directed spray and were effective as both post and pre-emergent herbicides. Only very low levels of residues of both sethoxydim and norflurazon were detected in boronia oil produced in crops treated with directed spray applications. Sethoxydim was effective as a cover spray for grasses whilst norflurazon showed potential as herbicide to be used in combination with other chemicals such as diuron, paraquat and diquat. Little contamination of boronia oils by herbicides with acidic moieties was found. This advantage, however, appears to be offset by the relatively poor weed control. Both pendimethalin and haloxyfop showed good weed control. Both, however, present problems with chemical residues in boronia oil and should only be used as a directed spray The stability of tebuconazole, monocrotophos and propiconazole in boronia under standard storage conditions was investigated. Field trials of tebuconazole and propiconazole were established in commercial boronia crops and the dissipation of both were monitored over time. The amount of pesticide detected in the oils was related to that originally present in the flowers from which the oils were produced.

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Experiments were conducted to determine whether the accumulation of terbacil residues in peppermint was retarding plant vigour. The level recorded in the peppermint leaves were comparatively low. It is unlikely that terbacil carry over is the cause for the lack of vigour in young peppermint plants. Boronia oils produced in 1996, 1997 and 1998 were screened for pesticides using the analytical methods developed. High levels of residues of propiconazole were shown to persist in crops harvested up until 1998. Field trials have shown that propiconazole residues should not present problems if the fungicide is used as recommended by the manufacturers. 1.2 Objectives ♦ Provide the industry, including the Standards Association of Australia Committee CH21, with a

concise practical reference, immediately relevant to the Australian essential oil industry ♦ Facilitate the transfer of technology from a research base to practical application in routine

monitoring programs ♦ Continue the development of analytical methods for the detection of metabolites of the active

ingredients of pesticide in essential oils. ♦ Validate the methods developed. ♦ Provide industry with data supporting assurances of quality for all exported products. ♦ Provide a benchmark from which Australia may negotiate the setting of a realistic maximum

residue limit (MRL) ♦ Determine whether the rate of uptake is relative to the concentration of active ingredient on the

leaf surface may establish the minimum application rates for effective pest control. 1.3 Methodology Three approaches were used to achieve the objectives set out above. ♦ Continue the development and validation of analytical methods for the detection of pesticide

residues in essential oils. Analytical methods were developed using gas chromatography high resolution mass spectrometry (GC HR MS), GC ECD, GC FPD and high pressure liquid chromatography with detection using MSMS.

♦ Provide industry with data supporting assurances of quality for all exported products. ♦ Coordinate research results into a comprehensive manual outlining practical approaches to the

development of analytical procedures One aspect of the commissioning of this project was to provide a cost effective analytical resource to assess the degree of the pesticide contamination already occurring in the essential oils industry using standard pesticide regimens. Oil samples from annual harvests were analysed for the presence of pesticide residues. Data from preceding years were collated to determine the progress or otherwise, in the application of best agricultural practice (BAP).

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2. Experimental Protocols & Detailed Results The experimental conditions and results are presented under the following headings: ♦ Method Development ♦ Monitoring of Commercial Harvests ♦ Production of a Manual 2.1 Method Development Method development focussed on the effectiveness of clean-up techniques, validation of existing techniques, the assessment of the application of GC ECD and FPD and high pressure liquid chromatography with ion trap MS, MS detection. 2.1.1 Clean-up Methodologies 2.1.1.i. Application of Disposable SPE cartridges in the clean-up of pesticide residues in essential oils Literature reviews provided limited information with regards to the separation of contaminants within essential oils. The retention characteristics of disposable C18 cartridges were trialed. Experiment 1 ; Aim : To assess the capacity of disposable C18 cartridges to the separation of boronia oil components. Experimental : Boronia concrete (49.8 mg) was dissolved in 0.5 mL of acetone and 0.4 mL of chloroform was added. 1mg of octadecane was added as an internal standard. A C18 Sep-Pak Classic cartridge (short body) was preconditioned with 1.25 mL of methanol, which was passed through the column at 7.5 mLmin-1, followed by 1.25 mL of acetone, at the same flow rate. The boronia sample was then applied to the column at 2 mLmin-1 flow and eluted with 1.25 mL of acetone / chloroform (5 / 4) and then eluted with a further 2.5 mL of chloroform. 5 fractions of 25 drops each were collected. The fractions were analysed by GC FID using the following parameters Analytical parameters GC Hewlett Packard 6890 column: Hewlett Packard 5MS 30m, i.d 0.32µm carrier gas instrument grade nitrogen injection volume: 1µL (split) injector temp: 250°C detector temp: 280°C inital temp: 50°C (3 min), 10°Cmin-1 to 270°C (7 mins) head pressure : 10psi. Results : Table 1 record the percentage volatiles detected in the fractions collected Fraction 1 2 3 4 5 % components eluting 18 67 13 2 %monoterpenes 15 63 6 %sesquiquiterpenes 33 65 2 %high M.W components 1 43 47 9

Table 1. Percentage volatiles eluting from SPE C18 cartridges Discussion : The majority of boronia components eluted on the solvent front, effecting minimal separation. This area of SPE clean-up of essential oils requires a wide ranging investigation, varying parameters such as cartridge type and polarity of mobile phase.

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Experiment 2. Aim : For the development of methods using LC MSMS without clean-up steps, the potential for oil components to accumulate on the reverse phase (RP) column must be assessed. The retention of essential oil components on SPE C18 cartridges, using the same mobile phase as that to be used in the LC system, would provide a good indication as to the risk of contamination of the LC columns with oil components. Experimental: Parsley oil (20-30 mg) was weighed into a GC vial. 200 µL of a 10 µgmL-1 solution (equivalent to 100mgkg-1 in oil) of each of sethoxydim, simazine, terbacil, prometryn, tebuconazole and propiconazole were used to spike the oil, which was then dissolved in 1.0 mL of acetonitrile. The solution was then slowly introduced to the C18 cartridge (Waters Sep Pac 'classic' C18 #51910) using a disposable luer lock, 10 mL syringe, under constant manual pressure, and eluted with 9 mLs of acetonitrile. Ten, 1 mL fractions were collected and transferred to GC vials. 1mg of octadecane was added to each vial and the samples were analysed by GC FID under the conditions described in experiment 1. The experiment was repeated using C18 cartridges which had been pre-conditioned with distilled water for 15 mins. Again, parsley oil, spiked with pesticides was eluted with acetonitrile and 5 x 1 mL fractions collected. Results: The majority of oil components and pesticides were eluted from the C18 cartridge in the first two fractions. Little to no separation of the target pesticides from the oil matrix was achieved. Table 2 lists the distribution of essential oil components in the fractions collected.

Fraction 1 2 3 4 5 % components eluting 18 67 13 2 %monoterpenes 15 63 6 %sesquiquiterpenes 33 65 2 %high M.W components 1 43 47 9 water conditioned % components eluting 35 56 8 1 %monoterpenes 30 68 2 0 %sesquiquiterpenes 60 39 1 0 %high M.W components 0 50 42 7

Table 2. Percentage volatiles eluting for SPE C18 cartridges

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Figure 1 shows a histogram of the percentage distribution of components from the oil in each of the four fractions.

Figure 1. Histogram of the percentage of volatiles of distilled oils in each of four fraction eluted

on SPE C18 cartridges (non-preconditioned)

Figure 2. Histogram of the percentage of volatiles of distilled oils in each of four fraction eluted on SPE C18 cartridges (preconditioned)

Discussion : The chemical properties of many of the target pesticides, including polarity, solubility in organic solvents and chromatographic behaviour, are similar to the majority of essential oil components. This precludes the effective separation of analytes from such matrices through the use of standard techniques, where the major focus is pre-concentration of pesticide residues from water or water based vegetative material. However, this experiment served to provide a good indication that under HPLC conditions, where a reverse phase C18 column is used in conjunction with acetonitrile / water based mobile phases, essential oil components do not remain on the column.

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2.1.1.ii. The Application of Solvent Partitioning to the Clean-up of Pesticide Residues in Essential Oils Previous clean-up techniques applied to the analysis of pesticide residues in boronia concretes were applied to distilled oils. Aim : To assess the effectiveness of solvent partition to the clean-up of pesticide residues in essential oil with detection by LC MSMS. Experimental : 20 - 30 mg of fennel oil, fortified with 200µL of a 10 µgmL-1 solution of sethoxydim, simazine, prometryn, tebuconazole and propiconzole, was weighed into a 1 mL GC vial. 200 µL of hexane was added, ensuring the oil was fully solvated. 800 µL of methanol was added and 2 drops of water were introduced to effect a partition. As no particulate matter was evident, no filtering was undertaken and the methanol / water fraction was sub-sampled for analysis using the new LC MSMS methods (page 36). In addition, the effluent form the LC column was diverted to a UV photodiode ray detector to access the amount of oil components remaining in the samples. Samples, fortified at equivalent levels and dissolved in 1mL methanol / water, were analysed concurrently. Analytical parameters LCMSMS : A Waters 2690 separation module (Alliance) system equipped with a NOVAPAK C18, 3.9 x 150 mm column was used to establish a mobile phase with gradient of 50:50 acetonitrile / 0.1M ammonium acetate buffer ramped to 90 : 10 over 15 minutes. A Finigan MAT LCQ was used to monitor the ions listed in table 3.

analyte ions retention monitored time

simazine 123.5-124.5 2.52 sethoxydim 281.5-282.5 4.08 prometryn 199.0-200.8 5.24 tebuconazole 307.5-308.5 5.32 propiconazole 158.5-159.5 6.58

Table 3. Ions monitored by LC MSMS for assessment of efficiency of solvent partition clean-up Results & Discussion ; The removal of non-polar essential oil components by solvent partition was minimal with the recovery of pesticides equivalent to that recorded for the essential oil components. As such, this clean-up method does not serve to increase detection limits in distilled oils. However, the removal of non-polar, high molecular weight components within essential oils prior to injection, will help to maintain LC column performance. Application of this technique should be of particular advantage in the analysis of solvent extracted essential oils such as those produced from boronia flowers and blackcurrant buds.

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2.1.2 Extension of Analytical Techniques 2.1.2 i. Assessment of GC ECD for the analysis of pesticide residues in essential oils Electron Capture Detectors (ECD) are specific to halogenated chemicals. Both distilled oils and solvent extracted oils are predominantly hydrocarbons and oxygenated hydrocarbons. The bulk of the matrix of an essential oil, contaminated with halogenated pesticide residues, should not register by ECD. As discussed previously, however, any one component of an essential oil can constitute over 10% of the oil, compared to the 0.0001% a pesticide contamination of 1 mgkg-1 would represent. Obviously co-elution of a major oil component with the target residue analyte would saturate the signal and mask the ECD response. The retention characteristics of the target analyte, relative to the major components of essential oils, is therefore of critical relevance. Gas chromatographic retention indices (Kováts' indices) relate the retention time of a particular analyte to the retention of a series of nCnH(2n+2) hydrocarbons. When considering the suitability of GC ECD to the analysis of a new target analyte, determining the retention characteristics relative to essential oil components presents as an obvious starting point. It is useful to have previously determined the retention indices of the components of the relevant essentials oils. The introduction of a new pesticide into a GC screen could then be assessed by first determining the retention indices for the new analyte and using the established indices for the essential oil components to predict the likelihood of the pesticide residue eluting in a time window clear of other major essential oil components. Retention Indices of Essential Oils Aim : The 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 easily detect a 1µL split injection of a 1 mgmL-1 solution. A 1 mgmL-1 solution of a range of CnH(2n+2) chemicals was prepared and analysed in the same column, and under identical conditions of pressure and temperature, as those to be used in the proposed GC ECD analysis. Analytical parameters Instrumentation Hewlett Packard 5890 gas chromatograph Hewlett Packard Flame Ionisation Detector Processing Software - HP Chem Injection: 1 µL, split automatic injections Column: 30 m HP 5MS, 0.22 mm id, 0.25 µm film thickness Carrier gas: Instrument grade nitrogen Head Press : 10 psi Oven Temp: 60°C (1min.) -20°C/min-290°C (10 min.) Injection Temp: 260°C Detector: FID 280°C Figure 3, 4, 5 and 6 are the GC FID chromatograms for injection of 20 mgkg-1 solutions of parsley, fennel, peppermint and boronia oils.

