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Journal of Chromatography A, 1216 (2009) 5092–5100

Contents lists available at ScienceDirect

Journal of Chromatography A

journa l homepage: www.e lsev ier .com/ locate /chroma

mproved extraction and clean-up of imidazolinone herbicides from soilolutions using different solid-phase sorbents�

ohammadkazem Ramezania,b, Nigel Simpsonc, Danielle Oliverd,∗, Rai Kookanad,urjeet Gill a, Christopher Prestona

School of Agriculture, Food & Wine, University of Adelaide, PMB 1, Glen Osmond, Adelaide, SA 5064, AustraliaPlant Protection Research Institute, P.O. Box 1454, Tehran 19395, IranVarian Australia, Pty., Ltd., 679 Springvale Road, Mulgrave, Melbourne, VIC 3170, AustraliaCSIRO Land and Water, PMB 2 Glen Osmond, Adelaide, SA 5081, Australia

r t i c l e i n f o

rticle history:eceived 5 January 2009eceived in revised form 22 April 2009ccepted 28 April 2009vailable online 3 May 2009

a b s t r a c t

Extraction and quantification of herbicide residues from soil are important in understanding thebehaviour of persistent herbicides. This research investigated extraction and clean-up methods for imi-dazolinone herbicides from soil and soil amended with organic material. A series of solvent mixes, pHconditions and sorbents was tested. Across three imidazolinone herbicides: imazapyr, imazethapyr and

eywords:olid-phase extractionmidazolinone herbicidesoil extraction solutions

imazaquin, 0.5 M NaOH extraction gave greater than 90% recovery from soil samples; however, 0.5 MNaOH:MeOH (80:20) resulted in higher recovery for imazaquin, but not for the other two herbicides. Ofthe sorbents tested, the use of chromatographic mode sequencing using C18 and SCX sorbents providedconsistent high (>85%) recovery of all three herbicides from soil and separation of the herbicides fromother soil components by high performance liquid chromatography (HPLC). These two methods will allowhigh recovery of these imidazolinone herbicides from soil and have the ability to detect these herbicides

oth

nantiomer soil extraction

without interference from

. Introduction

Imidazolinone herbicides have become widely used in agricul-ure because of their low application rates, decreased environ-

ental impact and selectivity in a wide range of crops. They arepplied either pre- or post-emergence as selective herbicides forroad spectrum control of broadleaf weeds and grasses in soybeanGlycine max (L.) Merr.) and several other legume crops [1,2]. Theyan also be used as non-selective wide spectrum herbicides in non-rop situations, as well as in forestry and plantation crops, such asubber and oil palm [3,4]. In addition, the imidazolinone herbicidesre used in Clearfield® (imidazolinone-tolerant crops) productionystems [5].

The imidazolinone herbicides are one of the five families oferbicides that inhibit the enzyme acetohydroxyacid synthase

AHAS), which is a key enzyme in the pathway responsible forhe biosynthesis of branched-chain amino acids in plants [6]. Allmidazolinone herbicides have a chiral imidazole moiety in their

olecular structure, but differ in the second heterocycle in the

� Work conducted at CSIRO Land and Water, PMB 2 Glen Osmond, SA 5081, Aus-ralia.∗ Corresponding author.

E-mail address: Danni.Oliver@csiro.au (D. Oliver).

021-9673/$ – see front matter. Crown Copyright © 2009 Published by Elsevier B.V. All rioi:10.1016/j.chroma.2009.04.080

er soil components.Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved.

structure (Fig. 1). Imazapyr and imazethapyr have a pyridine ringand imazaquin has a quinoline moiety [5]. A common characteristicof all imidazolinone herbicides is the presence of two enantiomersthat derive from the chiral centre of the imidazolinone ring. Theinhibitory activity of the R(+) enantiomer to AHAS is nearly eighttimes greater than that of the S(−) enantiomer [7].

The amphoteric nature (presence of both acidic and basic func-tional groups) of imidazolinone herbicides allows these herbicidesto exist in anionic, neutral or cationic states depending upon thepH of the environment [8,9]. The pKa values of imidazolinone her-bicides range from 1.3 to 3.9. When the soil pH is greater than theirpKa, these herbicides are usually present mainly in an anionic state(–COO−), while the acidic functional group (–COOH) is totally in anon-ionic form when the soil pH is two units lower than the mostacidic pKa [10,11]. Consequently, soil factors such as pH, organic car-bon content, and ionic strength may affect their persistence in theenvironment [12,13]. The imidazolinone herbicides are relativelypersistent in soil with half-lives ranging from 30 to 150 days andmay have carryover effects on subsequent crops grown [14–16].Moreover, it has been reported that the imidazolinone herbicides

show high potential for leaching because of their relatively low pKa

values (3.3–3.9) [17–22].The recommended application rates for imazapyr, imazethapyr

and imazaquin range from 100 to 200 g ha−1 [23]. These low appli-cation rates in field situations make residue analysis of these

ghts reserved.

