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Soil column extraction followed by liquid chromatography and electrospray ionization mass spectrometry for the efficient determination of aryloxyphenoxypropionic herbicides in soil samples at ng g−1 levels
Analytica Chimica Acta 375 (1998) 107±116
Soil column extraction followed by liquid chromatography and electrospray ionization mass spectrometry for the ef®cient determination of aryloxyphenoxypropionic herbicides in soil samples at ng gÿ1 levels Aldo LaganaÁ*, Giovanna Fago, Aldo Marino, Matteo Mosso Department of Chemistry, La Sapienza University, P.le Aldo Moro 5, 00185 Rome, Italy Received 5 March 1998; received in revised form 19 June 1998; accepted 28 June 1998
Abstract An analytical method is described for determination of ng gÿ1 levels of aryloxyphenoxypropionic herbicides in soil, which combines a simple technique for sample preparation and liquid chromatography/electrospray ionization mass spectrometry with negative ion mode of operation. This method involves a simple treatment of the sample via isolation and trace enrichment by soil column extraction (SCE) and clean-up with a Carbograph-1 cartridge. The effects of various extractant solutions and soil type, test of extractability for aged ®eld-treated soil and pesticide concentration on the performance of the SCE are investigated. Overall, the recoveries for each herbicide across all forti®ed samples (1±50 ng gÿ1) were 893.5% for ¯uazifop, 903.3% for haloxyfop and 893.1% for diclofop. To achieve good liquid chromatographic separation, acidi®cation of the eluent and subsequent postcolumn addition of a neutralization buffer are needed to avoid ion signal suppression. The detection limits (S/N3) obtained using the time-scheduled selected ion monitoring varied from 0.1 to 0.3 ng gÿ1. # 1998 Elsevier Science B.V. All rights reserved. Keywords: Liquid chromatography; Electrospray ionization; Mass spectrometry; Aryloxyphenoxypropionic herbicides; Soil column extraction
1. Introduction Aryloxyphenoxypropionic (ArPP) acids are a new class of herbicides used for the selective removal of most grass species. These compounds are most effective for post-emergence (foliar) application [1]. As *Corresponding author. Present address: Dipartimento di Chimica, UniversitaÁ ``La Sapienza'', Box no. 34-Roma 62, Piazzale Aldo Moro 5, 00185 Roma, Italy. fax: +39-6-490631; e-mail: [email protected] 0003-2670/98/$19.00 # 1998 Elsevier Science B.V. All rights reserved. PII S0003-2670(98)00461-9
shown in Fig. 1, the members of this class of herbicides have similarly structured features centered around a phenoxypropionic acid moiety and an attached aromatic system bearing a halogen moiety. Commercially available as ester derivatives in soil and in plants, the esters undergo fast hydrolysis to the free acids. Rapid farmer acceptance of post-emergence herbicides (POE) can be ascribed to several factors, such as reduced use of chemicals, improved weed control ef®ciency, environmental and safety bene®ts,
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Fig. 1. Structures and common names of aryloxyphenoxypropionic herbicides.
and soil conservation. In addition, recent advances in information systems for predicting crop damage on the basis of weed density, location, and soil type will favor the increased use of such compounds. A POE herbicide can be de®ned as a foliage-applied product used to control weeds which have emerged in competition with the developing crop. However, many of these herbicides still retain residual activity in the soil and can thus control late-germination weeds. There are relatively few methods available for the determination of ArPP herbicides in soil matrixes and the majority refer to the determination of ¯uazifop alone, the ®rst ArPP to appear on the market. Therefore, the procedures for isolation from soil described in the literature are usually based on the alkaline treatment of the samples, thus isolating the residues of the free acid together as a salt, followed by suitable analytical methods for the acid, usually liquid±liquid extraction and subsequent determination by means of liquid chromatography (LC) [2±4] or gas chromatography, after suitable derivatization [5]. Recently Zanco et al. [6] developed an analytical procedure based on solid phase extraction technology for the clean-up and concentration of Soxhlet soil extracts
containing ¯uazifop which involved the use of a phenyl-phase cartridge. Although sensitive, the method proved extremely complicated. In pesticide residue analysis sample preparation can be a time-consuming and error-prone step in the analytical procedure. In the case of soil samples the availability of an extraction procedure which suf®ciently isolates the pesticide residues from the matrix is crucial. A wide variety of methods and solvents have been used to extract pesticides from soil, including agitation of the soil with a solvent for various periods of time at various temperatures [7,8], sonication [9] and Soxhlet extraction [10]. More recently, the use of supercritical ¯uid extraction (SFE) [11±13], microwave-assisted extraction (MAE) [14±16] and accelerated solvent extraction (ASE) [17] have been investigated. ASE is a new extraction procedure that uses an organic solvent at high pressures and temperatures above the boiling point. Environmental analytical chemists are constantly seeking to improve procedures for determining pesticides, primarily by reducing analysis time and increasing accuracy and sensitivity. This paper gives the ®rst details of the soil column extraction (SCE) technique and the effect of experimental parameters on recovery. Soil type, extractant solvent, tests of extractability from samples with aged residue and pesticide concentrations were investigated to assess the effect of experimental conditions on the performance of the SCE. As the aim of the work was to attain lower levels of detection while still maintaining suf®ciently high selectivity in order to unequivocally identify and quantify selected target ArPPs in soil samples, we chose the combined techniques of LC with electrospray-mass spectrometry (ES-MS) under selected ion monitoring (SIM) conditions to detect three target ArPP herbicides isolated from soil samples. 2. Experimental 2.1. Chemicals Authentic aryloxyphenoxypropionic acids, namely ¯uazifop, (RS)-2-[4-(5-tri¯uoromethyl-2-pyridyloxy) phenoxy]propionic acid, haloxyfop, (RS)-2-[4-(3chloro-5-tri¯uoromethyl-2-pyridyloxy)phenoxy]pro-
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pionic acid, and diclofop, (RS)-2-[4-(2,4-dichlorophenoxy)phenoxy]propionic acid, were purchased from LabService (Bologna, Italy). For LC, distilled water was further puri®ed by passing it through a Milli-Q RG apparatus (Millipore, Bedford, MA). Methanol and acetonitrile of LC gradient grade were from Carlo Erba (Milan, Italy). Tri¯uoroacetic acid (TFA) and formic acid were purchased from Merck (Darmstadt, Germany). All other solvents were of reagent grade (Carlo Erba) and were used as received. Carbograph1, a graphitized carbon black (GCB), was kindly supplied by Carbochimica Romana (Rome). The particle size range was 37±150 mm. No particular precautions were taken in storing the GCB. The GCB extraction cartridges were prepared by ®lling large diameter (6 cm1.3 cm i.d.) syringe-like polypropylene tubes (Supelco, Bellefonte, PA) with 1 g of adsorbing material. Polyethylene frits were placed above and below the sorbent bed. Before processing the samples, the cartridge was washed with 6 ml of methylene chloride/methanol (80:20, v/v) acidi®ed with 50 mmol lÿ1 formic acid, 2 ml of methanol, and 15 ml of water acidi®ed with hydrochloric acid (pH2). 2.2. Preparation of ArPP standards The primary standard solutions at a concentration of 1 mg mlÿ1 were prepared by dissolving 100 mg of each ArPP in 100 ml of acetonitrile. Dilution (1:1000) of this standard solution was made using acetone to obtain a working standard solution (1 mg mlÿ1). Analytical standards for LC/ES-MS calibration were prepared from portions of the working standard solution by evaporating the solvent to dryness in a stream of nitrogen and dissolving the residue in acetonitrile/ water (80:20, v/v) acidi®ed with 100 mmol lÿ1 formic acid.