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Figure 3 - GC FID of distilled parsley oil

Figure 4. - GC FID of distilled peppermint oil

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Figure 5 - GC FID of distilled fennel oil

Figure 6 - GC FID of solvent extracted boronia oil

Table 4 records the retention times of a mixed, 1 mgmL-1 solutions of hydrocarbon standards ranging from C8H18 to C36H74. The Kovat indices are calculated using the formula t'R(A) - t'R(N) I ab = 100N + 100n -------------------- t'R(N+n) - t'R(N)

waxes and less volatile compounds

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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 - paraffin hydrocarbons of carbon numbers N and (N+ n) that are respectively smaller and larger then the adjusted retention times of the unknown, t'R(A) (ref 1.)

Standard Ret. Time Kovát's Peak (mins) indices C15H32 14.804 1500

C16H34 16.126 1600

C17H36 17.381 1700

C18H38 18.582 1800

C20H42 20.794 2000

C21H44 21.834 2100

C22H46 22.817 2200

C24H50 24.682 2400

C28H58 28.697 2800

C34H70 35.435 3400

Table 4. Retention times of CnHn+2 hydrocarbons The retention times of the major components and the calculated Kovats' indices are listed in table 5.

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Parsley ret. Kovat's Fennel ret. Kovat's

time indices time indices

α-pinene 6.40 674 α-pinene 6.33 667

β-pinene 7.13 743 α-phellandrene 7.45 773

mycrene 7.50 778 limonene 7.86 812

β-phellandrene 8.03 827 β-phellandrene 7.86 812

α-terpinolene 8.94 913 fenchone 9.07 925

menthatriene 9.47 962 estragole 10.71 1079 tetramethoxy- trans-anethole 12.52 1249

allyl benzene 15.57 1535 myristcin 16.53 1626 apiole 21.17 2061 Peppermint ret. Kovat's Boronia ret. Kovat's

time indices time indices

1,8-cineole 7.96 821 α-pinene 6.30 665

menthone 10.21 1032 β-pinene 7.02 733

menthol 10.71 1079 terpinolene 7.83 808 pulegone 11.49 1152 β-ionone 15.07 1488

isomenthol 12.22 1221 dodecyl acetate 15.40 1520 germacrene 14.26 1412 methyl jasmonate

isomer 16.41 1614

pipeterone 15.11 1492 heptadec-8-ene 17.37 1704 waxes & high M.W 23 - 34 2233-3266

Table 5. Kováts indices for major components of essential oils

Figures 7 to 10 record the GC ECD traces of injection of 20 mgkg-1 solutions of essential oils.

Figure 7. GC ECD of distilled parsley oil

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Figure 8. GC ECD of distilled peppermint oil

Figure 9. GC ECD of distilled fennel oil

13

Figure 10 GC ECD of solvent extracted boronia oil

Evident in figures 7 to 9 is that the component of the distilled essential oils which effect an ECD response, have Kovát's Indices less than 1500. The solvent extracted boronia oil shown in figure 10, however, has interfering components eluting thoughout the 35 minute run. Gas chromatography with detection by electron capture An acceptable limit of detection for the most basic screen would approach 1 mgkg-1. The loading capacity of standard non-polar columns such as HP1 or HP5MS, is in the vicinity of a 1 µL injection of a 20 mgmL-1 solution of an essential oil . 0.02µg of an active ingredient of a pesticide, in 20 mg of oil constitutes a 1 mgkg-1 solution. Without clean-up techniques, GC ECD would need to be able to detect 0.02 pg of analyte in a 1 µL injection. For each halogenated pesticide, then, a concentration of ~2 µg of analyte in 1mL of solution is equivalent to 100 mgkg-1 in a 20 mg sample of oil. 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. Table 6 lists the pesticides commonly used in the essential oil industry which contain at least one halogen in their molecular structure.

14

Halogenated, GC amenable pesticides tebuconazole procymidone glyphosate - derivatised propiconazole difenoconazole mecoprop - derivatised linuron ethofumesate MCPA - derivatised diuron dimethenamid Dicamba - derivatised simazine chlorpyrifos trichlopyr - derivatised oxyflurofen norflurazon clopyralid - derivatised chloroprofam haloxyfop esters fluroxypr - derivatised bromacil fluazifop esters terbacil

Table 6. Halogenated pesticides used within the essential oil industry Experimental: Acetone solutions (2 µgmL-1) of each halogenated, GC amenable pesticide were injected in a gas chromatograph with detection by ECD under the following conditions. Analytical parameters Instrumentation Hewlett Plackard 5890 gas chromatograph Hewlett Plackard Electron Capture Detector Processing Software - HP Chem Injection: 1 µL, split automatic injections Column: 30 m HP 5MS, 0.22 mm id, 0.25 µm film thickness Carrier gas: Instrument grade nitrogen Head Press: 10 psi Oven Temp: 60°C (1min.) -20°C/min-290°C (10 min.) Injection Temp: 260°C Detector: ECD 260°C Results ECD was found to be successful in the detection of terbacil, bromacil, haloxyfop ester, propiconazole, tebuconazole and difenaconzole. Figure 11 shows the chromatograms obtained by GC ECD.

15

Figure 11. GC ECD traces of 2 µgmL-1 solutions of halogenated pesticides

pesticide ret. Kovát's

time indices linuron 20.652 2013 ethofumesate 20.596 2007 simazine 18.099 1773 terbacil 19.147 1871 fluazifop ester 23.388 2270 fluroxypyr ester 24.951 2416 procymidone 22.146 2153 propiconazole 24.563 2380 tebuconazole 25.00 2421 difenaconazole 32.620 3136

Table 7. Kovát indices calculated for the pesticides shown in figure 11.

The effects of the matrix on the detection of halogenated pesticides were then assessed. Distilled oils (20 - 30 mg) were weighed into GC vials. Boronia extracts were warmed and mixed thoroughly to ensure an even distribution of all oil components. 20 - 30 mg of concrete or absolute were weighed into 2 mL GC vials. Vials were spiked with 10 µL of an acetone solution containing a 0.2 mgmL-1 mixture of each pesticide standard. Results

16

Figures 12 to 15 shows the GC ECD chromatograms for parsley, peppermint, fennel oils and boronia extracts.

Figure 12. GC ECD chromatogram of parsley oil fortified with 100 mgkg-1 mixed pesticide standard

Figure 13. GC ECD chromatogram of peppermint oil fortified with 100 mgkg-1 mixed pesticide

standard

17

Figure 14. GC ECD chromatogram of fennel oil fortified with 100 mgkg-1 mixed pesticide

standard

Figure 15. GC ECD chromatogram of boronia oil fortified with 100 mgkg-1 mixed pesticide

standard Method Validation for detection of pesticides by GC ECD in parsley, peppermint and fennel oils and boronia extracts.

18

Aim : From the results presented in figures 12 to15, a preliminary method validation experiment was designed to include simazine, terbacil, bromacil, haloxyfop and fluazifop esters, propiconazole, tebuconzole and difenaconazole. Experimental : 4 x 20 mg of each oil were spiked with standard solutions to produce a range of concentrations of 0.01 to 10 mgkg-1. Samples were analysed using the same parameters as listed on page 18. Repeat injections of each concentration of each oil were analysed to determine repeatability. Results : The detection limits, linearity as expressed by the r2 value of a linear regression, and the repeatability as expressed by the r.s.d.s are listed in table 8.

19

Pesticide ret. std. curve coefficients curve

fit detection limits

time x2 x r2 solvent fennel pep.mint parsley bor. r.s.d.

µgmL-1 %

simazine 18.02 1 - terbacil 18.92 2.29E-09 5.50E-05 1.000 0.02 1 0.5 1 5 12.2 bromacil 20.55 9.56E-10 8.39E-06 1.000 0.01 0.5 0.5 1 1 4.3 haloxyfop ester 22.05 6.34E-05 0.995 0.02 1 0.5 50 5 7.7 fluazifop ester 23.31 1 - - - - propiconazole 26.00 9.19E-09 1.85E-05 0.999 0.02 5 5 5 - 22.3 tebuconzole 21.92 5.12E-11 2.26E-06 0.997 0.01 0.5 0.5 1 5 2.2 difenaconazole 32.70 -2.92E-09 5.15E-05 0.998 0.02 1 1 1 1 13.7

Table 8. Detection limits and repeatability for the analysis of halogenated pesticides in essential

oils by GC ECD Discussion : Analysis of pesticide residues in essential oils by application of GC ECD are not specific enough to allow for an unequivocal identification of any contaminant. As a screen, ECD will only be effective in establishing that no gross contamination is present in oils in the absence of a co-eluting peak. In the situation where a peak is recorded with the correct elution characteristics, and which is enhanced when the sample is fortified with the target analyte, a second means of contaminant identification would be required, such as high resolution mass spectrometry. ECD, then, can only be used to rule out significant contamination and could not in itself be sufficient for a positive identification of pesticide contamination. 2.1.2.ii Assessment of GC FPD GC FPD was not found to be particularly suitable for the analysis of pesticide residues in essential oils without considerable clean-up procedures. Tests were undertaken with acephate, methedimos and monocrotophos. Poor chromatography, due to thermal degradation and poor interaction with the liquid phase of the GC columns, precluded this detection method in specific screens for the analysis of residues in essential oils. 2.1.2.iii 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, severe limitations imposed by the configuration of the system, precluded its application in the analysis of pesticide residues in essential oils without considerable clean-up techniques. Unlike other mass spectrometry systems, the effluent from the gas chromatograph elutes into the ionisation chamber without prior screening. In many mass spectrometers, only ions, and more often, only ions of a specific m/z ratio, enter directly into the ion trap. The introduction of a 1 µL split injection containing up to 20 µg of essential oil components has the potential to contaminate the benchtop GC MSMS system with 0.4 µg of eluant in a 1 : 50 split ratio. Excellent detection of pesticide residues in the first injection may be obtained, but within several injections the mass spectrometer would be seriously contaminated with essential oil components. Cleaning of the mass spectrometer requires several days work. Although several of the pesticides relevant to the essential oil industry were trialed in solvent solutions, with encouraging results, it was not considered practical to continue method development within the matrix of essential oils without comprehensive clean-up methodologies. 2.1.2.iv. Method Validation of the Analysis of Tebuconazole, Propiconazole, Simazine, Terbacil, Bromacil And Oxyflurofen in Boronia Extract by Gas Chromatography High Resolution Mass Spectrometry

20

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. Table 9 lists the weights of the standards dispensed. Analyte wt (mg)/25 mL mgmL-1 simazine 28.0 1.120 terbacil 24.3 0.972 oxyflurofen 26.3 1.052 bromacil 25.2 1.008 tebuconazole 27.2 1.088 propiconazole 27.0 1.080

Table 9. Weights of standards prepared for method validation 10µgmL-1 and 1 µgmL-1 solutions were prepared by diluting 250 µL and 25 µL of the 1mgmL-1 solutions respectively into 25mLs of acetone. Standard curves were prepared by weighing 22 x ~20 mg of boronia concrete into GC vials. Vial was fortified with the standard solutions. as listed in table 10, to allow for 6 replicate samples at 3 concentrations to establish repeatability. 5 µL of endosulfan solution was added to each vial as an internal standard. 200µL of hexane was added to each, the lids were placed firmly on top, without sealing, and the mixtures gently swirled to dissolve the concrete. The base of the vials was gently warmed by briefly placing on the base of oven where required.