M. Ramezani et al. / J. Chromatogr

hdc[lnTbnneh

smooth chromatogram with no significant exogenous peak inter-

TM

M

W

W

W

S

S

S

S

Fig. 1. Chemical structures of imazapyr, imazethapyr and imazaquin.

erbicides from soil problematic. Furthermore, extraction of imi-azolinone herbicides, particularly from soil, is difficult because ofo-extraction of other substances that interfere with chromatogram24], properties of the herbicides and the low analytical detectionimits required. Current methods for the extraction of imidazoli-one herbicides are time-consuming, labour-intensive and costly.he published methods for the extraction of imidazolinone her-icides from water and soil matrices are summarized in Table 1. A

umber of these methods were used for the extraction of imidazoli-one herbicides from water. Many of those methods used for thextraction from soils evaluated only one compound. One methodad good recovery for the extraction of three herbicides (imaza-

able 1ethods from the literature for the extraction of imidazolinone herbicides using SPE sorb

atrix Extraction solution SPE sorbent bed Instrum

ater naa Carbograph-1 HPLC-U

ater na Polystyrene LC–MSdivinylbenzene,SAX & alumina

ater na SAX & RP-102 styrene-divinyl benzene LC–MS

oil NH4HCO3 SAX & C18 HPLC-U(0.1 M, pH 5)

oil KH2PO4 Carbograph-1 LC-MS/M(0.01 M pH 8)

oil 0.5M NaOH C18 & SCX HPLC-U

oil Ca(OH)2 SPE disk & C18 HPLC-U

a na, not available.

. A 1216 (2009) 5092–5100 5093

pyr, imazethapyr and imazaquin) from soil, but this method wasdifficult to employ with larger soil samples (25 g) [25].

Due to the persistent nature of these herbicides in some soilsit is necessary to optimise analytical techniques to extract theseherbicides for analyses at suitable detection limits (<1 �g mL−1).Extraction of pesticides from environmental matrices is frequentlythe most time-consuming step in residue analysis. A wide varietyof methods and solvents has been used to extract pesticides fromwater and soil [24]. Over recent decades, there has been a trendtoward solid-phase extraction (SPE) as a substitute to the difficultand time-consuming liquid–liquid extraction (LLE) because SPE isfaster, uses less organic solvent and can provide higher recoveryof more polar pesticides [26,27]. Furthermore, since large volumesof samples can be loaded onto SPE sorbents, it is possible to con-centrate the analyte(s) of interest and increase the likelihood ofdetecting compounds. Optimisation of SPE column conditions andthe choice of sorbent are two key factors in the use of SPE forthe extraction of pesticides from water and soil. Both the physi-cal and the chemical properties of pesticides need to be consideredin method development. The best results using SPE sorbents areobtained when pesticides are strongly adsorbed by the sorbentfrom the aqueous sample, allowing percolation of a large volume ofsample and easy desorption of the pesticide by the solvent duringelution [28,29].

In the current study the amendment of soils with plant-derivedorganic materials (lupin residue Lupinus angustifolius) added fur-ther complexity to the detection of imidazolinone herbicides. Aspart of the larger study lupin crop residue was added to soils tostudy herbicide degradation. The lupin residue was added in orderto manipulate the C:N ratio in soils and thereby increase microbialactivity. Lupin crop residue was chosen because lupins are a legumecrop species often grown in rotation with cereals in Australia andlupin residue would therefore be present in many cropping soilswhere imidazolinone herbicides are used. The addition of lupin cropresidue to the soil created chromatographic problems, which led tothe development of the extraction procedure using SPE sorbents.It was necessary to develop an extraction procedure that removedthe additional organic material (mostly humic acids) to achieve a

ferences for the herbicides.The main objectives of this research, which are detailed in the

manuscript, were to investigate and evaluate: (1) the efficacy of dif-ferent solvents for the extraction of three imidazolinone herbicides

ents.

ent and detector Recovery ± RSD from SPE Herbicides References

V 89% ± 5.1% Imazapyr [37]ImazethapyrImazaquin

114% ± 9% Imazapyr [33]ImazethapyrImazaquin

73% ± 20% Imazapyr [34]ImazethapyrImazaquin

V na Imazapyr [38]

S 78–92% ± 4–5% Imazapyr [25]ImazethapyrImazaquin

V na Imazamox [32]

V na Imazethapyr [39]

5094 M. Ramezani et al. / J. Chromatogr. A 1216 (2009) 5092–5100

Table 2Selected physicochemical propertiesa of imazapyr, imazethapyr and imazaquin.

Herbicides Molecular weight Solubility in water (25 ◦C) (mg L−1) Vapour pressure (60 ◦C) (Pa) pKa1 pKa2 pKa3

Imazapyr 261.3 11270 <1.33 × 10−5 1.9 3.6 11.0II

fcsat

2

2

wt[adAQFpSEPbb

2

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2

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mazethapyr 289.3 1420mazaquin 311.3 60

a Reference [30].

rom soil; (2) the efficacy of a simple non-polar SPE method for pre-oncentration and extraction of imidazolinones from humic acidolutions and soil; and (3) the performance of different SPE sorbentsnd mechanisms for the clean-up of the aqueous soil extracts prioro analysis by high performance liquid chromatography (HPLC).