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2.3. Soils Soil samples were taken from three sites in Italy at depths of 0±30 cm. Bulk soil samples were ground and passed through a 2 mm sieve to remove stones and plant material. The soil was stored in a freezer prior to use. The three sampled soils consisted of loam from sites near Rome and Bologna. The pipette method of particle size analysis [18] was performed on each soil. Each soil was also analyzed for total organic carbon [19], cation-exchange capacity [20], and soil pH using a 1:1 soil/water ratio. The soils used were different in their organic matter content, soil pH levels, sand, silt, and clay content, and all three were characteristic of the Mediterranean area. The main characteristics of the three soils selected for this study are presented in Table 1. 2.4. Soil sample fortification Freshly forti®ed samples were prepared by adding an appropriate volume of standard working solution to 5 g of dried homogenized soil samples. Additional acetone was added until the solvent completely covered the soil particles. The bulk of the solvent was slowly evaporated to an air-dried level. The mixture was then thoroughly mixed for 1 h in a mechanical shaker. Samples with aged residues were prepared by spiking 50 g soil samples with an appropriate volume of a working standard. After overnight air drying, the sample was stored in the laboratory at ambient temperature. 2.5. Instrumental conditions Liquid chromatography was carried out with a Perkin-Elmer series 250 binary pump (Perkin-Elmer, Norwalk, CT) equipped with a Rheodyne 7125 injec-
Table 1 Physical and chemical properties of three common Italian soils Soil
Pomaia Coltano Cadriano
Particle size analysis Sand (wt%)
Silt (wt%)
Clay (wt%)
22.6 35.8 45.3
39.8 14.6 23.6
37.6 49.6 31.1
Organic matter content (wt%)
pH
Cation exchange capacity (meq/100 g of soil)
3.8 13.6 1.4
7.6 5.2 6.8
29.37 18.1 26.1
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tor having a 100 ml loop. The analytes were chromatographed on a 25 cm4.6 mm i.d. column ®lled with 5 mm (average particle size) LC-18 packing and a Supelguard precolumn, 2 cm4.6 mm i.d., both supplied by Supelco (Bellefonte, PA). Components were separated isocratically using a mobile phase of acetonitrile/water (80:20, v/v) acidi®ed with 100 mmol lÿ1 formic acid. The ¯ow rate of the mobile phase was 1 ml minÿ1. A 50 ml portion of column ef¯uent was diverted to the ES source. On leaving the column, a solution containing 50 mmol lÿ1 ammonium acetate (pH8.7) at a ¯ow rate of 0.05 ml minÿ1 was added to the mobile phase ¯ow which was directed towards the MS interface using an infusion pump (Perkin-Elmer). In this way, it was possible to partially neutralize the acidity of the mobile phase and to increase the ionization signal strength of the analytes. Electrospray MS was performed on a Perkin-Elmer/ Sciex API I single-stage quadrupole instrument equipped with an Ionspray interface (Sciex, Thornton, Canada). The mass spectrometer was operated in negative ion mode by applying a voltage of 4000 V to the capillary. The ori®ce voltage was set at 90 V and the interface temperature at 628C. Nitrogen was used as a curtain gas with a ¯ow rate of 1.1 l minÿ1 and air as a nebulizer gas with a pressure setting of 46 psi. Mass spectra collected in full-scan mode were obtained by scanning over the range 140±410 m/z in 2.6 s. Chromatograms were recorded under timescheduled SIM conditions; the selected m/z and the acquisition window used were respectively, 288±360 and 0±5.0 min, for haloxyfop, 254±326 and 5.0± 6.5 min, for ¯uazifop, and 254±326 and 6.5± 8.5 min, for diclofop. Peak area ratios for selected ions were determined automatically using the PE Sciex package MacQuan 1.1.2. 2.6. Soil column extraction and SPE clean-up A 5 g portion of soil was weighed onto aluminium foil and transferred quantitatively into an empty 61.3 cm cartridge (Supelco). The ArPP herbicides were extracted from the soil column by washing with 25 ml of methanol/ammonium acetate±ammonia (0.1 mol lÿ1, pH10) (50:50, v/v). The extract was diluted to 25 ml in a volumetric ¯ask and 10 ml of the extract was passed through a prepared 1 g Carbo-
graph-1 cartridge. The cartridge was then washed with 10 ml of water and 1 ml of methanol. After the cartridge was air-dried for 1 min a suitably drilled cylindrical PTFE piston with a conical indented base and a Luer tip was forced into the cartridge until it reached the upper frit. The cartridge was turned upside down, and back washed by passing through it 1 ml of methanol and 12 ml of methylene chloride/methanol (80:20, v/v), respectively. The ArPPs were then eluted with 6 ml of methylene chloride/methanol (80:20, v/v) acidi®ed with formic acid (50 mmol lÿ1). The extract was dried at 408C under a stream of nitrogen. The residue was reconstituted with 250 ml of acetonitrile/ water (80:20, v/v) acidi®ed with formic acid (100 mmol lÿ1). A 50 ml portion of the ®nal extract was injected into the LC column. 3. Results and discussion 3.1. General remarks In this study, soil type, solvent extraction system, tests of extractability from samples with aged residue (where the analyte had weeks to bind to active sites in the soil matrix), and pesticide concentration were evaluated to study the in¯uence of speci®c factors on the analytical procedure. ArPP herbicides selected for study were based on moderate estimated half-times (DT50) which ranged from six weeks for ¯uazifop to two to four months for diclofop and haloxifop. To judge the ef®ciency of the analytical procedure, recovery experiments were performed by spiking soil with different quantities of ArPP. Previous studies have indicated that reliable recovery data cannot be obtained from freshly spiked samples only [21]. Forti®cation procedures do not necessarily give a good indication of extraction ef®ciencies because it is more dif®cult to remove pesticides from soils following ®eld treatment than from a forti®ed soil sample. Therefore, extraction procedures evaluated by forti®cation should be considered critically. In the light of the above arguments, we tested the extractabililty of the ArPPs from aged soil samples. Since ArPPs are weak acids they exist primarily as anions in soil with pH>5. Iron and/or aluminium oxides and hydroxides, present in the soil, are mainly responsible for several anionic herbicides adsorption
A. LaganaÁ et al. / Analytica Chimica Acta 375 (1998) 107±116