21

Concentrations (mgmL-

1) no. 10µgmL-1 1µgmL-1 0.1µgmL-1 endosulfan

20 1 40 5 10 6 20 5 1 6 20 5 0.1 1 20 5 0.05 6 10 5 0.02 1 4 5 0 1 0 5 Table 10. Spiking protocol for the establishment of standard curves and repeatability

experiments Methanol (0.8 mLs) was added to each vial, which were then sealed and shaken vigorously to ensure complete mixing. After a settling period of ~ 5 minutes, the lids were removed and 4 drops of distilled water were added to each vial. The polar layer was filtered through cotton wool into 200 µL glass GC vial inserts for analysis. Recovery experiment A standard curve was also constructed in boronia solutions which had been subjected to the same partition clean-up step described above, but spiked after that process, using the same fortification levels as listed in table 10. Solvent only standard curve A standard curve was prepared by fortifying methanol / water solutions with standard pesticide solutions to concentrations equivalent to 0.05, 1, and 10 mgkg-1 in oils. 5µL endosulfan was added and the vials sealed for analysis. Analytical parameters Samples were analysed on a HP 5890 Gas Chromatograph directly coupled to a Kratos Concept ISQ Mass Spectrometer. The GC was equipped with a BPX5 fused silica capillary column (25m, 0.22 mm i.d., 0.25 µm film thickness). The carrier gas was helium. 1µl splitless injections of samples were analysed using a carrier gas flow program of 30 psimin-1 from 25 to 40 psi, held for 0.1 min, then at 30 psimin-1 to 25 psi, then at 1 psimin-1 to 35 psi. The GC injection temperature was 260°C and the oven temperature programmed from 60°C to 290°C at 20°Cmin-1. Ion monitored for terbacil was 161.0117 between 5:00 and 7:50 mins. Ion 194.9534 was monitored for internal standard, endosulfan, between 7:50 and 8:12 minutes. For oxyflurofen, ion 252.0398, was monitored between 8:12 to 9:00 minutes, tebuconazole, ion 250.0743, propiconazole, ion 259.0210, bromacil, ion 204.9613 and simazine, ion 201.0871. A dwell time of 300ms/ion and 50ppm voltage sweep were employed for all ions. Resolution of 10,000 (10% valley definition) and the ion m/z 242.9856 from perfluorokerosene was used as the lock mass for all analytes and the internal standard. Electron ionisation was undertaken at a source temperature of 210°C and an electron energy of 70eV, with an accelerating voltage of 5.3kV. Results : Table 11 records the detection limits and the relevant statistical data for the detection of the 6 analytes by GC high resolution MS.

analyte det. limit x coefficient r2 r.s.d. recovery mgkg-1 (at 1 mgkg-1) (at 1 mgkg-1)

tebuconazole 0.5 0.043 0.998 17 95 propiconazole 0.05 0.054 0.995 9 85 oxyflurofen 0.1 0.146 0.999 13 72 bromacil 0.05 0.046 1.000 12 82 terbacil 0.05 0.050 0.995 14 107 simazine 0.1 0.445 0.899 9 90

Table 11. Detection limits, recoveries and r.s.d.s for pesticides analysed by GC high resolution MS.

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2.1.2.v Method development using benchtop LC MSMS - Monocrotophos Monocrotophos is thermally unstable and analysis by GC often results in poor peak resolution and response. Liquid chromatography presents as an alternative method for the separation of this analyte from the components of essential oils. Aim : To develop an analytical technique for the detection of monocrotophos in essential oils. Experimental : Solutions of 56µgmL-1, 56ngmL-1 and 5.6ngmL-1 of monocrotophos in acetone were prepared. The 56µgmL-1 solution was taken up in the syringe fitted to the worm drive for continuous feeding into the MS. The flow rate was set at 3µLmin-1 and an MS method development established. First trialed was electrospray ionisation (ESI). As well as the parent ion of MW + H, another peak registered at MW + 23. A methanol adduct would produce a MW + 31 so this peak may have been the ion of the sodium salt. A tune file was established to optimise the collision energy (CE) and band width required to fragment the parent ion. However, the many adducts formed, possibly as a result of dimerisation, in addition to the target MW + H ion confused the signal. The APCI head was installed and the negative ions of the analyte investigated. For MS method development using the APCI a flow of 0.8mLmin-1 was established from the LC, to be used purely as a carrier. This was to ensure that the method developed did not need major readjustment when the LC separation of the analyte was established. A capillary was connected into the flow path via a T-junction immediately prior to the MS intake. The establishment of a MSMSMS method of ions 224, 194 and 127 was optimised. The parent ion, MWH+, which was fragmented at a collision energy of 11%, with a band width of 3 amu, was 224. The MS2 was ion 192.9 in a 5 amu window, which was fragmented at 12% CE. The MS of the two fragmentations are attached. Ghosting of the ions in the system persisted at levels of 3 x e3, due to the high level of analyte used in the development. The worm drive was disconnected and the analyte introduced by way of injection of 20µL of the 56 µLmL-1 solution. From these trials it was determined that detection could be achieved to around 5ngmL-1. In extractions of 10 mg of boronia extract in 1mL of solvent, this may be calculated as follows

(0.005µg/10 000µg) x 1000000 = 0.5ppm An LC run was established using isocratic 100% methanol. An injection of 20µL of the 56ngmL-1 solution of monocrotophos was injected. The analyte eluted on the solvent front at around 2 to 3 minutes. Literature reviews identified the suitablity of an aqueous, 60% methanol containing a 0.1M ammonium acetate buffer, mobile phase (ref 2). Monocrothophos is unstable in low molecular weight alcohols and so the mobile phase selected was acetonitrile / ammonium acetate buffer. A 0.5% ammonium acetate buffer in distilled water was prepared and filtered. Several ratios of solvents were trialed. It was difficult to retard the elution of monocrotophos , however a ratio of 40% acetonitrile 60% ammonium acetate buffer gave an acceptable retention time of 1.42 minutes. The tune file was re-optimised using this carrier makeup. A 20µL injection of the 50ngmL-1 acetone solution was undertaken. MS3 (collision induced fragmentation of parent ions and subsequent daughter ions) analysis was undertaken, monitoring for the fragmentation of the parent ion 224 and the daughter, daughter ions 193 and 127. Figure 16 shows the repeat injections of the standard over 20 minutes.

23

Figure 16. Precision trial for the analysis of monocrotophos by LC MS3

Despite the poor peak shape, the precision of the experiment expressed as residual standard deviation (R.S.D.) was 12.6%. Residual standard deviation (R.S.D.) = (standard deviation / mean) x 100. Peak shape and response values were improved by monitoring the MS2 fragmentation. Figure 17 shows the precision experiment for the 20µL injection of the 50ngmL-1 solution monitoring only ions 224 and 194. The RSD has been improved to 5.6%.

Figure 17. Precision trial for the analysis of monocrotophos by LC MS2 Analysis for monocrotophos in the matrix of the boronia extract was trialed. Sample Preparation 3 x ~20mg of boronia extract, produced on Bruny Island, where monocrotophos has not been previously applied, were weighed into 5mL vials and spiked with 0.224 and 0.0112 µg of monocrotophos using a 56 µgmL-1 acetone solution. Each of the concretes was dissolved in 200µL of hexane, with warming. 1mL of distilled water was added and the mixture was shaken vigorously. The 2 phases were allowed to settle and the lower, aqueous phase drawn carefully off with a pipette and transferred to a glass insert in an HPLC vial. 10µL of the 10ppm sample (0.224µg/mL) aqueous solution was injected. A small peak at retention time 2.61 was recorded. A MS3 repeat analysis was conducted to confirm the peak at 1.49 mins. as being monocrotophos. To improve the selectivity the next sample of 5ppm (0.011µgmL-1) was monitored for the parent ion and two fragments. The two fragments 98 and 194 were monitored. No peak was recorded for ion 98 with a retention time of 2.61 minutes. What was evident, however, was that the peak monitored has an improved shape with no splitting. The introduction of the analyte in the matrix of distilled water has served to focus the sample onto the column. Consequently, the 5 mgkg-1 boronia sample was partitioned into water prior to injection and again, the improvement in peak shape was evident.

24

Standards, (10 µL injections of a 112 ngmL-1 solution) dissolved directly in water, were analysed. The solution was further diluted to 1:100 and 1:500 with water. Precision tests were undertaken for these 2 solutions by repeat injections over 14 minutes. The repeat injections gave a R.S.D. of 4.5 and the linear regression describing the areas related to the range of concentrations had an 'r2' value of 0.93. Repeat injection of the boronia samples, extracted in water and analysed under identical conditions gave an R.S.D of 8.0%. Discussion : From these experiments it was evident that monocrotophos may be detected to very low levels in boronia extract. For the analysis of monocrotophos in the matrix of boronia leaves, it is anticipated that the background noise will be greatly increased , as many sugars and water soluble components will be co-extracted.

25

2.1.2.vi Method development using benchtop LC MSMS - general screen Preliminary work-up Solutions were diluted to 10µgmL-1 in acetone and introduced into the MS ionisation source by direct infusion using a continuous flow worm drive. ESI was first trialed. Table 12 shows the response and notes on the response of the analytes included in the trial.

ESI parent ion daughter ion collision isolation notes energy width (amu)

linuron NR no response oxyflurofen NR no response propiconazole 342 159 21 6 prometryn 242.1 200.1 20 5 tebuconazole 308.1 NR sethoxydim 328 282 17 5 simazine 202 124 18 4 endosulfan NR bromacil NR terbacil 215.2 (-ive) 159.1 15 6 haloxyfop ester 433.9 316 25 6 fluazifop ester 384 328 19 5 fluroxypyr ester 369 254.9 27 6

Table 12. Response of pesticides by MS with ionisation with ESI

For the chemicals trialed, 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. Overall, there was very little difference between the sensitivity for ESI and APCI. For analytes such as tebuconazole, the MW ion was detected but no daughter ions were produced at the collision energies trialed, such that MSMS could not be used to increase the specificity and lower background noise. 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, ESI appears to have limited application for a wide ranging screen. All the chemicals were trialed using APCI in the negative and positive mode. Tale 13 shows the general parameters established.