. Experimental

.1. Chemical, reagents and apparatus

Standards of imazapyr, imazethapyr and imazaquin (99% purity)ere purchased from Sigma–Aldrich (Australia). Selected proper-

ies of imazapyr, imazethapyr and imazaquin are given in Table 230]. Solvents (HPLC grade) including hexane (HEX), 2-propanol,nd AR grade solvents of methanol (MeOH), acetonitrile (ACN), andichloromethane (DCM) were purchased from Biolab (Australia).ll solutions were prepared with ultra pure deionised water (Milli-water, Millipore). The humic acids sample was purchased from

luka Chemie (Buchs, Switzerland). Whatman GF/C (70 mm, 1.2 mmore) filters were purchased from Biolab (Australia). A range ofPE sorbents including Varian Bond Elut-C18 (C18), Varian Bondlut SCX (SCX), Varian Bond Elut PPL (PPL), Phenomenex NH2 andhenomenex X-AW (AW) were assessed and properties of these sor-ents are given in Table 3. Phosphate buffer solutions were preparedy mixing appropriate stock solutions of K2HPO4 and KH2PO4.

.2. Preparation of standard stock solutions

Stock solutions (1000 �g mL−1) of the three herbicides, imaza-yr, imazethapyr and imazaquin, were made individually in ACN.rom the stock solution a 100 �g mL−1 herbicide standard solutionf individual herbicides was prepared into 100 mL ACN, which wasurther diluted to 0.5, 1, 2.5, 5 and 10 �g mL−1 and stored at −4 ◦C.

.3. Fortification of humic acid solution and soil samples

A humic acid stock solution (30 �g mL−1) was prepared by dis-olving humic acid in Milli-Q water using a sonication bath, filteringhrough a 0.45 �m filter and kept in the dark at 4 ◦C (pH 6.8). Thisoncentration of humic acid solution was chosen because it dis-

olved completely in the water while at higher concentrations arecipitate formed. The total organic carbon concentration of thenfiltered solution was 19 mg L−1 and of the filtered solution was7.9 mg L−1. Three replicates of imazapyr, imazethapyr and imaza-uin (40 �L) were prepared in this humic acid solution and used

able 3tructure and some properties of SPE sorbents assessed for efficacy of extraction ofmidazolinone herbicides from soil solutions.

orbent Supplier Sorbent structure

Based structure Area (m2 g−1)

PL Varian Polymeric 70018 Varian Octadecyl bonded silica 462CX Varian Benzene sulfonic acid bonded silica 503-AW Phenomenex Styrene-divinylbenzene naa

H2 Phenomenex Aminopropyl bonded silica naa

a na, not available

<1.33 × 10−5 2.1 3.9<1.33 × 10−5 3.8

to assess the efficacy of the PPL sorbent to remove the selectedherbicide.

Unless otherwise indicated, the following procedure was usedfor all extraction studies. For each experiment, three replicates ofa sandy loam soil (10 g) were weighed into separate centrifugetubes and fortified with each herbicide separately by adding 100 �Lof 100 �g mL−1 to give the required fortification concentration of1 �g g−1. A blank or unspiked soil sample, to which 200 �L of Milli-Q water was added, was also run in triplicate with each experiment.After fortification, solvents were allowed to evaporate and then thesoils were homogenized using a vortex mixture for 1 min and keptat 4 ◦C for 48 h before extraction.

2.4. Evaluation of the efficacy of various solutions to extractimidazolinones from soil

Different extraction solutions have been reported in the liter-ature using batch extraction for the extraction of imidazolinoneherbicides from soils. In a preliminary experiment the efficacy ofa range of extraction solutions including 0.1 M potassium chlo-ride (KCl), 0.5 M sodium hydroxide (NaOH), 0.01 M NaOH and 0.5 MNaOH:MeOH (80:20) to extract imazapyr, imazethapyr and imaza-quin from soil was investigated. Triplicate soil samples (5 g) thatwere either amended with 2 g lupin (L. angustifolius) residue (<1 mm) or non-amended were each spiked with imidazolinone her-bicides at 1 �g g−1 and kept at 4 ◦C for 48 h before extraction. Thesoil (0–15 cm) used in this experiment had been collected in thefield, air-dried and sieved <2 mm before the lupin residue wasadded. The soil was a sandy loam with a pHw of 5.2, an organiccarbon content of 1.9% and clay (<0.002 mm) of 19.2% and a sandcontent (0.02–2 mm) of 67.8%. In this part of the experiment dif-ferent ratios of soil and extraction solutions namely, 1:2, 1:3, and1:4, were used for extraction of soil samples to find the opti-mal conditions for maximum recovery of the herbicides from thesoil.