[22±25]. Shea [26] postulated that weak acid herbicides adsorption is due to H-bonding with soil organic matter. Similarly ArPP anionic species can form interactions due to charge transfer bonds between their benzene or pyridyloxy substituents and organic matter in the soil. Interactions can even take place at the clay exchange surface because of the presence of impurities or for ligand exchange between ArPP carbonyl group and water molecules involved in the cation hydration. 3.2. Soil column extraction The selection of the extraction process may be one of the most important factors in the optimization of ArPP analysis, therefore several solvent systems were tested with respect to both extraction ef®ciency and compatibility with the overall procedure. Since several studies have shown that the mobility of herbicides of the weak acid type increases with increasing soil pH [27], we decided to evaluate two common organic solvents used in soil analysis and different aqueous buffered solutions of pH between that of water and strong bases on the three soils forti®ed to a level of 10 ng gÿ1. The data shown in Table 2 point to the importance of achieving a pH
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greater than 7 to maximize ArPP extractability, and pH10, 0.1 mol lÿ1 ammonium acetate±ammonia was selected for the remainder of the study in order to validate the method. Within organic solvents (Table 3) acetonitrile resulted in no analyte recovery. It is likely that in the absence of water, the carboxyl group of ArPP may form hydrogen bonds with exchange sites of soil compounds. The acetonitrile is unable to displace ArPP, from the soil surface because of its low capacity for forming hydrogen bonds, which renders it inef®cient in the competition for desorption. Satisfactory recoveries were obtained using extractants (methanol or aqueous buffers) able to attach themselves quite strongly via hydrogen bonds to the acidic adsorption sites of the exchanger and thus compete for desorption. Moreover methanol/ ammonium acetate±ammonia (0.1 mol lÿ1, pH10) (50:50, v/v) maximized ArPP extractability from the three different soils. The differences in composition of the three soil samples investigated did not appreciably in¯uence the results of the extractability experiments. Soil samples were spiked with an appropriate standard solution to give forti®ed samples at 1, 10, and 50 ng gÿ1 to investigate the effect of herbicide concentration on recovery. The results (Table 4) indicate
Table 2 Evaluation of different extractants from spiked (10 ng gÿ1) soils Soil
Mean recoverya (%)RSDb CH3OH
CH3CN
H2 O
NH4OAc±NH3 (0.1 M pH10)
CH3OH/NH4OAc±NH3 (0.1 M pH10) (50:50, v/v)
Pomaia FLZ HAL DICL
723.3 753.7 763.1
0 0 0
552.7 592.4 503.0
813.6 774.0 583.2
924.1 963.1 902.1
Coltano FLZ HAL DICL
683.5 702.7 712.8
0 0 0
522.6 523.1 482.8
823.8 712.9 522.9
872.1 903.2 882.5
Cadriano FLZ HAL DICL
753.7 793.4 773.0
0 0 0
602.5 552.7 572.9
884.0 823.2 652.8
983.6 943.8 934.1
FLZfluazifop; HALhaloxyfop; DICLdiclofop. Mean values from six determinations. b Relative standard deviation. a
A. LaganaÁ et al. / Analytica Chimica Acta 375 (1998) 107±116
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Table 3 Evaluation of ammonium acetate/ammonia extractant at different pH values Soil
Mean recoverya (%) RSDb
Table 5 Mean recovery (%)relative standard deviation (n6) from samples of Pomaia soil fortified at 10 ng gÿ1 level extracted after different times Compound
Recovery (%)RSD
pH7
pH8
pH9
pH10
pH11
Pomaia FLZ HAL DICL
692.9 653.2 502.5
693.4 693.5 463.4
802.8 742.2 502.5
813.3 772.8 583.6
803.9 753.7 533.3
Fluazifop Haloxifop Diclofop
Coltano FLZ HAL DICL
652.4 613.3 452.7
693.2 673.9 472.9
743.9 673.7 502.4
823.1 712.9 522.6
803.8 703.0 522.2
cessed as described in Section 2. These studies did not reveal any great differences in ArPP extractability as shown in Table 5.
Cadriano FLZ HAL DICL
703.3 682.7 552.7
682.9 702.4 542.9
773.2 813.7 602.5
883.8 823.5 653.0
863.5 843.6 633.1
3.3. SPE clean-up of extracts
FLZfluazifop; HALhaloxyfop; DICLdiclofop. a Mean values from six determinations. b Relative standard deviation.
no signi®cant in¯uence of analyte concentration on recovery and show that irreversible adsorption at very low herbicide concentrations did not take place on the surfaces of the sorbent and the other materials constituting the trap. Overall, the recoveries expressed as the averagerelative standard deviation (RSD) (n6) for each herbicide across all forti®ed samples were 893.5% for ¯uazifop, 903.3% for haloxyfop and 893.1% for diclofop. Samples with an aged residue were analyzed to simulate recovery from actual ®eld samples. Samples of soil forti®ed at the 10 ng gÿ1 level were extracted at different times (0, 1, 8, 15 and 30 days) with the mixture of methanol/ammonium acetate±ammonia (0.1 M pH10) (50:50, v/v) and subsequently pro-
0
1 day
8 days
15 days
30 days
924.1 963.1 902.1
893.7 912.9 883.2
902.6 903.3 882.4
924.0 943.5 913.7
863.4 893.7 853.5
The authors recently used Carbograph-1, a graphitized carbon black, for ArPP determination in aqueous samples [28]. The adsorption of these substances took place with samples having a neutral pH. Coextraction of interfering substances was potentially larger when the Carbograph-1 cartridge was used than with a polymeric sorbent. Since, the tested solvent with best results for soil was found to be the mixture containing 50% methanol and 50% aqueous buffered solution, the effect of %methanol and the pH on the retention capacity of a Carbograph-1 cartridge was also examined. Recovery of ArPP herbicide was determined from a solution of methanol/ammonium acetate± ammonia (0.1 mol lÿ1 pH10) (50:50, v/v) containing variable concentrations of the pesticides investigated. Using a Carbograph-1 cartridge extraction, preconcentration and fractionation into classes were possible by means of differential elution. The presence of active centers bearing a positive charge enabled Carbograph-1 to behave as both a nonspeci®c and an
Table 4 Mean recovery (%)relative standard deviation (n6) from fortified soil samples at different spike levels Compound
Recovery (%)RSD Pomaia (ng gÿ1)
Fluazifop Haloxifop Diclofop
Coltano (ng gÿ1)
Cadriano (ng gÿ1)
1
10
50
1
10
50
1
10
50
803.7 822.9 833.2
924.1 963.1 902.1
933.7 943.3 953.0
793.9 802.7 813.2
872.1 903.2 882.5
903.0 953.7 883.6
863.3 843.6 853.5
983.6 943.8 934.1
964.1 973.6 952.9
A. LaganaÁ et al. / Analytica Chimica Acta 375 (1998) 107±116