26

APCI parent ion daughter ion collision isolation notes

energy width (amu)

linuron 251 182 19 6 poor response oxyflurofen 363 315.9 21 5 poor response propiconazole 344 159.2 21 6 prometryn 242.1 200.1 tebuconazole 308 sethoxydim 328 282 simazine 202 124 endosulfan NR bromacil 261 (-ive) 205 19 5 terbacil 215 161.3 19 5 haloxyfop ester NR fluazifop ester NR fluroxypyr ester NR

Table 13. Detection of pesticide analytes by MS using ionisation with APCI

Initial results indicated that APCI has limited application for the detection of linuron, oxyflurofen and the esters of the acid moiety pesticides. Again, poor results were obtained for tebuconazole, with no daughter ions generated. However, excellent results were obtained for prometryn, sethoxydim, simazine, bromacil and terbacil. However, compared to ESI, APCI was slightly more sensitive for the commonly used pesticides, tebuconazole and propiconazole, and showed a response, though poor, to linuron and oxyflurofen. In addition, APCI is the preferred ionisation method for the following reasons, ♦ APCI uses less nitrogen gas compared to ESI, making overnight runs less costly: ♦ 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 blowing the cell which is pressure sensitive. Chromatography The chromatographic characteristics of the triazole pesticides, propiconazole and tebuconazole were poor. In addition, under the mobile phase conditions tested, bromacil, simazine and terbacil have similar retention times. In addition, terbacil has a higher response in the negative mode by APCI. The MS trap has the capability to switch from the positive mode, to the negative several times within a single run. However, the similar elution characteristics of simazine, which is ionised in the positive mode, precluded the monitoring of these three pesticides in the same screen as there is sufficient separation to allow for switching between the ionisation modes. Overall, however, the mobile phase has general application to most of the pesticides trialed. Honing of the rate of phase change, adjustments of the pH to allow for better resolution of the haloxyfop esters and triazine pesticides and trials of alternative mobile phases are all aspects which require further experimentation. Inclusion of acephate, carbaryl, cyanazine, dicamba, dimethoate, difenoconazole, ethofumesate, glyphosate, pirimicarb, pendimethalin, procymidone, mcpa, monocrophohos and propazine. Standards of all the target analytes were prepared to concentrations of 10µgmL-1 in acetone and each were introduced into the MS via the continuous flow worm drive with the mobile phase of 50/50 0.1M ammonium acetate buffer/acetonitrile flowing from the LC at 0.8mLmin-1 to provide a background. The ionisation potential, M + H ion, daughter ion, collision energy (CE) and band width were determined for each compound. Target compounds which had good ionisation potential in positive APCI mode were then analysed by LC MS using the original standard mobile phase profile.

27

Analytes that ionised in the negative APCI mode were incorporated into a separate screen which included bromacil, terbacil, and the esters of the fluazifop and haloxyfop acids. Table 14 lists the parameters established for each analyte.

analyte M.W. parent daughter band C.E. notes ion ion width

acephate 183 184 143 3 13 buffer adduct=loss signal carbaryl 201 202 145 4 20 good ionisation but lost in LC

cyanazine 240 242 214,216 5 18 pot. Int. std. dicamba 220 220 175,177 8 17 daughter ion weak, try -ive dimethoate 229 230 199 4 20 good ionisation but lost in LC difenaconazole 405 408 337 6 22 good signal - double peak

ethofumesate 286 287 259,241,207 4 20 3 daughters similar inten.=poor glyphosate 169 no ion. possible ESI or -ive APCI pirimicarb 238 239 182,195 3 22 strong signal pendimethalin 281 282 212 4 20 strong signal but lost in LC procymidone 283 284(-) 161,163 6 23 weak signal-alternative LC MCPA 200 200(-) 141,143 5 18 poor signal mecoprop 214 141,143 4 18 +ive & -ive monocrotophos 224 193,167,98 5 22 strong signal & LC peak propazine 229 231 1898 5 18

Table 14. Detection of pesticide analytes using positive APCI

The results listed above formed the basis for the inclusion of monocrotophos, pirimicarb, propazine and difenaconazole into the standard screen already established. Acephate, carbaryl, dimethoate, ethofumesate and pendimethalin all require further work for enhanced ionisation and / or improved elution profiles. Negative ionisation mode for APCI gives improved characteristics for dicamba, procymidone, MCPA and mecoprop, but this ionisation mode is compromised by the acetate buffer. More work into an improved mobile phase profile is required for detection by APCI in the negative mode. Method validation The thirteen pesticides included in this general screen were monocrotophos, simazine, cyanazine, pirimicarb, propazine, sethoxydim, prometryb, tebuconazole, propiconazole, , difenaconazole and the esters of fluroxypyr, fluazifop and haloxyfop.. Bromacil and terbacil were not included as both require negative ionisation and elute within the same time window as simazine, which requires positive ionisation. Cycling the MS between the two modes was not practical. The method validation was conducted for three oils, peppermint, parsley and fennel. Aim : To validate the methods established for a general screen for pesticides by LC MSMS using ionisation by APCI in the positive mode. Experimental: Four of each of 200µL (~200mg) of each of peppermint, fennel and parsley oil were dispensed with an Eppendorf into GC vials and 800µL of analG acetone was added. Stock solutions (1mgmL-1) of the 10 pesticides were prepared in volumetric flasks in acetone which were then diluted to 100, 10 and 1µgmL-1 standard solutions. Oils were fortified with solutions of the mixture of 13 standards pesticides to the equivalent of 0.1, 0.5, 1.0 and 10 mgkg-1. All fortified oils were spiked with 20µL of 100µgmL-1 solutions of cyanazine as an internal standard. The oils were analysed by LCQ using the following conditions. Analytical parameters LCQ : A Waters 2690 separation module (Alliance) system equipped with a NOVAPAK C18, 3.9 x 150 mm column was used to establish a mobile phase with gradient of 50:50 acetonitrile / 0.1M

28

ammonium acetate buffer ramped to 90 : 10 over 15 minutes. A Finigan MAT LCQ was used to monitor the ions listed in table 15 within the relevant time windows.

analyte ions retention monitored time

monocrotophos 192.5-193.5 1.52 simazine 123.5-124.5 2.52 cyanazine 212.0-218.0 2.45 pirimicarb 181.5-182.5 3.06 propazine 187.5-188.5 4.02 sethoxydim 281.5-282.5 4.08 prometryn 199.0-200.8 5.24 tebuconazole 307.5-308.5 5.32 propiconazole 158.5-159.5 6.58 difenaconazole 336.5-337.5 7.78

Table 15. Ions monitored by LC MSMS

29

Results : Figure 18 shows the LC MSMS trace of a typical analytical run. RT: 0.01 - 5.17

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

Time (min)

0

50

100

0

50

100

0

50

100

0

50

100

0

50

100monocrotophos

RT: 1.49AA: 4137884

simazine

RT: 2.56MA: 2522163

RT: 2.45MA: 1986999

pirimicarbRT: 3.13AA: 9862826

propazineRT: 4.11MA: 23349203

NL: 7.47E5

m/z= 192.5-193.5 F: + c SRM ms2 224.00 [ 165.50 - 194.50]

NL: 4.14E5

m/z= 123.5-124.5 F: + c SRM ms2 202.00 [ 122.50 - 125.50]

NL: 3.52E5m/z= 212.0-218.0 F: + c SRM ms2 242.00 [ 212.50 - 217.50]

NL: 1.42E6m/z= 181.5-182.5 F: + c SRM ms2 239.00 [ 181.00 - 196.00]

NL: 2.90E6m/z= 187.5-188.5 F: + c SRM ms2 231.00 [ 186.50 - 189.50]

RT: 1.74 - 10.27

2 3 4 5 6 7 8 9 10

Time (min)

0

50

100

0

50

100

0

50

100

0

50

100

0

50

100

sethoxydim

RT: 4.09MA: 10313193

prometrynRT: 5.18MA: 30994994

folicurRT: 5.54MA: 8354032

tiltRT: 6.68MA: 2375398

difenoconozoleRT: 7.62MA: 32135613

NL: 1.13E6

m/z= 281.5-282.5 F: + c SRM ms2 328.00 [ 280.50 - 283.50]

NL: 4.19E6m/z= 199.0-200.8 F: + c SRM ms2 242.00 [ 198.50 - 201.50]

NL: 8.64E5m/z= 307.5-308.5 F: + c SIM ms [ 306.50 - 309.50]

NL: 2.03E5m/z= 158.5-159.5 F: + c SRM ms2 344.00 [ 157.50 - 160.50]

NL: 1.96E6m/z= 336.5-337.5 F: + c SRM ms2 408.00 [ 335.00 - 339.00]

Figure 18. Analysis of pesticides by LC MSMS with ionisation by APCI in the positive mode.

The tables below present the detection limit achieved, the coefficients for the standard curves, the 'r2' value and the relative standard deviation at the 1 mgkg-1 detection level in fennel, parsley and peppermint respectively.

30

Fennel analyte det. limit x coefficient r2 %rsd

mgkg-1 (at 1 mgkg-1) monocrotophos 0.1 0.932 1.000 3 simazine 0.1 0.585 0.999 6 pirimicarb 0.1 2.12 1.000 7 propazine 0.1 4.858 1.000 4 sethoxydim 0.1 2.493 1.000 4 prometryn 0.1 6.869 1.000 6 tebuconazole 0.5 1.926 0.999 7 propiconazole 0.5 0.509 0.999 5 difenoconazole 0.1 7.248 1.000 3

Table 16. Method validation for pesticides in fennel by LC MSMS using ionisation with APCI in

the positive mode

Parsley analyte det. limit x coefficient r2 %rsd


Table 17. Method validation for pesticides in parsley by LC MSMS using ionisation with APCI in the positive mode

Peppermint analyte det. limit x coefficient r2 %rsd


Table 18. Method validation for pesticides in peppermint by LC MSMS using ionisation with

APCI in the positive mode LCQ provides an effective screen for the screening of the ten analytes, with detection limits to 0.01 mgkg-1 for analytes excluding propiconazole and tebuconazole which can only be detected to levels of 0.5 mgkg-1. The type of oil analysed had minimal effect on the response function as expressed by slope of the standard curve. 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. These levels are in the order of 0.3 mgkg-1. Over all method development experiments, we have consistently found contamination of most parsley oils with this pesticide. Location of an oil free of prometryn contamination will allow for a repeat of this validation experiment.

31

In table 19 the r.s.d.s or percentage coefficient of variation (%c.v.) for the method validations are recorded. Predictably, the r.s.d.s increases as the detection limit is approached.

10 mgkg-1 1 mgkg-1 0.5 mgkg-1 0.1mgkg-1 Fennel monocrotophos 5 3 11 48 simazine 8 6 15 28 pirimicarb 7 7 9 4 propazine 7 4 4 14 sethoxydim 6 4 13 17 prometryn 7 6 5 19 folicur 4 7 12 tilt 7 5 13 difenoconozole 6 3 8 15 Parsley monocrotophos 6 9 9 12 simazine 4 4 8 15 pirimicarb 4 8 2 21 propazine 4 6 3 14 sethoxydim 6 9 6 20 prometryn 2 6 5 7 folicur 1 13 19 tilt 4 19 21 difenoconozole 4 6 5 11 Peppermint monocrotophos 4 7 9 14 simazine 4 11 20 38 pirimicarb 4 4 5 14 propazine 2 3 34 8 sethoxydim 6 5 10 22 prometryn 4 6 5 8 folicur 6 16 15 tilt 5 11 26 difenoconozole 3 4 5 7

Table 19. %R.S.D. for analysis of pesticides in essential oils using LC MSMS with ionisation by

APCI in the positive mode. 2.1.2.vii. Method development using benchtop LC MSMS - acidic moiety pesticides The pesticides which have an carboxylic acid moiety such as fluazifop, haloxyfop and fluroxypyr, present several complications in any analytical method development. The commercial preparations usually have the carboxylic acid in the ester form, which is hydrolysed to the active acidic form on contact with soil and vegetation. In addition, the esters may be present in several forms, such as the etotyl or butyl ester. The potential to use the acidic moiety of these pesticides in an aqueous based extraction of essential oils would not be effective in extracting any residues which are still in the ester form. As a general purpose screen, it was considered possible to effect an extraction with a highly caustic aqueous solution to, not only extract the acids from the matrix of the essential oil, but also to hydrolyse any remaining esters present, so as to fully assess the contamination of oil samples.