2.5. Solid-phase extraction clean-up

2.5.1. Optimising solution pH for herbicide extraction from waterand humic acid solutions using PPL sorbents

Bond Elut PPL sorbents are packed with highly cross-linked andchemically modified styrene divinyl benzene copolymer. The choiceof this sorbent for the extraction of imidazolinone herbicides wasbased on previous studies where 12 phenols (weak acids) were suc-cessfully extracted from drinking water [31]. The optimal solutionpH for maximum recovery of imidazolinone herbicides from waterand humic acid solutions was assessed in triplicate in a preliminaryexperiment using 100 mL of water spiked with 1 �g mL−1 of onlyimazapyr with pH adjusted to 2, 5.5, 7 or 9. Details of conditioningthe PPL sorbents and elution are given in Table 4.

To examine the effect of humic acid on the extraction of the her-bicides from the PPL sorbents, triplicate solutions of 30 �g mL−1

(100 mL) of humic acid were spiked individually with 100 �L of thestandard working solution. After mixing for 2 min, samples wereacidified to pH 2.3 with 6N hydrochloric acid (HCl) and kept at 4 ◦Cfor extraction 48 h later. The extraction procedure was the sameas outlined above for the preliminary experiment. The efficacy of

M.Ram

ezanietal./J.Chrom

atogr.A1216

(2009)5092–5100

5095

Table 4Summary of the treatment of the SPE sorbents for the extraction of the imidazolinone herbicides from water, humic acid solutions or soil extracts.

Solution Water and humic acid solutions NaOH soil extracts

Sorbent PPL PPL X-AW Chromatographic modesequencing using NH2 andPPL

Chromatographic modesequencing using C18 andSCX

SCX

Prime 2 × 3 mL DCM 2 × 3 mL DCM 1 × 5 mL MeOH:formicacid (98:2)

1 × 5 mL 1% acetic acid inMeOH

1 × 5 mL MeOH 1 × 5 mL HEX

2 × 3 mL MeOH 2 × 3 mL MeOH 1 × 5 mL MeOH3 × 2 mL H2O(pH 2) 3 × 2mL H2O (pH 2) 1 × 2 mL H2O 1 × 5 mL 1% acetic acid in

water1 × 5 mL H2O 1 × 5 mL H2O

Load Spiked water samplesor soil extracts

Filtered 0.5 M NaOHextracts adjusted to pH 2

40 mL × filtered 0.01 MNaOH extract

40 mL × filtered 0.5 MNaOH extract

40 mL × filtered 0.5 MNaOH extract

MeOH: H2O (1:1) elutedfrom C18 cartridge

Rinse – – 1 × 5 mL H2O – 1 × 5 mL H2O1 × 5 mL MeOH

Dry Dry under vacuum Dry under vacuum Dry under vacuum – Dry under vacuum Dry under vacuum

Elute 2 × 3 mL DCM A, B or C 6 mL ×MeOH:NH4OH(98:2)

Clear eluate (0.5 M NaOH)was collected and loadedonto PPL which was treatedas outlined

20 mL × MeOH:H2O (1:1) 1 × 20 mL phosphate buffer(pH6.5)

(A) 2 × 3 mL DCM Then load onto SCX afterSCX was primed asoutlined

(B) 2 × 3 mLDCM:isopropanol (80:20)(C) ethylacetate:isopropanol(80:20)

Final Preparation Add 4 mL × 2-propanol,evaporate undernitrogen to 2 mL

Evaporate under nitrogento 2 mL

Evaporate to drynessunder N2

As detailed in text

Redissolve in ACN:H2O(1:1)

5096 M. Ramezani et al. / J. Chromatogr

Ff

Pom

2

fowqwbe

ndtowsbuTcpStTtt

ig. 2. Flow chart of methodology for the extraction of imidazolinone herbicidesrom soil using different SPE sorbents.

PL sorbents to retain the imazaquin enantiomers with and with-ut humic acid (30 �g mL−1) was also evaluated using the sameethod.

.5.2. Clean-up of NaOH soil extracts using SPE sorbentsThe highest recoveries of the imidazolinones from soil were

ound using NaOH so only this extractant was considered whenptimizing the clean-up processes. Triplicate soil samples (10 g)ere spiked separately with imazapyr, imazethapyr and imaza-

uin herbicides at the concentration of 1 �g g−1. When the solventsere completely evaporated, the soil samples were homogenized

y Vortex for approximately 1 min and left for a week at 4 ◦C beforextraction as detailed in Fig. 2.