anion exchange sorbent. Anionic ArPP herbicides were speci®cally adsorbed on the Carbograph-1 surface via electrostatic forces and they could be desorbed only by adding a displacing agent such as formic acid to the organic solution. Carbograph can be used in a manner similar to ion exchangers with the stepwise elution of acidic compounds on the basis of their pKa by properly selecting displacing agents. 3.4. LC/ES-MS analysis The method previously reported [28] for the determination of six ArPPs in water samples involved a gradient elution in order to obtain the best separation with an overall run time of 25 min. This was found to be unnecessary to separate the three compounds considered here and the isocratic conditions used allowed the total run time to be reduced to about 8 min. For determination of acidic compounds the addition of a modi®er to the LC eluent served to improve separation conditions. In LC/ES-MS, such modi®ers may signi®cantly affect the type of molecular ion species observed as well as the signal intensity. It was possible to obtain an improvement in sensitivity in the determination of acidic herbicides by LC/ES-MS in the NI mode by the postcolumn addition of a base [29], tripropylamine. Postcolumn pH neutralization was required in order to form ions in solution and to facilitate the charging of droplets. Weak ion signals for the analytes considered were obtained when the LC/ES-MS instrumentation was operated using a C18 silica column with an acidic mobile phase (100 mM formic acid). Equimolar amounts of tripropylamine or different quantities of ammonium acetate were assessed with a view to optimizing the signal intensity. This set of experiments was performed in the full-scan mode by setting the ori®ce voltage to 90 V and scanning the quadrupole mass ®lter from 140 to 410 m/z in 2.6 s scanÿ1 and injecting 40 ng of each ArPP. The total ion current for each analyte was monitored for signal-to-noise ratio (S/N) by measuring peak heights against the average background noise. Measurements were made in triplicate. Contradictory results have been reported by various authors [30,31] concerning the effect of analyte ion intensity when large acid concentrations are added to the mobile phase. Ikonomou et al. [30] observed a decrease in the ion signal on the addition of a strong acid concentration in excess of
113
10 mmol lÿ1 to the electrosprayed solution. Our results, on the other hand, are in agreement with those of Zhou and Hamburger [31] who observed a steady increase in ion intensity upon increasing the formic acid concentration upto 25 mmol lÿ1. The best results were obtained using an eluent ¯ow rate of 1 ml minÿ1 and keeping the postcolumn addition of buffer to a ¯ow rate of 0.05 ml minÿ1, where ammonium acetate completely neutralizes the mobile phase. Increasing acetate concentration produced no signi®cant increase in sensitivity [32] and decreased the robustness of the method. At neutral pH, the herbicides are ionized in order to promote [MÿH]ÿ anion formation. By LC/ES-MS, each ArPP generated an [MÿH]ÿ ion as a base peak for the entire group of acidic herbicides. In addition, characteristic fragment ions from moieties of the ArPP group and adducts with formate and acetate were formed under the ES conditions applied: i.e. [MÿCH3CH2COO]ÿ, ÿ [MÿHCOO] and [MCH3COO]ÿ ions. The tentatively identi®ed ions formed and their relative abundance obtained by LC/ES-MS in the NI mode are shown in Table 6. For an unambiguous identi®cation of these compounds in environmental samples, it is recommended that at least two structure-signi®cant ions be used for compounds during SIM experiments Table 6 Main ions and their relative abundance (RA) for ArPP herbicides under ES-MS using NI mode of operationa Compound
Mwb
m/z and tentative ions
Haloxifop
361
360, 288, 406, 420,
[MÿH]ÿ [MÿCH3CH2COO]ÿ [MHCOO]ÿ [MCH3COO]ÿ
100 21 18 11
Fluazifop
327
326, 254, 372, 386,
[MÿH]ÿ [MÿCH3CH2COO]ÿ [MHCOO]ÿ [MCH3COO]ÿ
100 17 10 10
Diclofop
327
326, 254, 372, 386,
[MÿH]ÿ [MÿCH3CH2COO]ÿ [MHCOO]ÿ [MCH3COO]ÿ
100 15 12 7
a
RA
Conditions: extraction voltage, 90 V; carrier stream, CH3CN/H2O (80:20, v/v) acidified with HCOOH, 100 mmol lÿ1, postcolumn addition of CH3COONH4 50 mM (pH8.7). b Nominal mass.
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[33]. In our case, the quasi-molecular ion and the [Mÿ72] ion were used; the latter can be attributed to the losses of the propionic carboxylic acid moiety. The linear dynamic range and the absolute sensitivity of the ES-MS detector were estimated by injecting in triplicate a known, variable amount of ArPP and measuring the peak area ratios of the chromatograms relative to m/z 288, 360 (haloxyfop), 254, 326 (¯uazifop) and 254, 326 (diclofop). The response was linear for all compounds in the concentration range 0.2±40 ng mlÿ1 and the relative slopes and R2 values (n5) were respectively, 1.57 and 0.998 for haloxyfop, 2.57 and 0.999 for ¯uazifop, and 1.97 and 0.998 for diclofop. The variability of triplicates ranged from 6.2% to 9.8%. The signal-to-noise ratio (S/N) from the lowest injected amount of ArPP suggests the absolute limits of detection (S/N3) of the ES-MS detector
were about 0.1 ng for ¯uazifop, and 0.2 ng for haloxifop and diclofop. 3.5. Soil analysis Soil samples from three different sources were spiked with the appropriate standard solution to give forti®ed samples at 10 ng gÿ1. A typical chromatogram depicting full scan conditions for the Pomaia soil is shown in Fig. 2. Quanti®cation was performed by external calibration under SIM conditions. Considering the sample preparation procedure involved in the method, the limit of detection (LOD) was calculated using a signal-to-noise ratio of 3 (the ratio of the peak intensity under SIM conditions and the intensity of the noise was used) and varied from 0.1 to 0.3 ng gÿ1 for the three herbicides. A chromatogram using SIM
Fig. 2. Chromatogram obtained from Pomaia soil sample spiked with the ArPP herbicides at the level of 10 ng gÿ1 each under full-scan conditions. (1) fluazifop, (2) haloxyfop and (3) diclofop. For the list of ions monitored, see Table 2. For other experimental conditions, see Section 2.
A. LaganaÁ et al. / Analytica Chimica Acta 375 (1998) 107±116
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Fig. 3. Chromatogram obtained from Pomaia soil sample spiked with the ArPP herbicides at the level of 1 ng gÿ1 each using time scheduled SIM conditions under NI mode. (1) fluazifop, (2) haloxyfop and (3) diclofop. For the experimental conditions, see Section 2.
under NI mode for the Pomaia soil sample (Fig. 3) spiked at 1 ng gÿ1 is close to the detection limit. The sensitivity is improved over previous methods since no satisfactory analytical method has been available for the determination of ArPPs at concentration levels as low as 0.1 ng gÿ1 in soils. 4. Conclusions A simple isolation and enrichment procedure and LC/ES-MS method has been developed to routinely quantify ArPP at low ng gÿ1 concentrations. The method gave reproducible and accurate results for three different soil samples. The sensitivity and selectivity of SCE followed by LC/ES-MS for ArPP analysis greatly reduces the amount of extract that must be processed through clean-up procedures. As a consequence, the complexity of the analytical method and the sample preparation time are reduced. Use of SCE makes the sample preparation method ef®cient and economical.