32

Hydrolysis of Esters and Extraction of Pesticides with Acidic Moieties Aim : To hydrolyse esters of pesticide residues and extract acids from fortified oils using a pH based aqueous extraction / back extraction to remove target analytes from the matrix. Experimental : trial 1. Acetone solutions of haloxyfop, fluazifop and fluroxypyr esters, at concentrations of 1 mgmL-1 were further diluted with acetone and varying concentrations of 0.1M ammonium acetate were added. The solutions were left to stand for 2 weeks. However, very little free acids was formed under these conditions. However, the small amount of acids that were produced were used to establish the retention times and likely response behaviour of the acids under the standard LC conditions. The hydrolysed standard solutions were introduced into the LC using the following protocol and parameters previously established. Analytical parameters LCQ : A Waters 2690 separation module (Alliance) system equipped with a NOVAPAK C18, 3.9 x 150 mm column was used to establish a mobile phase with gradient of 50:50 acetonitrile / 0.1M ammonium acetate buffer ramped to 90 : 10 over 15 minutes. A Finigan MAT LCQ was used to monitor the ions listed in table 12 within the relevant time windows. Results : The retention times using the above LC protocol gave promising results with the parent esters eluting much later than those recorded for the other analytes to be targeted by positive APCI. The sensitivity for haloxyfop and fluazifop esters were adequate. Sensitivity was poor for fluoxypyr. Response of the acid hydrolysis products were excellent in negative ion ESI MSMS. trial 2 The extraction of oil samples fortified with fluazifop, haloxyfop and fluroxypyr acids with a NaOH solution was trialed. Equal volumes 0.1M NaOH and fennel oil showed that variations within the batches of fennel oil extracted, effected differing degrees of emulsification and a degree of saponification. Salt was added to the emulsions to effect a clean partition with little success. However, the layers were sufficiently delineated to allow for a phase separation and when the aqueous layer was acidified with HCl, back extracted with dichloromethane and evaporated to dryness, an oily residue remained. The problems encountered through this process, however, precluded its practical application. To optimise the ratio of caustic to oil , varying ratios of 0.1M NaOH were trialed. Experimental : Table 20 records the ratios of fennel oil and caustic trialed. Samples were placed in a 50 mL test tube, vortexed then centrifuged at 200 r.p.m. for 15 minutes.

volume of NaOH observations oil (mLs) 0.1M

2 10 white creamy suspension in oil layer, aqueous discoloured 3 8 white creamy suspension in oil layer, aqueous discoloured 4 6 white creamy suspension in oil layer, aqueous discoloured 5 10 white creamy suspension in oil layer, aqueous discoloured

NaOH 0.01M

2 10 good separation 3 8 good separation 4 6 aqueous layer cloudy 5 10 excellent separation

Table 20. Separation characteristics of fennel oil and caustic.

33

Trial 3. Each of 5mL samples of peppermint oil were fortified to give 0.01, 0.1, 1.0 and 10.0 mgkg-1 of haloxyfop acid and ester. The samples were prepared in 50 mL screw cap glass test tubes and 5mL of 0.01M NaOH was added. The tubes were rolled for 3 hours and the aqueous layers were separated and washed with 2.5 mL of hexane. The aqueous layers were acidified with 1 mL 0.1M HCl and extracted with 2 x 2.5 mL of DCM which was then evaporated to dryness. The samples were taken up in 100 µL of acetone for an analysis by LCQ. Analytical parameters LCQ : A Waters 2690 separation module (Alliance) system equipped with a NOVAPAK C18, 3.9 x 150 mm column was used to establish a mobile phase with gradient of 50:50 acetonitrile / 0.1M ammonium acetate buffer ramped to 90 : 10 over 15 minutes. A Finigan MAT LCQ was used to monitor the ions listed in table 21within the relevant time windows. Results Haloxyfop acid was detected to the 1 mgkg-1 level with some trace levels detected in the 0.1 mgkg-1 sample. No hydrolysis of the parent ester to the acid was apparent. trial 4. 1mL of 0.01M NaOH was fortified to 10 µgmL-1 and 50 µgmL-1 with DCM solutions. Two drops of ethanol were added to ensure mixing of standards and aqueous layer. The samples were vortexed for 2 x 30 second bursts then allowed to stand overnight. The extracts were acidified and extracted with 1mL of DCM then evaporated to dryness under a stream of nitrogen. Samples were analysed by direct injection into the MS . Both the ester and the acid were present for all 3 chemicals. The method was not quantitative as the different ionisation potential of the esters and the acids were not known. However, it was estimated that less than 30% of the esters were hydrolysed to the acidic form. Table 21 records the ions monitored and the relevant retention times. analyte MW MS MSMS ret. time band width

& C.E. fluazifop-butyl 383 384 (+) 328 11.1 5 amu 19% fluazifop acid 327 326 (-) 254 3 amu 17% haloxyfop ethoxyethyl 433/435 434/436 (+) 316/318 10 6 amu 25% haloxyfop acid 361/363 360/362 (-) 288/290

361 288/290 4 amu 26% fluroxypyr methyl heptyl 366/368/370 367/369/371 (+) 255/257 12.7 6 amu 18% fluroxypyr acid 255/256/258 253/255/257 (-) 233/235 6 amu 17%

Table 21. MS parameters for the detection of pesticides with acidic moieties

Discussion ESI was tested. Preliminary results indicate that ESI is unsuitable for haloxyfop and fluroxypyr ester. Fluazifop possessed good ionisation characteristics using ESI with responses approximately thirty times that recorded for haxloyfop. The significant difference presented the suspicion that the haloxyfop standard had deteriorated. Solid probe high resolution mass spectrometry was used to confirm that both analytes were at similar concentrations. Subsequent analysis, however, revealed that the haloxyfop ester standard contained both the ethoxy ethyl and methyl esters. Mobile Phase Development Poor chromatography and response necessitated improved mobile phase and the effect of pH on elution characteristics was considered the most critical parameter. The inclusion of acetic acid resulted in improved peak resolution.

34

Internal standards The herbicides 2,4D and 2,4,5T were proposed as suitable internal standards and both were introduced in the MS using direct infusion. Table 22 lists the MS characteristics of both. analyte molecular M.W. parent ion daughter isolation collision comments

formula (-ive) ion width energy

2,4D C8H6Cl2O3 221 219 161 6 20% parent ion same as dicamba

but stronger daughter ion

2,4,5T C8H5Cl3O3 255 257, 255, 253 199, 197, 195 8 20% parent and daughter clusters

Table 22. Ionisation of 2,4-D & 2,4,5-T using negative APCI Method validation for the analysis of pesticides with acidic moieties by LC MSMS Aim : To validate the methods for the detection of dicamba, fluroxypyr, MCPA, mecoprop and haloxyfop using LC MSMS Experimental : To obtain a sufficient quantity of oil, a range of commercially produced oils were combined to produce a blended oil with uniform characteristics. 20 x 5 mL of each of peppermint, fennel and parsley oils were dispensed into 50 mL test tubes. 1 mL of 0.01M NaHCO3 (adjusted to pH 10 with NaOH) was added and the mixtures were vortexed for 4 x 15 secs bursts. The emulsions were transferred to 13 x 100 mm borosiliate glass tube and centrifuged at 2500 rpm for 15 mins. The aqueous layer was transferred to a 10 x 75 mm borosilicate glass tube using a pasteur pipette, to facilitate separation of the partitions, 1mL of hexane was added and the mixture was vortexed for 2 x 15 sec. bursts to remove residual oil contaminants. The mixture was centrifuged at 2000 rpm for 5 mins. and 250µL of the aqueous layer was quantitatively transferred to a GC vial insert. 5 µl of a 10 µgmL-1 solution of 2,4,5-T was added as an internal standard. LCQ : A Waters 2690 separation module (Alliance) system equipped with a NOVAPAK C18, 3.9 x 150 mm column was used to establish a mobile phase with gradient of 50:50 acetonitrile / 0.1M ammonium acetate buffer ramped to 90 : 10 over 15 minutes. A Finigan MAT LCQ was used to monitor the ions listed in tables 21 and 22 within the relevant time windows. results : Several points of note in the practicality of the extraction include ♦ difficulties in recovering 250 µl of the 1mL of buffer used to extract fennel oil - excluding hexane

and residual oil from the sub-sample was problematic ♦ the varying nature of the density of parsley oil presented as an emulsion in this experiment - an

increase in rates of centrifugation to 2500 rpm for 40 minutes improved phase separation minimally

♦ the saturation of the aqueous parsley extract with NaCl effected phase separation, however, this rendered the samples incompatible with LC MS and all parsley samples were excluded.

Figure 19 shows a typical LC MSMS trace for the analysis of the pesticides. Table 23 records the detection limits and repeatabilities for the methods developed for fennel and peppermint distilled oils, whilst table 24 lists the r.s.d.s for each analyte over a range of concentrations.

35

RT: 0.01 - 11.81

1 2 3 4 5 6 7 8 9 10 11

Time (min)

0

1000

100

0

100

0

100

0

1000

100

0

100 dicamba2.16

2.53

fluroxypyr2.50

2.91

2,4D4.13

4.44

MCPA4.51

2,4,5-T5.86

mecoprop6.02

haloxyfop8.89

NL: 1.56E6

m/z= 218.3-219.3+220.3-221.3 F: - c SIM ms [ 218.00 - 284.00]

NL: 1.73E6m/z= 232.5-233.5+234.5-235.5 F: - c SRM ms2 254.00 [ 232.00 - 236.00]

NL: 2.02E6

m/z= 160.7-161.7+162.7-163.7 F: - c SRM ms2 221.00 [ 160.00 - 164.00]

NL: 2.57E6m/z= 140.7-141.7+142.7-143.7 F: - c SRM ms2 200.00 [ 140.00 - 144.00]

NL: 5.76E5

m/z= 194.7-195.7+196.7-197.7+198.7-199.7 F: - c SRM ms2 255.00 [ 193.00 - 201.00]

NL: 1.85E6m/z= 140.7-141.7+142.7-143.7 F: - c SRM ms2 214.00 [ 140.00 - 144.00]

NL: 3.15E6m/z= 287.7-288.7+288.7-289.7 F: - c SRM ms2 361.00 [ 287.00 - 291.00]

Figure 19. LC MSMS chromatogram for the analysis of dicamba, fluroxypyr, MCPA, mecoprop

and haloxyfop

analyte det. limit x coefficient r2 %rsd Fennel mgkg-1 (at 1 mgkg-1)

dicamba 0.1 4.25E-06 0.999 2 fluroxypyr 0.1 3.20E-06 0.995 7 MCPA 0.01 2.28E-06 0.992 4 mecoprop 0.01 3.99E-06 0.995 6 haloxyfop 0.01 3.24E-06 0.997 6 Peppermint

dicamba 0.1 3.86E-06 0.999 13 fluroxypyr 0.01 3.28E-06 0.996 15 MCPA 0.01 2.78E-06 0.998 18 mecoprop 0.01 6.89E-06 0.999 20 haloxyfop 0.01 1.63E-06 0.999 14

Table 23. Detection limits for the analysis of dicamba, fluroxypyr, MCPA, mecoprop and

haloxyfop in peppermint and fennel distilled oils

10 mgkg-1 1 mgkg-1 0.5 mgkg-1 0.1mgkg-1 Fennel

dicamba 9 2 16 fluroxypyr 8 7 8 MCPA 9 4 6 22 mecoprop 11 6 8 42 haloxyfop 12 6 5 50 Peppermint

dicamba 5 13 42 fluroxypyr 2 15 10 9 MCPA 5 18 6 31 mecoprop 4 20 18 7 haloxyfop 8 14 13 51