The efficacy of different SPE sorbents to extract the imidazoli-one herbicides from the NaOH soil extracts was determined asetailed in Table 4. Initially the PPL sorbent was assessed but dueo the existence of interfering compounds on the chromatogramsbtained following the elution from the PPL sorbent, particularlyith imazapyr, and the low recovery of herbicides, a variety of SPE

orbents were then examined. The protocols used for the SPE sor-ents assessed, namely X-AW, chromatographic mode sequencingsing NH2 followed by PPL and C18 followed by SCX, are given inable 4. When the C18 sorbent was assessed approximately 15 g ofelite was added to the extracts to facilitate filtration and then the

rocedure as described in Fig. 2 was applied. After elution from theCX cartridge with the phosphate buffer the solution was acidifiedo pH 2 and the herbicides partitioned three times with 15 mL DCM.he solvent was evaporated under a gentle stream of nitrogen gaso dryness and the herbicides were redissolved in 2-propanol andhen analysed using HPLC.

. A 1216 (2009) 5092–5100

2.6. HPLC analysis

2.6.1. Reversed-phase HPLC for imidazolinone herbicidesThe final extracts of herbicides, dissolved in ACN, were anal-

ysed by reversed-phase HPLC using an Agilent 1100 HPLC equippedwith a quaternary pump, vacuum degasser, diode array detec-tor, autosampler, a column oven and an Altima C18 column(250 mm × 4.6 mm I.D., 5 �m particle size). Data were processedusing the commercially available Agilent ChemStation software.The HPLC operating conditions were: an isocratic mobile phaseof 55:45 acetonitrile:1% acetic acid in HPLC grade water; a flowrate of 1 mL min−1; a column oven temperature of 25 ◦C; and aUV–vis detector set at 240 nm. The retention times of imazapyr,imazethapyr and imazaquin in this system were 2.92, 4.39, and5.53 min, respectively. The detection limit of imazapyr, imazethapyrand imazaquin was 0.5 �g mL−1.

2.6.2. Normal-phase HPLC for imazaquin enantiomersNormal-phase HPLC was used for the detection of the imaza-

quin enantiomers. For normal-phase separation, the final sampleswere dissolved in n-HEX:2-propanol (1:1, v/v). The Agilent 1100HPLC was used for normal-phase analyses with the following con-ditions: a Chiralcel OJ [cellulose tri(4-methylbenzoate)] column(250 mm × 4.6 mm I.D., 10 �m particle size); an isocratic mobilephase of 65:35:0.1 n-HEX:2-propanol:trifluoroacetic acid (TFA); aflow rate of 1 mL min−1; and a UV–vis detector set at a wavelengthof 240 nm. The retention times of S(−) and R(+) enantiomers ofimazaquin were 9.3 and 10.5 min, respectively. The detection limitfor each of the enantiomers of imazaquin was 0.5 �g mL−1.

2.7. Statistical analysis

Percentage recovery data were log-transformed to normalizetheir distribution before analysis of variance (ANOVA). However,the untransformed data are presented. ANOVA was used to evalu-ate differences between recovery rates (%) of extraction solutionsand SPE cleaning methods. The significant differences of recoverymeans were compared by least significant difference (LSD) meth-ods at a confidence level of P < 0.05 using the statistical package,GenStat (version 8.2, Rothamsted Experimental Station).

3. Results and discussion

3.1. Evaluation of the efficacy of extraction solutions to recoverimidazolinone herbicides from soil

There were no significant differences found (P < 0.05) betweenlupin-amended and non-amended treatments within the soils forthe extraction of imazapyr, imazethapyr and imazaquin. All sol-vent systems gave more than 70% recovery of herbicides from soil(Table 5). However, there were significant differences (P < 0.05)between extraction solutions in their ability to extract the herbi-cides from soil.

The lowest mean recovery of imazapyr, imazethapyr and imaza-quin from non-amended soil was found using 0.1 M KCl. Therecoveries were 73.3, 76.8 and 88.3%, respectively, which could bedue to the low pH (5.9) of this extraction solution. Low extractionefficiency of water for the extraction of imidazolinone herbicidesfrom soil has been noted previously and has been attributed to thelow pH of the extraction solution [32].

The pKa values of the selected imidazolinone herbicides range

from 3.6 to 3.9 (Table 2), so the use of extraction solutions withalkaline pH would increase solubility by deprotonating the herbi-cides. Furthermore, at pH 5.9 the anionic form of these herbicideswill tend to be repulsed from the colloids produced by suspensionof the humic acids [33]. The use of alkaline aqueous solutions for

M. Ramezani et al. / J. Chromatogr. A 1216 (2009) 5092–5100 5097

Table 5Evaluation of different solutions for extraction of imidazolinone herbicides from soila.