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[14] V. Lopez-Avila, R. Young, W.F. Beckert, Anal. Chem. 66 (1994) 1097. [15] V. Lopez-Avila, R. Young, N. Teplitsky, J. AOAC Int. 79 (1996) 142. [16] C. Molins, E.A. Hogendoorn, H.A.G. Heusinkveld, D.C. van Harten, P. van Zoonen, R.A. Baumann, Chromatographia 43 (1996) 527. [17] B.E. Richter, B.A. Jones, J.L. Ezzel, N.L. Porter, Anal. Chem. 68 (1996) 1033. [18] P.R. Dy, in: C.A. Black et al. (Eds.), Methods of Soil Analysis, Agron. Monogr. 9 ASA, CSSA, and SSSA, Part 1, Madison, WI, 1965. [19] J.D. Snyder, J.A. Trofymow, Commun. Soil Sci. Plant Anal. 15 (1984) 587. [20] H.D. Chapman, in: C.A. Black et al. (Eds.), Methods of Soil Analysis, Agron. Monogr. 9 ASA, CSSA, and SSSA, Part 1, Madison, WI, 1965. [21] H. Steinwandter, Fresenius' Z. Anal. Chem. 327 (1987) 309. [22] G. Wehtje, R. Dickens, J.W. Wilcut, B.F. Hajek, Weed Science 35 (1987) 858.
[23] D.A. O'Connor, J.U. Anderson, Soil Sci. Soc. Am. Proc. 38 (1974) 433. [24] R.J. Atkinson, A.M. Posner, J.P. Quirk, J. Phys. Chem. 71 (1967) 550. [25] C.J.B. Mott, in: D.J. Greenland, M.B.H. Hayes (Eds.), The Chemistry of Soil Processes, Wiley, New York, 1981, p. 179. [26] P.J. Shea, Weed Science 34 (1986) 474. [27] R.H. Veeh, W.P. Inskeep, F.L. Roe, A.H. Ferguson, J. Environ. Qual. 23 (1994) 542. [28] A. LaganaÁ, G. Fago, A. Marino, J. Chromatogr. 796 (1998) 309. [29] S. Chiron, S. Papilloud, W. Haerdi, D. BarceloÂ, Anal. Chem. 67 (1995) 1637. [30] M.G. Ikonomou, A.T. Blades, P. Kebarle, Anal. Chem. 63 (1991) 1989. [31] S. Zhou, M. Hamburger, Rapid Commun. Mass Spectrom. 9 (1995) 1516. [32] G. Wang, R.B. Cole, Anal. Chem. 66 (1994) 3702. [33] A. Di Corcia, C. Crescenzi, E. Guerriero, R. Samperi, Environ. Sci. Technol. 31 (1997) 1658.
Soil column extraction followed by liquid chromatography and electrospray ionization mass spectrometry for the ef®cient determination of aryloxyphenoxypropionic herbicides in soil samples at ng gÿ1 levels Aldo LaganaÁ*, Giovanna Fago, Aldo Marino, Matteo Mosso Department of Chemistry, La Sapienza University, P.le Aldo Moro 5, 00185 Rome, Italy Received 5 March 1998; received in revised form 19 June 1998; accepted 28 June 1998
Abstract An analytical method is described for determination of ng gÿ1 levels of aryloxyphenoxypropionic herbicides in soil, which combines a simple technique for sample preparation and liquid chromatography/electrospray ionization mass spectrometry with negative ion mode of operation. This method involves a simple treatment of the sample via isolation and trace enrichment by soil column extraction (SCE) and clean-up with a Carbograph-1 cartridge. The effects of various extractant solutions and soil type, test of extractability for aged ®eld-treated soil and pesticide concentration on the performance of the SCE are investigated. Overall, the recoveries for each herbicide across all forti®ed samples (1±50 ng gÿ1) were 893.5% for ¯uazifop, 903.3% for haloxyfop and 893.1% for diclofop. To achieve good liquid chromatographic separation, acidi®cation of the eluent and subsequent postcolumn addition of a neutralization buffer are needed to avoid ion signal suppression. The detection limits (S/N3) obtained using the time-scheduled selected ion monitoring varied from 0.1 to 0.3 ng gÿ1. # 1998 Elsevier Science B.V. All rights reserved. Keywords: Liquid chromatography; Electrospray ionization; Mass spectrometry; Aryloxyphenoxypropionic herbicides; Soil column extraction
1. Introduction Aryloxyphenoxypropionic (ArPP) acids are a new class of herbicides used for the selective removal of most grass species. These compounds are most effective for post-emergence (foliar) application [1]. As *Corresponding author. Present address: Dipartimento di Chimica, UniversitaÁ ``La Sapienza'', Box no. 34-Roma 62, Piazzale Aldo Moro 5, 00185 Roma, Italy. fax: +39-6-490631; e-mail: [email protected] 0003-2670/98/$19.00 # 1998 Elsevier Science B.V. All rights reserved. PII S0003-2670(98)00461-9
shown in Fig. 1, the members of this class of herbicides have similarly structured features centered around a phenoxypropionic acid moiety and an attached aromatic system bearing a halogen moiety. Commercially available as ester derivatives in soil and in plants, the esters undergo fast hydrolysis to the free acids. Rapid farmer acceptance of post-emergence herbicides (POE) can be ascribed to several factors, such as reduced use of chemicals, improved weed control ef®ciency, environmental and safety bene®ts,
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Fig. 1. Structures and common names of aryloxyphenoxypropionic herbicides.