Table 24. R.S.D.s obtained in the analysis of dicamba, fluroxypyr, MCPA, mecoprop and

haloxyfop in peppermint and fennel distilled oils

36

2.1.2.viii. Method development using benchtop LC MSMS - quaternary ammonium salts Paraquat and diquat are ammonium quaternary salts which are very soluble in water. The analysis of these two compounds using LC MSMS was investigated. Aim: To develop extraction protocols and LC MSMS methods for the detection of paraquat and diquat in essential oils. Experimental : Acetone solutions of paraquat and diquat (1mgmL-1) were diluted to 10 µgmL-1 and introduced into the MS using a continuous flow worm driven syringe. Both ESI and APCI were trialed to determine optimum source and tune file parameters. Table 24 presents the parameters optimised for ESI.

analyte M.W. MS MSMS isolation width & C.E. paraquat 186 185.1 171.3 3 amu 17% diquat 184 183.1 157.2 1.5 amu 17.1%

Table 24. Fragmentation ions, isolation width and collision energies for paraquat and diquat

LC Method Development 1 mL of fennel oil, spiked, were extracted with 1 mL of distilled water in a 5 mL screw cap scintillation vial. The aqueous layer was washed with 1mL of hexane and 1mL of DCM and the extract was introduced into the MS by direct infusion at 0.8 mLmin-1 into the eluent from the LC column of ammonium acetate buffer/acetonitrile. ESI - Response for both paraquat and diquat using ESI was excellent, however, sensitivity was compromised by shorting across the ESI source effected by the ammonium acetate in the buffer. Acetonitrile was replaced with methanol. The TIC was recorded for an extracted oil sample. Since very little background noise was recorded, the introduction of samples directly into the MS presented the most efficient and rapid way for analysis. This avoided the need for specialised columns or the inclusion modifiers in the mobile phase to instigate ion exchange, normally required for the LC of paraquat and diquat. It was determined that direct loop injections of 20µL of extracted oil samples could be introduced into a 70/30 methanol/0.1M ammonium acetate buffer isocratic LC eluant, post LC column. A series of oils spiked with a range of paraquat / diquat standards were extracted in 1 mL of water to establish a standard curve. Concentrations ranged from 0.05 to 1 mgkg-1. However, response levels at low concentrations showed continuous decay over successive injections such that linearity and sensitivity were compromised. Several explanations for the depletion of target analyte, including adsorption onto the teflon plungers and dilution of the injection volume with hexane. The hexane wash was removed from the extraction protocol and the scintillation vials, which had plastic screw caps, were replaced with test tubes. A TIC was conducted on oil samples extracted without the hexane wash. Very few oil components remained in the aqueous extract. At concentration levels of 0.05 mgkg-1 diquat was easily detected, however, despite the measures instigated, the linearity of the standard curve, though somewhat improved, was still unacceptable at low levels of analyte. Paraquat was not detected at low concentration levels and reproducibility and linearity of the standard curve could not be established. Technical information on paraquat and diquat report minimal degradation of both chemicals in aqueous solutions with paraquat having a half life of over 23 weeks at low concentrations in water. However, fresh standards were prepared to exclude the possibility of deterioration of the standard as the cause for lack of reproducibility.

37

The figure below shows the decline in response for paraquat and diquat over 40 minutes, with the area recorded for paraquat decreasing by 68%.

RT : 0.01 - 47.76

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46

T im e (m in)

0

10

20

30

40

50

60

70

80

90

100

RT : 9.42AA: 839050

RT : 38.78AA: 713276RT: 5.66

AA: 795152RT : 33.60AA: 673516

RT : 24.56AA: 740456

RT : 41.49AA: 622433

RT : 11.85AA: 712836

RT: 18.18AA: 668402

RT : 14.74AA: 734053

RT: 21.48AA: 612331

RT: 30.89AA: 557462

RT : 36.40AA: 585527

RT: 27.04AA: 618054

RT: 44.98AA: 611718

RT: 2.35AA: 583405

RT: 42.73AA: 133825

NL:1.29E5T IC F : + p SRM m s2 183.00 [ 156.00 - 159.00]

RT : 0.01 - 47.76

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46

T im e (m in)

0

10

20

30

40

50

60

70

80

90

100

RT: 4.17M A: 1863466

RT: 7.47M A: 1441081

RT : 16.69AA: 1108409

RT : 29.35AA: 810076

RT : 13.07AA: 1329360

RT: 23.07AA: 904116

RT: 10.68M A: 1207703 RT: 26.10

AA: 868281RT : 19.86AA: 965801 RT : 34.77

AA: 614841RT: 40.27AA: 606398RT: 32.15

AA: 758317RT : 42.80AA: 509166 RT: 46.33

AA: 547145

NL: 1.22E5

m /z= 156.0-173.0 F : + p SRM m s2 186.00 [ 170.00 - 173.00]

Figure 20. Stability of paraquat and diquat over time A new diquat standard gave a three fold increase in response compared to the existing standard. However, little improvement was observed for paraquat and it was proposed that paraquat, and to some degree, diquat, adsorbs onto glass and other surfaces. Literature searches revealed other research groups had encountered the phenomenom of adsorbtion of quaternary ammonium salts onto glassware. Kaniansky et al., (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. These were eliminated by spiking samples with diethylenetriamine (DETA). DETA was found to preferentially accumulate on the surfaces of sample containers, competitively binding on the adsorption sites. Experimental All glassware to be used in the paraquat diquat analysis were washed in a 5% solution of 0.1M DETA and then left to drain and partially dry. 3 x 5 mL of fennel oil in 10 mL glass tubes were spiked with 0.05,0.5 and 2.5 µg of paraquat to give concentrations in the oil equivalent to ~0.01, 0.1 and 0.5 mgkg-1. A further 3 x 5 mL of fennel oil were spiked with equivalent concentrations of diquat. The oils were extracted with 2mL of distilled water and vortexed for 3 x 20 second bursts. The tubes were centrifuged at 2500 rpm for 15 minutes and the aqueous layer was pippetted into smaller 10 x 74 mm kimbal tubes. The solution was washed with 0.75 mL of DCM. The experiment was repeated with glassware not previously washed with DETA solution. Paraquat and diquat solutions were used to fortify 2 mL water samples at levels equivalent to water extracted oil samples assuming 100% recovery to estimate the recoveries of the oil extraction experiments. 5 µL, 50µL and 250 µL of a 10, 100 and 500 µgmL-1 solutions were used to spike each of 2 mL of distilled water.

diquat

paraquat

38

LCQ parameters A Waters 2690 separation module (Alliance) system equipped with a NOVAPAK C18, 3.9 x 150 mm column was used to establish a isocratic mobile phase of 70:30 methanol / water. A Finigan MAT LCQ was used to monitor the parent ion, 186 for paraquat with the daughter ion in range 170 to 173 and the parent ion, 186 for diquat with the daughter ion range of 156 to 159. Manual injections to fill 25 µL loop were interposed into the LC mobile phase after the column, directly into the MSMS. Results Figure 21 presents a representative chromatogram of the repeat injection of extracts of paraquat fortified in fennel oil at the level of 0.01 mgkg-1 prepared in glassware pre-treated with DETA and with no pre-treatment.

RT : 0.01 - 2.98

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8

T im e (m in)

0

10

20

30

40

50

60

70

80

90

100

RT: 1.76AA: 27172

RT : 0.34M A: 16945 RT: 1.30

M A: 27974RT : 2.40AA: 17233

RT: 0.80M A: 2711

RT : 2.63M A: 1925

NL:7.51E3T IC F : + p SRM m s2 183.00 [ 156.00 - 159.00]

RT : 0.01 - 2.98

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8

T im e (m in)

0

10

20

30

40

50

60

70

80

90

100

RT: 1.83AA: 6641

RT : 0.22M A: 5102

NL: 2.42E3

m /z= 156.0-173.0 F : + p SRM m s2 186.00 [ 170.00 - 173.00]

Figure 21. Paraquat 4Jul 10ppb fresh spl with DETA & paraquat 4Jul 10ppb fennel fresh spl no

DETA The table below presents the areas for repeat injections of the fennel oil extracts.

paraquat diquat parent ion 186 183 daughter range 170-173 156-159 mgkg-1 + DETA - DETA + DETA - DETA

0.01 23234 5860 15028 12206 17824 4145 32109 6302 35127 9840 32640 20810

mean 25253 7671 std. Dev. 7986 3273

R.S.D. 32 43 + deta / -deta 3.3 0.1 186155 149364 144892 54473

206904 112825 126952 44854 273962 106703 146982 43708 236205 107986 99221 48236 183563 97298 102230 70337 231997 88943

39

173454 mean 213177 110520 124055 52322 std. Dev. 36064 20859 22701 10907 R.S.D. 17 19 18 21 + deta / -deta 1.9 2.4

0.5 374669 763011 505016 490703 474457 438389 420107 407946 385911

mean 432032 525012 std. Dev. 56317 162305 R.S.D. 13 31 + deta / -deta 0.8

linear regression r2 0.999 0.998 0.986 0.984

Table 25. Effect of the inclusion of DETA on the repeatability of analysis of paraquat and

diquat. The inclusion of a DETA solution pre-treatment of all glassware has effected ♦ a 3.3 fold increase in paraquat response at 0.01 mgkg-1 fortification level; ♦ a 2.4 fold increase in diquat response at the 0.1 mgkg-1 fortification level; ♦ an increase in the repeatability of the analysis for paraquat as expressed by the relative standard

deviation (R.S.D.), lowered from 43 to 32%; ♦ an increase in the linearity of the standard curve at low concentrations as expressed by the

improved 'r square' values for both paraquat and diquat. In addition to this experiment 100ppb samples of paraquat and diquat in fennel oil, prepared in glassware pre-treated and not pre-treated with DETA solutions and which had been stored for 2 weeks, were compared to freshly prepared standard extracts. Results are presented in table 26.

0.1 mgkg-1 paraquat diquat

parent ion 186 183 daughter range 170-173 156-159

+ DETA - DETA + DETA - DETA 2 weeks 8866 bdl 16903 bdl

< 12 hours 213177 110520 124055 52322

*bdl - below detection limit Table 26. Effect of DETA on the stability of paraquat and diquat standards

Results in table 26 confirm that despite the improved response and reproducibility afforded by the use of glassware pre-treated with DETA, depletion of both paraquat and diquat in solutions necessitates the preparation of standards and samples on the day of analysis. Measures undertaken to eliminate the adsorption problem improved, somewhat, for the analysis of diquat at low levels but still the responses for paraquat analysis were inconsistent. Blank injections of DETA solutions resulted in quite high responses for both paraquat and diquat as the DETA displaced adsorbed analyte from previous runs from the walls of the loop and transfer tubes. The mobile phase was changed to 100% 100 µM DETA so that all active sites within the system remained primed. The ion for DETA (ion 104) was monitored and signal intensity increased over time from 1.2 x e4 to 2 x e6, possibly as a result of the DETA slowly saturating the active sites. The change in mobile phase initially improved the response for both analytes, but the peak for paraquat showed significant tailing. The mobile phase was changed to 50:50 DETA / methanol, which

40

immediately improved the elution profile for paraquat. Ghosting problems persisted and the desorption of analytes from the metal interfaces within the switching valve were proposed as a possible source of the analytical error. This could be bypassed if the LC automated sampler could be included in the system. With the new mobile phase and pre-conditioned systems, both diquat and paraquat could be detected using the automated LC injection system with short flow paths, bypassing LC columns. However, the peak for paraquat tailed significantly, a problem which worsened over time. The inclusion of acetic acid into the mobile phase did not alleviate this problem and some stripping of the DETA priming was evident. Finally, after the return to the 50 : 50 DETA : methanol mobile phase, the metallic injection loop was replaced with teflon tubing. In conjunction with manual injections this proved to be the most effective and reproducible system for the analyse of paraquat and diquat. Method Validation Aim : To determine the repeatability of the detection of paraquat and diquat using LC MSMS Experimental : 16 x 5 mL of fennel oil were weighed into 10 mL glass tubes previously washed in a 5% solution of and dried. 12.5 µL of a 10 µgmL-1 paraquat / diquat solution, 10 and 50 µL of a 50 µgmL-1 and 50 µL of 100µgmL-1 were used to spike each of 5 mL of fennel oils. The spiking was repeated four times at each concentration to determine repeatability of the method. The oils were extracted with 2mL of 0.1M DETA solution and vortexed for 3 x 20 second bursts. The tubes were centrifuged at 2500 rpm for 15 minutes and the aqueous layer was pipetted into smaller 10 x 74 mm kimbal tubes. The solution was washed with 0.75 mL of DCM. Analytical parameters A Waters 2690 separation module (Alliance) system equipped with a NOVAPAK C18, 3.9 x 150 mm column was used to establish a isocratic mobile phase of 50 : 50 DETA : methanol mobile phase. A Finigan MAT LCQ was used to monitor the parent ion, 186 for paraquat with the daughter ion in range 170 to 173 and the parent ion, 186 for diquat with the daughter ion range of 156 to 159. Manual injections to fill 25 µL teflon loop were interposed into the LC mobile phase after the column, directly into the MSMS. Each injection was repeated 6 times for each of the four repeats of each fortification level and the areas averaged. Results : Table 27 records the results obtained.