Extractant Mean recovery (%) ± standard deviation

Imazapyr Imazethapyr Imazaquin

Amended Non-amended Amended Non-amended Amended Non-amended

0.1 M KCl 81.2 ± 9.2a 73.3 ± 7.8a 80.2 ± 12.7a 76.8 ± 10.6a 91.3 ± 9.1a 88.3 ± 5.4a0.01 M NaOH 89.5 ± 2.8b 87.9 ± 2.8a 87.6 ± 3.1b 86.6 ± 3.1ab 95.2 ± 4.1ab 93.2 ± 4.1ab0.5 M NaOH:MeOH (80:20) 92.6 ± 2.4cb 90.6 ± 2.4 b 80.9 ± 2.9a 79.9 ± 2.9ab 105.7 ± 3.2c 102.6 ± 3.2c0 93.4

t (P < 0

ttsuo

iff0ihb

cbwfiaptfn

3iu

idPpiwh

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was similar to that obtained using 100% DCM. Furthermore, thecoloured substances that caused the interfering peaks on the chro-matogram were still present. Likewise, there was no improvementin the recovery efficiency when a PPL sorbent with a bigger sorbent

.5 M NaOH 96.8 ± 3.6c 93.4 ± 5.8bc

a Mean recoveries with different letters within columns are significantly differen

he extraction of acidic herbicides is desirable because it minimiseshe use of harmful solvents and is simple to prepare. Additionally,uch a solution permits the use of non-polar SPE sorbents for clean-p of the extracts without the extra step of dilution or evaporationf solvents.

The 0.1 M KCl solution had significantly lower (P < 0.05) recover-es than the 0.5 M NaOH and 0.5 M NaOH:MeOH (80:20) solutionsor imazapyr and imazaquin in non-amended soils. No dif-erence was observed between the recoveries obtained using.5 M NaOH:MeOH (80:20) and 0.5 M NaOH for imazapyr and

mazethapyr, although for imazaquin a significantly (P < 0.05)igher recovery was obtained with 0.5 M NaOH:MeOH (80:20) inoth amended and non-amended soils.

There was no significant difference between the extraction effi-iency of 0.01 M and 0.5 M NaOH for imazethapyr and imazaquin,ut greater reproducibility of extraction efficiency was observedhen 0.5 M NaOH was used as extraction solution. The major dif-culty encountered using 0.5 M NaOH was co-extraction of humicnd fulvic acids from the soil, which generated a large unresolvedeak at the beginning of the chromatograms. The optimal condi-ions for the extraction of imazapyr, imazethapyr and imazaquinrom soil was a soil:solution ratio of 1:4 using 0.5 M NaOH (dataot shown).

.2. Optimisation of solution pH for the extraction ofmidazolinone herbicides from water and humic acids solutionsing PPL sorbents

The optimum solution pH at which imidazolinone herbicides aren the appropriate form for retention by the PPL sorbent was initiallyetermined using imazapyr in water. The retention of imazapyr onPL sorbents rapidly decreased to less than 10% with increasingH from 2 to 5.5 and decreased further at pH 9 (Fig. 3), indicat-

ng that maximum retention of the imazapyr on the PPL sorbentas obtained when the sample solution was acidified to below theerbicide pKa (3.6).

The recoveries of three herbicides from acidified humic acidolutions ranged from 94.3% to 123.4% with average RSD of value.9%. The percent recoveries above 100% were related to a matrixnhancement effect, which has been reported for imidazolinoneerbicides and other pesticides. For example, it has been reportedhat sample matrix effects increased instrument response by as

uch as 200% over the pure standards for the samples spiked withulfonylurea, imidazolinone and sulfonamide herbicides [34]. Thismpact is greater for polar pesticides and is influenced by manyactors including pesticide nature, the kind of matrix, and the con-entration of matrix and/or pesticides [35]. The chromatograms ofhe three imidazolinone herbicides obtained after clean-up with

PL sorbent shows the interfering peaks around 2.9 min that madeesolution of imazapyr difficult (Fig. 4a).

The PPL sorbent was highly effective for the extraction of imaza-uin enantiomers in pure water and in the presence of humic acids.he recovery of the S(−) enantiomer of imazaquin from the PPL

± 5.6c 91.2 ± 8.2b 98.2 ± 4.8b 96.7 ± 7.2b

.05).

sorbent was 98.7% from Milli-Q water and 103.5% from 30 �g mL−1

of humic acid solution. The recovery of the R(+) enantiomer was99.8% from Milli-Q water and 107.8% from 30 �g mL−1 of humicacid solution.

3.3. Optimisation of extraction of imidazolinone herbicides from0.5 M NaOH soil extracts using PPL sorbents

Since high recoveries were found for the extraction of imida-zolinone herbicides from water and humic acid solution using PPLsorbents, this cartridge was tested for the extraction of these her-bicides from soil extracts. After extraction of the imidazolinoneherbicides from soil with 0.5 M NaOH, as described earlier, theclear supernatants were passed through the PPL sorbents and theneluted using 5 mL DCM. The chromatogram for the 0.5 M NaOHsoil extract (spiked at 1 �g g−1) and 10 �g mL−1 of standard solu-tion of herbicides are shown in Fig. 4b. The chromatogram of theDCM extract eluted from the PPL sorbent showed interfering peaksbetween 2 and 3.8 min where imazapyr eluted (Fig. 4b), which madequantification of this herbicide difficult. Recoveries of imazapyr,imazethapyr and imazaquin from 0.5 M NaOH soil extracts usingPPL sorbent were significantly different (P < 0.05) at 10.9%, 69.3%and 75.4%, respectively.