and soil conservation. In addition, recent advances in information systems for predicting crop damage on the basis of weed density, location, and soil type will favor the increased use of such compounds. A POE herbicide can be de®ned as a foliage-applied product used to control weeds which have emerged in competition with the developing crop. However, many of these herbicides still retain residual activity in the soil and can thus control late-germination weeds. There are relatively few methods available for the determination of ArPP herbicides in soil matrixes and the majority refer to the determination of ¯uazifop alone, the ®rst ArPP to appear on the market. Therefore, the procedures for isolation from soil described in the literature are usually based on the alkaline treatment of the samples, thus isolating the residues of the free acid together as a salt, followed by suitable analytical methods for the acid, usually liquid±liquid extraction and subsequent determination by means of liquid chromatography (LC) [2±4] or gas chromatography, after suitable derivatization [5]. Recently Zanco et al. [6] developed an analytical procedure based on solid phase extraction technology for the clean-up and concentration of Soxhlet soil extracts
containing ¯uazifop which involved the use of a phenyl-phase cartridge. Although sensitive, the method proved extremely complicated. In pesticide residue analysis sample preparation can be a time-consuming and error-prone step in the analytical procedure. In the case of soil samples the availability of an extraction procedure which suf®ciently isolates the pesticide residues from the matrix is crucial. A wide variety of methods and solvents have been used to extract pesticides from soil, including agitation of the soil with a solvent for various periods of time at various temperatures [7,8], sonication [9] and Soxhlet extraction [10]. More recently, the use of supercritical ¯uid extraction (SFE) [11±13], microwave-assisted extraction (MAE) [14±16] and accelerated solvent extraction (ASE) [17] have been investigated. ASE is a new extraction procedure that uses an organic solvent at high pressures and temperatures above the boiling point. Environmental analytical chemists are constantly seeking to improve procedures for determining pesticides, primarily by reducing analysis time and increasing accuracy and sensitivity. This paper gives the ®rst details of the soil column extraction (SCE) technique and the effect of experimental parameters on recovery. Soil type, extractant solvent, tests of extractability from samples with aged residue and pesticide concentrations were investigated to assess the effect of experimental conditions on the performance of the SCE. As the aim of the work was to attain lower levels of detection while still maintaining suf®ciently high selectivity in order to unequivocally identify and quantify selected target ArPPs in soil samples, we chose the combined techniques of LC with electrospray-mass spectrometry (ES-MS) under selected ion monitoring (SIM) conditions to detect three target ArPP herbicides isolated from soil samples. 2. Experimental 2.1. Chemicals Authentic aryloxyphenoxypropionic acids, namely ¯uazifop, (RS)-2-[4-(5-tri¯uoromethyl-2-pyridyloxy) phenoxy]propionic acid, haloxyfop, (RS)-2-[4-(3chloro-5-tri¯uoromethyl-2-pyridyloxy)phenoxy]pro-
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pionic acid, and diclofop, (RS)-2-[4-(2,4-dichlorophenoxy)phenoxy]propionic acid, were purchased from LabService (Bologna, Italy). For LC, distilled water was further puri®ed by passing it through a Milli-Q RG apparatus (Millipore, Bedford, MA). Methanol and acetonitrile of LC gradient grade were from Carlo Erba (Milan, Italy). Tri¯uoroacetic acid (TFA) and formic acid were purchased from Merck (Darmstadt, Germany). All other solvents were of reagent grade (Carlo Erba) and were used as received. Carbograph1, a graphitized carbon black (GCB), was kindly supplied by Carbochimica Romana (Rome). The particle size range was 37±150 mm. No particular precautions were taken in storing the GCB. The GCB extraction cartridges were prepared by ®lling large diameter (6 cm1.3 cm i.d.) syringe-like polypropylene tubes (Supelco, Bellefonte, PA) with 1 g of adsorbing material. Polyethylene frits were placed above and below the sorbent bed. Before processing the samples, the cartridge was washed with 6 ml of methylene chloride/methanol (80:20, v/v) acidi®ed with 50 mmol lÿ1 formic acid, 2 ml of methanol, and 15 ml of water acidi®ed with hydrochloric acid (pH2). 2.2. Preparation of ArPP standards The primary standard solutions at a concentration of 1 mg mlÿ1 were prepared by dissolving 100 mg of each ArPP in 100 ml of acetonitrile. Dilution (1:1000) of this standard solution was made using acetone to obtain a working standard solution (1 mg mlÿ1). Analytical standards for LC/ES-MS calibration were prepared from portions of the working standard solution by evaporating the solvent to dryness in a stream of nitrogen and dissolving the residue in acetonitrile/ water (80:20, v/v) acidi®ed with 100 mmol lÿ1 formic acid.
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2.3. Soils Soil samples were taken from three sites in Italy at depths of 0±30 cm. Bulk soil samples were ground and passed through a 2 mm sieve to remove stones and plant material. The soil was stored in a freezer prior to use. The three sampled soils consisted of loam from sites near Rome and Bologna. The pipette method of particle size analysis [18] was performed on each soil. Each soil was also analyzed for total organic carbon [19], cation-exchange capacity [20], and soil pH using a 1:1 soil/water ratio. The soils used were different in their organic matter content, soil pH levels, sand, silt, and clay content, and all three were characteristic of the Mediterranean area. The main characteristics of the three soils selected for this study are presented in Table 1. 2.4. Soil sample fortification Freshly forti®ed samples were prepared by adding an appropriate volume of standard working solution to 5 g of dried homogenized soil samples. Additional acetone was added until the solvent completely covered the soil particles. The bulk of the solvent was slowly evaporated to an air-dried level. The mixture was then thoroughly mixed for 1 h in a mechanical shaker. Samples with aged residues were prepared by spiking 50 g soil samples with an appropriate volume of a working standard. After overnight air drying, the sample was stored in the laboratory at ambient temperature. 2.5. Instrumental conditions Liquid chromatography was carried out with a Perkin-Elmer series 250 binary pump (Perkin-Elmer, Norwalk, CT) equipped with a Rheodyne 7125 injec-
Table 1 Physical and chemical properties of three common Italian soils Soil
Pomaia Coltano Cadriano
Particle size analysis Sand (wt%)
Silt (wt%)
Clay (wt%)
22.6 35.8 45.3
39.8 14.6 23.6
37.6 49.6 31.1
Organic matter content (wt%)
pH
Cation exchange capacity (meq/100 g of soil)
3.8 13.6 1.4
7.6 5.2 6.8
29.37 18.1 26.1