41

diquat

mgkg-1 µg mean r.s.d.

0.0 0 0 0.1 0.5 9528314 14 0.5 2.5 14776066 6 1.0 5 18640057 8

paraquat mgkg-1 µg mean r.s.d.

0.0 0 0 0.1 0.5 4494099 14 0.5 2.5 14490788 12 1.0 5 17807392 10

Table 27. Repeatability of the detection of paraquat and diquat using LC MSMS Discussion : The results in table 27 indicate a non-linear relationship between analyte concentration and peak area for the analysis of paraquat and diquat by LC MSMS. A curve fit describing the shape of the curve with a second order polynomial gave a r2 value of 0.971 and 1.000 for paraquat and diquat respectively. At low concentrations adsorption problems seem more pronounced for paraquat, such that the response for this analyte is half that seen for diquat and the 0.1 mgkg-1 level. The problems encountered for the detection of paraquat and diquat at levels of 1 mgkg-1 have to the greater extent been overcome. Problems affecting linearity of the response at low levels have yet to be resolved. However, method development is continuing, and it is hoped that this methodology will be presented as specific, sensitive, reproducible and robust. 2.1.2.ix. Method development using benchtop LC MSMS - dithiocarbamates The standard method for the analysis of dithiocarbamate pesticides, that is headspace analysis of carbon disulfide produced by the acidic stannous chloride digestion of dithiocarbamates with analyse by GC FPD, is not suitable for the analysis of essential oils, as sulfur chemicals, endogenous to essential oil crops, interfere. Gustafsson and Thompson (1981) developed a HPLC UV method for the determination of dithiocarbamates using a caustic EDTA extraction, followed by methylation with methyl iodide. This method was adapted with the intention of using detection by LC MSMS. Aim : To develop an anaytical methodology for the detection of mancozeb residues in essential oil. Experimental : When mancozeb is extracted in a EDTA / sodium hydroxide solution, the salt produced is disodium N, N'-ethylenebis(dithiocarbamate), which is transferred across the aqueous organic interface into a methyl iodide hexane / chloroform solution with the phase transfer reagent, , tetrabutylammonium hyrdrogen sulfate.

The disodium N, N'-ethylenebis(dithiocarbamate) was first synthesised using the method described by Gustafsson and Thompson (1981). The standard was dissolved in acetone and introduced into the MS via the worm drive syringe effecting direct infusion into the MS at a flow of 0.8 mLmin-1. The parent ion was 240.8 with a collision energy of 12% to produce the daughter ion at 192.8. The analyte was then introduced into

42

the LC to be chromatographed on a NOVAPAC C18 cloumn with a 50/50 methanol / 0.1M ammonium acetate buffer mobile phase with a gradient of 90/10 over 15 minutes. The analyte eluted at 5.2 minutes. Mancozeb was dissolved in an EDTA caustic solution and the methylation process undertaken using methyl iodide, chloroform and hexane with tetrabutylammonium hyrdrogen sulfate as the phase transfer reagent. When introduced into the LC Q system it became immediately apparent that tetrabutylammonium hyrdrogen sulfate contaminated the entire LC MS system, such that any signal from the target analyte was masked. Alternatives to the phase transfer reagent are now being investigated. The separation of the ethylenebis(dithiocarbamate) salt from the aqueous phase using ion exchange chromatography or specialised emporlite discs, to allow for direct methylation without the need for phase transfer reagent, is the most favoured line of research. 2.2 Monitoring of Harvests 2.2.1. Monitoring of 1998 / 1999 Boronia Harvest Aim : To screen oils produced in the 1998 / 1999 season Experimental : A standard curve was established by fortifying a boronia extract, known to be free of pesticide contamination. 10 to 20 mg was sub-sampled into GC vials and spiked with the standard solutions of propiconazole to produce the equivalent of 20, 1, 0.1 and 0.5 mg/kg concentrations of the residues in the oils. The standard curve solutions were dissolved in 100 µL of hexane, 900µL of methanol was added and the solutions were filtered into 300µL GC vial glass inserts. The selected boronia extracts (~10 to 15 mg) were weighed into GC vials after warming and mixing thoroughly. All samples and standards were spiked with 0.94 µg of endosulfan as an internal standard. 100µL of hexane was added to each vial and, where necessary, the hexane was gently warmed to increase the solubility of the oils in the solvent. 900µL of methanol was added to precipitate out the waxes. The solutions were filtered through glass wool into 300 µL glass GC vial inserts. All samples were subject to analysis by high resolution GC MS. Analytical parameters Samples were analysed on a HP 5890 Gas Chromatograph directly coupled to a Kratos Concept ISQ Mass Spectrometer. The GC was equipped with a HP5MS fused silica capillary column (30m, 0.22mm i.d., 0.25µm film thickness). 1µl splitless injections of samples were analysed using a carrier gas flow program of 30 psimin-1 from 25 to 40 psi, held for 0.1 min, then at 30 psimin-1 to 25 psi, then at 1 psimin-1 to 35 psi. The GC injection temperature was 260°C and the oven temperature programmed from 60°C to 290°C at 20°Cmin-1. Ions monitored were 194.9534 for internal standard, endosulfan and ion 259.0210 for propiconazole. A dwell time of 300ms/ion and 50ppm voltage sweep were employed for all ions. Resolution of 10,000 (10% valley definition) and the ion m/z 242.9856 from perfluorokerosene was used as the lock mass for all analytes and the internal standard. Electron ionisation was undertaken at a source temperature of 210°C and an electron energy of 70eV, with an accelerating voltage of 5.3kV. Results : Table 28 lists the results for the monitoring of propiconazole in boronia extract for the 1998 / 1999 harvest season

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variety sample mgkg-1

clone 3 Bor 750 1.1 Bor 758 0.5 Bor 764 0.3 Bor 779 0.6 Bor 807 0.9

clone 5 Bor 748 1.3 Bor 771 0.5 Bor 809 0.8

clone 17 Bor 745 0.4 Bor 749 0.8 Bor 765 0.6 Bor 770 0.6

clone 250 Bor 743 0.6 Bor 756 1.2 Bor 811 0.6 Bor 760 1.1

blends Bor 763 1.4 Bor 801 0.5 Bor 740 10.5

Table 28. Propiconazole residues detected in boronia extract from the 1998 / 1999 harvest

2.1.2. Monitoring of 1999 / 2000 Essential Oil Harvest Aim : To screen extracts produced in the 1999 / 2000 season Experimental & Results: Several analysis were conducted through the year 2000 using gas chromatography high resolution mass spectrometry. The preparation and analytical parameters used for the analysis of boronia extracts are as described for the 1998 / 1999 season. Table 29 lists the levels of pesticide contamination detected.

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Sample propiconazole tebuconazole oxyflurofen prometryn terbacil simazine number mgkg-1 mgkg-1 mgkg-1 mgkg-1 mgkg-1 mgkg-1

Aug. - 1999 EOT#5 19.8 0 0.7 8.6 EOT#3 17.0 15.1 0.5 0.0 Nov. 1999 EOT 2721 1.7 EOT 8346 26.5 EOT 3339 182.8 Feb. - 2000 Bor 741 0.4 bdl bdl bdl bdl bdl Bor 851 0.1 bdl bdl bdl bdl bdl Bor 855 0.1 bdl bdl bdl bdl bdl Bor 864 8.7 bdl bdl bdl 0.1 0.2 Bor 866 0.7 bdl bdl bdl bdl bdl Bor 872 1.1 bdl bdl bdl bdl bdl Bor 873 0.2 bdl bdl bdl bdl bdl Bor 874 7.8 bdl bdl bdl bdl bdl Bor 878 0.4 bdl bdl bdl bdl bdl Bor 882 0.3 bdl bdl bdl bdl bdl Bor 906 0.6 bdl bdl bdl bdl bdl Bor 909 10.4 bdl bdl bdl bdl bdl Bor 921 1.2 bdl bdl bdl bdl bdl Bor 955 35 bdl bdl bdl 0.5 bdl

Table 29. Pesticide residues detected in extracts harvested in 1999 / 2000

Table 30 lists the results obtained for distilled oils from the same season. Oils were analysed by gas chromatography, high resolution mass spectrometry, however, the partition of oils between hexane and methanol was not necessary. Distilled oils were weighed into GC vials (20 - 30 mg), spiked with 0.9 µg of endosulfan, dissolved in 1mL of chloroform and analysed under the conditions previously described.

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Parsley extracts

Sample propiconazole tebuconazole oxyflurofen prometryn terbacil simazine sethoxydim

number mgkg-1 mgkg-1 mgkg-1 mgkg-1 mgkg-1 mgkg-1 mgkg-1

pa7 bdl bdl bdl bdl bdl < 0.02 bdl pa128 bdl bdl bdl bdl bdl < 0.02 bdl pa42 bdl bdl bdl bdl bdl < 0.02 bdl EOT730 bdl bdl bdl bdl < 0.1 < 0.02 bdl

Peppermint extracts

p45 bdl bdl bdl bdl bdl < 0.02 bdl p58 bdl bdl bdl bdl bdl < 0.02 bdl p12 bdl bdl bdl bdl bdl bdl bdl p11 bdl bdl bdl bdl bdl bdl bdl p130 bdl bdl bdl bdl bdl < 0.02 bdl p118 bdl bdl bdl bdl bdl bdl bdl

Fennel oils

f151 bdl bdl bdl bdl bdl bdl bdl f393 bdl bdl bdl bdl bdl < 0.02 bdl f188 bdl bdl bdl bdl bdl bdl bdl f105 bdl bdl bdl bdl bdl bdl bdl EOT731 bdl bdl bdl bdl bdl bdl bdl EOT732 bdl bdl bdl bdl bdl bdl bdl

Table 30. Pesticide residues in steam distilled essential oils

2.1.3. Monitoring of 2000 / 2001 Essential Oil Harvest Aim : To screen oils produced in the 2000 / 2001 season Experimental & Results : The new LC MSMS analytical methodologies were used for the screening of the 2000 / 2001 harvests. The analytical parameters and sample preparations were as described on page 35. Pesticides tested for in the screen include monocrotophos, simazine, cyanazine, pirimicarb, sethoxydim, prometryn, tebuconazole, propiconazole, difenaconazole and the esters of fluazifop and haloxyfop. The only pesticide detected in the oils analysed was prometryn. Table 31 lists the oils and levels of contamination obtained.