To decrease the effect of soil co-extractives on imazapyr resolu-tion and to improve herbicide recovery, several solvents were testedin the elution step. None of the solvent mixtures (DCM:isopropanol(80:20), ethyl acetate:isopropanol (80:20) and HEX:isopropanol(80:20)) improved the recovery for the three herbicides, which

Fig. 3. Effect of sample pH on retention of imazapyr by PPL sorbent (n = 3).

5098 M. Ramezani et al. / J. Chromatogr

Fig. 4. Chromatogram of imazapyr, imazethapyr and imazaquin (a) after elutionfrom the PPL sorbent. The three herbicides were initially in a humic acid solution(New

b(

3e

tpfitft

30 �g mL−1); (b) in a standard solution (10 �g mL−1) (dotted line) and in a 0.5 MaOH soil extract cleaned up with the PPL sorbent (solid line); and (c) in 0.5 M NaOHxtract of a spiked soil sample (1 �g g−1) after chromatographic mode sequencingith a C18 and a SCX sorbent.

ed (6 mL, 500 mg) was used, compared with the smaller sorbent3 mL, 200 mg) (data not shown).

.4. Extraction of imidazolinone herbicides from NaOH soilxtracts using X-AW sorbents at alkaline pH

Some researchers have reported anion exchange sorbents forhe extraction of acidic herbicides with no acidification at neutralH [28]. Others have used strong anion exchange resin sorbents

or isolating 12 herbicides including imazapyr, imazethapyr andmazaquin from surface and ground water [34]. They indicatedhat this sorbent removed much of the dissolved organic carbonrom the sample and imidazolinone herbicides were retained onhe sorbent. Other extraction procedures for removing imidazoli-

. A 1216 (2009) 5092–5100

none herbicides from soil have acidified the extraction solutionto approximately pH 2 to obtain good retention of the herbicideson the SPE sorbents [32,33]. However, acidification of the solu-tion pH is an additional step in the extraction procedure that istime-consuming and can cause the precipitation of co-extractedsubstances, leading to loss of resolution of the imidazolinone her-bicides in chromatography.

Owing to large interferences close to the retention time ofimazapyr (2.9 min, Fig. 4b) and low recoveries of imazapyr andimazethapyr using the PPL sorbent, an anion exchange sorbent(X-AW) was assessed for its efficacy to retain the herbicides. Themain objective of this experiment was to develop a method to usefor the extraction of imazapyr, imazethapyr and imazaquin fromsoil extracts with no pH adjustment before loading onto the SPEsorbent. Therefore, this experiment used 0.01 M NaOH instead of0.5 M NaOH for extraction of the herbicides. The X-AW sorbentcontains a polymer-based anion exchange sorbent with base par-ticles of styrene divinylbenzene (SDB), which is modified with adiamine group. The supplier recommends this sorbent for extrac-tion of weak acids, which can be loaded at the pH range of 6–8, asthese sorbents are more retentive for polar acidic herbicides thansilica [28].

The soil extracts were loaded directly onto the X-AW sorbentwith no pH adjustment because the pH value of 0.01 M NaOH wasalways less than 10. Although the size of the interfering peaks atthe retention time of imazapyr (2.92 min) decreased when X-AWsorbent was used, there was still a significant unresolved peakat the beginning of the chromatograms after the clean-up. Therecovery results of three imidazolinone herbicides using the X-AWsorbent were significantly (P < 0.05) different and were 15.6 ± 12.3%,43.8 ± 9.8% and 52.8 ± 11.8% for imazapyr, imazethapyr and imaza-quin, respectively (Table 6).

The recovery of the three herbicides, particularly imazapyr, wasgreatly increased by decreasing the sample loading flow rate from3 mL min−1 to 0.5 mL min−1, since decreasing the flow rate wouldlead to increased contact time between the herbicides and the sor-bent. At the slower flow rate the recovery for imazapyr increasedfrom 15.6% to 32.2%, whereas for imazethapyr recovery increasedfrom 43.8% to 56.8% and for imazaquin from 52.8% to 61.3%.Although the use of the X-AW sorbent was found to be beneficialto the clean-up of the sample matrix, it appeared to be insuf-ficient for effective extraction of imidazolinone herbicides fromsoil solutions. Consequently systems employing chromatographicmode sequencing using two SPE sorbents that offer different reten-tion mechanisms were assessed for their efficacy for removing theimidazolinone herbicides from the soil extracts.