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tor having a 100 ml loop. The analytes were chromatographed on a 25 cm4.6 mm i.d. column ®lled with 5 mm (average particle size) LC-18 packing and a Supelguard precolumn, 2 cm4.6 mm i.d., both supplied by Supelco (Bellefonte, PA). Components were separated isocratically using a mobile phase of acetonitrile/water (80:20, v/v) acidi®ed with 100 mmol lÿ1 formic acid. The ¯ow rate of the mobile phase was 1 ml minÿ1. A 50 ml portion of column ef¯uent was diverted to the ES source. On leaving the column, a solution containing 50 mmol lÿ1 ammonium acetate (pH8.7) at a ¯ow rate of 0.05 ml minÿ1 was added to the mobile phase ¯ow which was directed towards the MS interface using an infusion pump (Perkin-Elmer). In this way, it was possible to partially neutralize the acidity of the mobile phase and to increase the ionization signal strength of the analytes. Electrospray MS was performed on a Perkin-Elmer/ Sciex API I single-stage quadrupole instrument equipped with an Ionspray interface (Sciex, Thornton, Canada). The mass spectrometer was operated in negative ion mode by applying a voltage of 4000 V to the capillary. The ori®ce voltage was set at 90 V and the interface temperature at 628C. Nitrogen was used as a curtain gas with a ¯ow rate of 1.1 l minÿ1 and air as a nebulizer gas with a pressure setting of 46 psi. Mass spectra collected in full-scan mode were obtained by scanning over the range 140±410 m/z in 2.6 s. Chromatograms were recorded under timescheduled SIM conditions; the selected m/z and the acquisition window used were respectively, 288±360 and 0±5.0 min, for haloxyfop, 254±326 and 5.0± 6.5 min, for ¯uazifop, and 254±326 and 6.5± 8.5 min, for diclofop. Peak area ratios for selected ions were determined automatically using the PE Sciex package MacQuan 1.1.2. 2.6. Soil column extraction and SPE clean-up A 5 g portion of soil was weighed onto aluminium foil and transferred quantitatively into an empty 61.3 cm cartridge (Supelco). The ArPP herbicides were extracted from the soil column by washing with 25 ml of methanol/ammonium acetate±ammonia (0.1 mol lÿ1, pH10) (50:50, v/v). The extract was diluted to 25 ml in a volumetric ¯ask and 10 ml of the extract was passed through a prepared 1 g Carbo-
graph-1 cartridge. The cartridge was then washed with 10 ml of water and 1 ml of methanol. After the cartridge was air-dried for 1 min a suitably drilled cylindrical PTFE piston with a conical indented base and a Luer tip was forced into the cartridge until it reached the upper frit. The cartridge was turned upside down, and back washed by passing through it 1 ml of methanol and 12 ml of methylene chloride/methanol (80:20, v/v), respectively. The ArPPs were then eluted with 6 ml of methylene chloride/methanol (80:20, v/v) acidi®ed with formic acid (50 mmol lÿ1). The extract was dried at 408C under a stream of nitrogen. The residue was reconstituted with 250 ml of acetonitrile/ water (80:20, v/v) acidi®ed with formic acid (100 mmol lÿ1). A 50 ml portion of the ®nal extract was injected into the LC column. 3. Results and discussion 3.1. General remarks In this study, soil type, solvent extraction system, tests of extractability from samples with aged residue (where the analyte had weeks to bind to active sites in the soil matrix), and pesticide concentration were evaluated to study the in¯uence of speci®c factors on the analytical procedure. ArPP herbicides selected for study were based on moderate estimated half-times (DT50) which ranged from six weeks for ¯uazifop to two to four months for diclofop and haloxifop. To judge the ef®ciency of the analytical procedure, recovery experiments were performed by spiking soil with different quantities of ArPP. Previous studies have indicated that reliable recovery data cannot be obtained from freshly spiked samples only [21]. Forti®cation procedures do not necessarily give a good indication of extraction ef®ciencies because it is more dif®cult to remove pesticides from soils following ®eld treatment than from a forti®ed soil sample. Therefore, extraction procedures evaluated by forti®cation should be considered critically. In the light of the above arguments, we tested the extractabililty of the ArPPs from aged soil samples. Since ArPPs are weak acids they exist primarily as anions in soil with pH>5. Iron and/or aluminium oxides and hydroxides, present in the soil, are mainly responsible for several anionic herbicides adsorption
A. LaganaÁ et al. / Analytica Chimica Acta 375 (1998) 107±116
[22±25]. Shea [26] postulated that weak acid herbicides adsorption is due to H-bonding with soil organic matter. Similarly ArPP anionic species can form interactions due to charge transfer bonds between their benzene or pyridyloxy substituents and organic matter in the soil. Interactions can even take place at the clay exchange surface because of the presence of impurities or for ligand exchange between ArPP carbonyl group and water molecules involved in the cation hydration. 3.2. Soil column extraction The selection of the extraction process may be one of the most important factors in the optimization of ArPP analysis, therefore several solvent systems were tested with respect to both extraction ef®ciency and compatibility with the overall procedure. Since several studies have shown that the mobility of herbicides of the weak acid type increases with increasing soil pH [27], we decided to evaluate two common organic solvents used in soil analysis and different aqueous buffered solutions of pH between that of water and strong bases on the three soils forti®ed to a level of 10 ng gÿ1. The data shown in Table 2 point to the importance of achieving a pH
111
greater than 7 to maximize ArPP extractability, and pH10, 0.1 mol lÿ1 ammonium acetate±ammonia was selected for the remainder of the study in order to validate the method. Within organic solvents (Table 3) acetonitrile resulted in no analyte recovery. It is likely that in the absence of water, the carboxyl group of ArPP may form hydrogen bonds with exchange sites of soil compounds. The acetonitrile is unable to displace ArPP, from the soil surface because of its low capacity for forming hydrogen bonds, which renders it inef®cient in the competition for desorption. Satisfactory recoveries were obtained using extractants (methanol or aqueous buffers) able to attach themselves quite strongly via hydrogen bonds to the acidic adsorption sites of the exchanger and thus compete for desorption. Moreover methanol/ ammonium acetate±ammonia (0.1 mol lÿ1, pH10) (50:50, v/v) maximized ArPP extractability from the three different soils. The differences in composition of the three soil samples investigated did not appreciably in¯uence the results of the extractability experiments. Soil samples were spiked with an appropriate standard solution to give forti®ed samples at 1, 10, and 50 ng gÿ1 to investigate the effect of herbicide concentration on recovery. The results (Table 4) indicate
Table 2 Evaluation of different extractants from spiked (10 ng gÿ1) soils Soil
Mean recoverya (%)RSDb CH3OH
CH3CN
H2 O
NH4OAc±NH3 (0.1 M pH10)
CH3OH/NH4OAc±NH3 (0.1 M pH10) (50:50, v/v)
Pomaia FLZ HAL DICL
723.3 753.7 763.1
0 0 0
552.7 592.4 503.0
813.6 774.0 583.2
924.1 963.1 902.1
Coltano FLZ HAL DICL
683.5 702.7 712.8
0 0 0
522.6 523.1 482.8
823.8 712.9 522.9
872.1 903.2 882.5
Cadriano FLZ HAL DICL
753.7 793.4 773.0
0 0 0
602.5 552.7 572.9
884.0 823.2 652.8
983.6 943.8 934.1
FLZfluazifop; HALhaloxyfop; DICLdiclofop. Mean values from six determinations. b Relative standard deviation. a
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Table 3 Evaluation of ammonium acetate/ammonia extractant at different pH values Soil
Mean recoverya (%) RSDb
Table 5 Mean recovery (%)relative standard deviation (n6) from samples of Pomaia soil fortified at 10 ng gÿ1 level extracted after different times Compound
Recovery (%)RSD
pH7
pH8
pH9
pH10
pH11
Pomaia FLZ HAL DICL
692.9 653.2 502.5
693.4 693.5 463.4
802.8 742.2 502.5
813.3 772.8 583.6
803.9 753.7 533.3
Fluazifop Haloxifop Diclofop
Coltano FLZ HAL DICL
652.4 613.3 452.7
693.2 673.9 472.9
743.9 673.7 502.4
823.1 712.9 522.6
803.8 703.0 522.2
cessed as described in Section 2. These studies did not reveal any great differences in ArPP extractability as shown in Table 5.