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sample prometryn fennel oils mgkg-1

eot 768 0.00 eot 769 0.92 eot 771 0.00 f11 0.00 f217 0.00 f278 0.00 f400 0.00 f464 0.00 f95 0.00 l904 0.00

peppermint oils

p107 0.00 p112 0.00 p120 0.00 p23 0.00 p79 0.00 parsley oils

pa172 1.01 pa173 0.96 pa174 0.83 pa176 0.26 pa20 1.09 pa43 0.99

Table 31. Levels of prometryn residues detected in steam distilled oils in the 2000 / 2001 harvest In addition, the oils listed in table 31 were tested for residues of paraquat and diquat. The extraction protocol was not successful for parsley oils, which formed a viscous emulsion which could not be resolved by centrifugation or salting out. No paraquat or diquat residues were detected in the fennel or peppermint samples analysed. Discussion : Contamination of boronia extract with propiconazole continues to present problems. The harvests from the year 2000 present with residue levels as high as 10 mgkg-1. The other recurring contaminant is prometryn, which repeatedly has been detected in parsley oil. The active ingredient of Gesaguard, prometryn is a systemic herbicide. Industry is currently reviewing its pest management protocols. Overall the results for the industry monitored, the levels of pesticide residues are below detectable limits. Close monitoring of application regimens has conferred a level of responsible pest management within the essential oil industry. 2.3 Production of Manual Accompanying this report is a copy of the manual entitled 'Approaches to the Analyses of Pesticide Residues in Essential Oils.' The manual is designed to allow intending analysts to determine methodologies and equipment most suited to the chemical type of the pesticide of interest or the applicability of the type of analytical equipment generally available. The manual has been a dynamic project, with the development of methodologies continuing throughout the relevant stages of production.

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3. Conclusions Effective clean-up methodologies for medium polarity pesticides in essential oils continues to be elusive. However, aqueous extractions of distilled oils using carbonate buffer, has been successful in the pre-concentration of pesticides with acidic moieties such as dicamba, mecoprop, MCPA, fluazifop, haloxyfop and fluroxypyr. Paraquat and diquat have also been successfully extracted from the matrix of distilled oils, with the clean-up technique allowing for direct injection of extracts into the MS, bypassing the LC system. The capacity of disposable C18 cartridges to the separation of boronia extract components was found to be limited with the majority of essential oil components eluting on the solvent front. Laboratory supplies companies have invested considerable funds to the development of SPE, and a comprehensive assessment of the wide range of phases now available should be instigated. ECD was found to be successful in the detection of the majority of halogenated pesticides trialed, with detection limits extending to 0.1 to 1 mgkg-1 in some matrices. However, analysis of pesticide residues in essential oils, by application of GC ECD is not specific enough to allow for a unequivocal identification of any contaminant. As a screen, ECD will only be effective in establishing that no gross contamination is present in oils in the absence of a peak eluting within a given time frame. In the situation where a peak is recorded with the correct elution characteristics and which is enhanced when the sample is fortified with the target analyte, a second means of contaminant identification would be required. ECD, then, can only be used to rule out significant contamination and could not in itself be sufficient for a positive identification of pesticide contamination. Benchtop GC MSMS was assessed. The lack of flexibility of the system accessed at the CSL at the University of Tasmania precluded the development of methodology without comprehensive clean-up techniques. The elution of all components from the GC column, into the mass spectrum would quickly lead to detector contamination. Method validation for the detection of 6 common pesticides in boronia extract using GC high resolution mass spectrometry was completed. Detection limits ranged from 0.05 to 0.1 mgkg-1 with r.s.d.s of between 9 and 17%. Excellent linearities were recorded for all analytes with the exception of simazine. High resolution mass spectrometry continues to be a highly sensitive and specific method for the screening of essential oils but with the high capital outlay required, and the high level of expertise needed for its continued operation and maintenance, the methodology is unlikely to be widely adopted. The most encouraging method development was in the area of high pressure liquid chromatography with detection using ion trap mass spectrometry. Analytical techniques using atmospheric pressure chemical ionisation in the positive mode were developed for monocrotophos, simazine, cyanazine, pirimicarb, propazine, sethoxydim, prometryb, tebuconazole, propiconazole, , difenoconazole and the esters of fluroxypyr, fluazifop and haloxyfop. The method validation was conducted for three oils, peppermint, parsley and fennel. Detection limits ranged from 0.1 to 0.5 mgkg-1 within the matrix of all essential oils trialed with a linear relation ship established between pesticide concentration and peak height (r2 greater than 0.997) and repeatibilities as described by the relative standard deviation ranging from 3 to 19 %. The type of oil analysed had minimal effect on the response function as expressed by the slope of the standard curve. Acephate, carbaryl, dimethoate, ethofumesate and pendimethanlin all required further work for enhanced ionisation and / or improved elution profiles. Method development of LC MSMS included the assessment of ESI and negative APCI. For the medium polarity chemicals trialed, ESI has limited application. No response was recorded for some of the most commonly used pesticides in the essential oil industry, such as linuron and oxyflurofen. However, overall, there was very little difference between the sensitivity for ESI and APCI. However,

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APCI was slightly more sensitive for the commonly used pesticides, tebuconazole and propiconazole, and showed a response, though poor, to linuron and oxyflurofen. Analytes that ionised in the negative APCI mode were incorporated into a separate screen which included bromacil, terbacil, and the esters of the fluazifop and haloxyfop acids. Negative ionisation mode for APCI gave improved characteristics for dicamba, procymidone, MCPA and mecoprop, but this ionisation mode was compromised by the acetate buffer. The replacement of acetonitrile with methanol slowed the elution of the analytes from the C18 LC column. The inclusion of acetic acid improved peak resolution. 2,4-D and 2,4,5-T were assessed as suitable internal standards. The LC MSMS method for the detection of dicamba, fluroxypyr, MCPA, mecoprop and haloxyfop in peppermint and fennel distilled oils underwent the validation process. Detection limits ranged from 0.01 to 0.1 mgkg-1 The parent esters of the pesticides which have an carboxylic acid moiety such as fluazifop, haloxyfop and fluroxypyr, present several complications in analytical method development. Detection using ESI was tested. Preliminary results indicate that ESI is unsuitable for haloxyfop and fluroxypyr ester. Fluazifop possessed good ionisation characteristics using ESI with responses approximately thirty times that recorded for haxloyfop. Poor chromatography and response necessitated improved mobile phase and the effect of pH on elution characteristics was considered the most critical parameter. Extraction protocols and LC MSMS methods for the detection of paraquat and diquat in essential oils were developed. ESI produced excellent responses for both paraquat and diquat, however, sensitivity was compromised by arcing across the ESI source effected by the ammonium acetate in the buffer. Acetonitrile was replaced with methanol reducing the arcing effect. Extraction methodology using aqueous phases was developed. Extraction with carbonate buffer proved to be the most effective in terms of recovery and robustness. A total ion chromatogram of the LC run of an aqueous extract of essential oil was recorded and detection using a photodiode array detector confirmed that very little essential oil matrix was co-extracted. The low background noise indicated that samples could be introduced directly into the MS. This presented a most efficient and rapid way for analysis of paraquat and diquat, avoiding the need for specialised columns or modifiers to be included in the mobile phase to instigate ion exchange. Lack of reproducibility and deterioration of signal over time made it evident that paraquat and diquat adsorb onto glass and other surfaces. This problem was eliminated to some degree by spiking samples with diethylenetriamine (DETA), which preferentially accumulates on the surfaces of sample containers, competitively binding on the adsorption sites. All glassware used in the paraquat diquat analysis was washed in a 5% solution of 0.1M DETA and then left to drain and partially dry. The inclusion of a DETA solution pre-treatment of all glassware effected ♦ a 3.3 fold increase in paraquat response at 0.01 mgkg-1 fortification level; ♦ a 2.4 fold increase in diquat response at the 0.1 mgkg-1 fortification level; ♦ an increase in the repeatability of the analysis for paraquat as expressed by the relative standard

deviation (R.S.D.), lowered from 43 to 32%; ♦ an increase in the linearity of the standard curve at low concentrations as expressed by the

improved 'r2' values for both paraquat and diquat. The mobile phase was changed to 50:50 DETA / methanol, which immediately improved the elution profile for paraquat. Ghosting problems persisted and the desorption of analytes from the metal interfaces within the switching valve were proposed as a possible source of the analytical error. The stainless steel tubing on the switching valve was replaced with teflon, further improving reproducibility. Method validation was undertaken of the analysis of paraquat and diquat using the protocols established. The relationships between analyte concentration and peak area were not linear at low concentrations, but were best described by a second order polynomial. The r2 values for the curve fits for paraquat and diquat were 0.971 and 1.000 respectively. At low concentrations, adsorption problems seem more pronounced for paraquat, such that the response for this analyte is half that seen for diquat and the 0.1 mgkg-1 level.

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The development of a method for the detection of the dithiocarbamate, mancozeb was commenced. An adaptation of a HPLC UV method used by Gustafsson and Thompson (1981), using a caustic EDTA extraction, followed by methylation with methyl iodide was modified with the intention of using detection by LC MSMS. The intended product of extracted and derivatised mancozeb, disodium N, N'-ethylenebis(dithiocarbamate) was first synthesised. A LC method with detection using MSMS was successfully completed. For the analysis of extracted mancozeb, however, using as a methylation agent, a phase transfer reagent was required to shuttle the salt across the aqueous interface into an organic phase for methylation. The phase transfer reagent trialed was , tetrabutylammonium hyrdrogen sulfate, which when introduced into the LC Q system, contaminated the entire system, such that any signal from the target analyte was overwhelmed. Alternatives to the phase transfer reagent are now being investigated including ion exchange chromatography. Monitoring of harvests was undertaken for the years spanning 1998 to 2001. Problems continued for residues of propiconazole in boronia in the 1998 / 1999 year with levels to 1 mgkg-1 still being detected. Samples in subsequent years were screened for a range of pesticide residues including tebuconazole, simazine, terbacil, bromacil, sethoxydim, prometryn, oxyflurofen and the esters of the pesticides with acidic moieties. Screening was extended in the harvest year of 2000 / 2001, to include the pirimicarb, difenaconazole, herbicides with acidic moieties and paraquat and diquat. Apart from propiconazole the other recurring contamination of essential oils produced from commercial harvest resulted from the use of prometryn, which presented residues in parsley oil.

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Implications & Recommendations ♦ Method development : The benchtop high pressure liquid chromatography system, configurated

with a mass spectrometer, presents as the most robust and specific analytical equipment in the detection of pesticide residue in essential oils. It has a wide range of applications, and with sufficient methodology work-up, has the potential to cover most pesticide types.

♦ Monitoring of essential oil harvests : The continued monitoring of commercial harvests has

shown that in most respects the recommended application rates have not been exceeded for most of the pesticides for which screens have been developed. The absence of gross contamination indicates responsible use of pest control agents. However, problems still exist with propiconazole in boronia and contamination of parsley samples with prometryn at levels of around 1 mgkg-1.

♦ Methods manual : The accompanying methods manual is designed to present assessments of

methodology and application of analytical equipment for the detection of pesticides in essential oils.

Bibliography Gustafsson, K. H., Thompson, R. A. High-pressure liquid chromatographic determination of fungicidal dithiocarbamates. J. Agric. Food Chem., 1981, 29, 729 – 732 Kaniansky, D., Ivanyi, F. and Onuska, F. I. On-Line Isotachophoretic Sample Pretreatment in Ultratrace Determination of Paraquat and Diquat in Water By Capillary Zone Electrophoresis. Analytical Chemistry, 1994, 66 11 1817-1824

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