3.5. Extraction of imidazolinone herbicides from NaOH soilextracts using chromatographic mode sequencing with NH2 andPPL sorbents

A previous study using NH2 for the extraction of a sulfony-lurea herbicide, triasulfuron from amended soils found that thissorbent eliminated much of the co-extracted organic carbon fromthe sample [36]. The authors reported average recovery of triasul-furon from compost-amended soils for this method of 86.8 ± 0.9%.In the current study the NH2 sorbent retained co-extracting humicsubstances from 0.5 M NaOH solution at pH 2, while the imida-zolinone herbicides were not retained on this sorbent. Therefore,0.5 M NaOH soil extracts were initially loaded onto the NH2 sor-bent and then passed through the PPL sorbent. Although there

was a considerable extraneous peak at the beginning of the chro-matograms when this clean-up was used, no interfering peaks wereobserved on the chromatogram at the retention times of the her-bicides of interest (data not shown). The recovery of imazapyr atthe concentration of 1 �g g−1 was 64.5%. Recovery was higher for

M. Ramezani et al. / J. Chromatogr. A 1216 (2009) 5092–5100 5099

Table 6Recovery of imidazolinone herbicides in the extracts of soils spiked at 1 �g g−1 (n = 3) after passing through different SPE sorbents.

SPE sorbent(s) Imazapyr Imazethapyr Imazaquin

Recovery (%) RSD (%) Recovery (%) RSD (%) Recovery (%) RSD (%)

PPL 10.9a 9.2 69.3a 8.4 75.4a 8.9X-AW 15.6a 12.3 43.8b 9.8 52.8b 11.8N 76.6cC 89.7d

M .05).

itPi

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eSamewt8Tn

arbtcuhfstrawphawe

4

eTtrNPtanceo

[

[

[[[[

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[

[

[[[[[[

H2 + PPL 64.5b 10.618 + SCX 85.4c 7.2

ean recoveries with different letter within column are significantly different (P < 0

mazethapyr at 76.6% and highest for imazaquin at 82.7%. Althoughhis was an improvement in % recovery compared with using thePL or X-AW sorbents alone the recoveries were still considerednadequate.

.6. Extraction of imidazolinone herbicides from NaOH soilxtracts using chromatographic mode sequencing with C18 andCX sorbents

The related compound, imazamox, has been successfullyxtracted from soil using a combination of C18 and cation exchangePE mechanisms [32]. Consequently a similar approach wasssessed in this study. It was found that using chromatographicode sequencing with a combination of C18 and SCX sorbents to

xtract imidazolinone herbicides from 0.5 M NaOH soil extractsas efficient: the mean recoveries from the soil extracts using

he C18 and SCX sorbents in series were 85.4 ± 7.2% for imazapyr,9.7 ± 5.8% for imazethapyr and 92.6 ± 8.2% for imazaquin (Table 6).he combination of sorbents was also the most effective in elimi-ating co-extractants (Fig. 4c).

The mechanism of the extraction following this protocol issequential combination of non-polar and cation exchange

etentions. The first extraction on C18 retains the imidazolinone her-icides and a variety of other, mostly anionic, organic matter fromhe soil while inorganic material and small, highly polar organicompounds pass through unretained. Under the elution conditionssed (1:1 MeOH:water) organic compounds including the analytes,umic acids and other moderately polar analytes are eluted while

atty acids and other lipid-like species are left uneluted on the C18orbent. By placing a SCX sorbent directly below the C18 at the elu-ion stage, conditions are set-up for a very selective cation exchangeetention to occur. Consequently, any species co-eluted with thenalytes will only retain on the SCX if it exists in a 50% MeOH/50%ater mixture as a pure cation. This simple set-up therefore com-letely eliminates the bulk of co-extracted components such asumic acids, tannins and sugars. Final elution from the SCX yieldedclean extract suitable from the sandy loam for further analysis,hether the original sample was largely mineral or highly organic-

nriched.

. Conclusion

In this study the efficacy of different aqueous solutions forxtraction of imidazolinone herbicides from soils was evaluated.he recovery of imazapyr, imazethapyr and imazaquin was greaterhan 70% in the different extraction solutions assessed. The bestecoveries and most reproducible results were obtained with 0.5 MaOH. Evaluation of different solid-phase sorbents showed that thePL sorbent is an efficient sorbent for isolation and quantification ofhese herbicides in water and humic-amended solutions when used

t pH 2. This sorbent can be used for the extraction of imidazoli-one herbicides from aqueous solutions including those with highoncentrations of humic substances. When used with NaOH soilxtracts, a number of co-extracted substances were also retainedn the sorbent, which decreased the recovery of imazethapyr and

[[[

[

12.3 82.7c 9.75.8 92.6d 8.2

imazaquin and made it difficult to quantify imazapyr. Using chro-matographic mode sequencing which involves two SPE sorbentsin series allowed removal of the co-extracting substances, whichresulted in high recovery of herbicides and clear quantification. Theuse of chromatographic mode sequencing with C18 and SCX sor-bents provided consistently high (>85%) herbicide recovery withlow RSD. This system may be used with confidence in the quantifi-cation of imidazolinone herbicides. This study also demonstratedthat PPL sorbents were effective for the extraction of imazaquinenantiomers (>95% recovery) from water even when humic acidswere present.

Acknowledgements

The senior author is grateful to the Agricultural Research andEducation Organisation of Iran for funding his studies in Australia.The authors also wish to thank Mr Michael Karkkainen for assis-tance with organic analyses.

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