Cadriano FLZ HAL DICL
703.3 682.7 552.7
682.9 702.4 542.9
773.2 813.7 602.5
883.8 823.5 653.0
863.5 843.6 633.1
3.3. SPE clean-up of extracts
FLZfluazifop; HALhaloxyfop; DICLdiclofop. a Mean values from six determinations. b Relative standard deviation.
no signi®cant in¯uence of analyte concentration on recovery and show that irreversible adsorption at very low herbicide concentrations did not take place on the surfaces of the sorbent and the other materials constituting the trap. Overall, the recoveries expressed as the averagerelative standard deviation (RSD) (n6) for each herbicide across all forti®ed samples were 893.5% for ¯uazifop, 903.3% for haloxyfop and 893.1% for diclofop. Samples with an aged residue were analyzed to simulate recovery from actual ®eld samples. Samples of soil forti®ed at the 10 ng gÿ1 level were extracted at different times (0, 1, 8, 15 and 30 days) with the mixture of methanol/ammonium acetate±ammonia (0.1 M pH10) (50:50, v/v) and subsequently pro-
0
1 day
8 days
15 days
30 days
924.1 963.1 902.1
893.7 912.9 883.2
902.6 903.3 882.4
924.0 943.5 913.7
863.4 893.7 853.5
The authors recently used Carbograph-1, a graphitized carbon black, for ArPP determination in aqueous samples [28]. The adsorption of these substances took place with samples having a neutral pH. Coextraction of interfering substances was potentially larger when the Carbograph-1 cartridge was used than with a polymeric sorbent. Since, the tested solvent with best results for soil was found to be the mixture containing 50% methanol and 50% aqueous buffered solution, the effect of %methanol and the pH on the retention capacity of a Carbograph-1 cartridge was also examined. Recovery of ArPP herbicide was determined from a solution of methanol/ammonium acetate± ammonia (0.1 mol lÿ1 pH10) (50:50, v/v) containing variable concentrations of the pesticides investigated. Using a Carbograph-1 cartridge extraction, preconcentration and fractionation into classes were possible by means of differential elution. The presence of active centers bearing a positive charge enabled Carbograph-1 to behave as both a nonspeci®c and an
Table 4 Mean recovery (%)relative standard deviation (n6) from fortified soil samples at different spike levels Compound
Recovery (%)RSD Pomaia (ng gÿ1)
Fluazifop Haloxifop Diclofop
Coltano (ng gÿ1)
Cadriano (ng gÿ1)
1
10
50
1
10
50
1
10
50
803.7 822.9 833.2
924.1 963.1 902.1
933.7 943.3 953.0
793.9 802.7 813.2
872.1 903.2 882.5
903.0 953.7 883.6
863.3 843.6 853.5
983.6 943.8 934.1
964.1 973.6 952.9
A. LaganaÁ et al. / Analytica Chimica Acta 375 (1998) 107±116
anion exchange sorbent. Anionic ArPP herbicides were speci®cally adsorbed on the Carbograph-1 surface via electrostatic forces and they could be desorbed only by adding a displacing agent such as formic acid to the organic solution. Carbograph can be used in a manner similar to ion exchangers with the stepwise elution of acidic compounds on the basis of their pKa by properly selecting displacing agents. 3.4. LC/ES-MS analysis The method previously reported [28] for the determination of six ArPPs in water samples involved a gradient elution in order to obtain the best separation with an overall run time of 25 min. This was found to be unnecessary to separate the three compounds considered here and the isocratic conditions used allowed the total run time to be reduced to about 8 min. For determination of acidic compounds the addition of a modi®er to the LC eluent served to improve separation conditions. In LC/ES-MS, such modi®ers may signi®cantly affect the type of molecular ion species observed as well as the signal intensity. It was possible to obtain an improvement in sensitivity in the determination of acidic herbicides by LC/ES-MS in the NI mode by the postcolumn addition of a base [29], tripropylamine. Postcolumn pH neutralization was required in order to form ions in solution and to facilitate the charging of droplets. Weak ion signals for the analytes considered were obtained when the LC/ES-MS instrumentation was operated using a C18 silica column with an acidic mobile phase (100 mM formic acid). Equimolar amounts of tripropylamine or different quantities of ammonium acetate were assessed with a view to optimizing the signal intensity. This set of experiments was performed in the full-scan mode by setting the ori®ce voltage to 90 V and scanning the quadrupole mass ®lter from 140 to 410 m/z in 2.6 s scanÿ1 and injecting 40 ng of each ArPP. The total ion current for each analyte was monitored for signal-to-noise ratio (S/N) by measuring peak heights against the average background noise. Measurements were made in triplicate. Contradictory results have been reported by various authors [30,31] concerning the effect of analyte ion intensity when large acid concentrations are added to the mobile phase. Ikonomou et al. [30] observed a decrease in the ion signal on the addition of a strong acid concentration in excess of
113
10 mmol lÿ1 to the electrosprayed solution. Our results, on the other hand, are in agreement with those of Zhou and Hamburger [31] who observed a steady increase in ion intensity upon increasing the formic acid concentration upto 25 mmol lÿ1. The best results were obtained using an eluent ¯ow rate of 1 ml minÿ1 and keeping the postcolumn addition of buffer to a ¯ow rate of 0.05 ml minÿ1, where ammonium acetate completely neutralizes the mobile phase. Increasing acetate concentration produced no signi®cant increase in sensitivity [32] and decreased the robustness of the method. At neutral pH, the herbicides are ionized in order to promote [MÿH]ÿ anion formation. By LC/ES-MS, each ArPP generated an [MÿH]ÿ ion as a base peak for the entire group of acidic herbicides. In addition, characteristic fragment ions from moieties of the ArPP group and adducts with formate and acetate were formed under the ES conditions applied: i.e. [MÿCH3CH2COO]ÿ, ÿ [MÿHCOO] and [MCH3COO]ÿ ions. The tentatively identi®ed ions formed and their relative abundance obtained by LC/ES-MS in the NI mode are shown in Table 6. For an unambiguous identi®cation of these compounds in environmental samples, it is recommended that at least two structure-signi®cant ions be used for compounds during SIM experiments Table 6 Main ions and their relative abundance (RA) for ArPP herbicides under ES-MS using NI mode of operationa Compound
Mwb
m/z and tentative ions
Haloxifop
361
360, 288, 406, 420,
[MÿH]ÿ [MÿCH3CH2COO]ÿ [MHCOO]ÿ [MCH3COO]ÿ
100 21 18 11
Fluazifop
327
326, 254, 372, 386,
[MÿH]ÿ [MÿCH3CH2COO]ÿ [MHCOO]ÿ [MCH3COO]ÿ
100 17 10 10
Diclofop
327
326, 254, 372, 386,
[MÿH]ÿ [MÿCH3CH2COO]ÿ [MHCOO]ÿ [MCH3COO]ÿ
100 15 12 7
a
RA
Conditions: extraction voltage, 90 V; carrier stream, CH3CN/H2O (80:20, v/v) acidified with HCOOH, 100 mmol lÿ1, postcolumn addition of CH3COONH4 50 mM (pH8.7). b Nominal mass.
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[33]. In our case, the quasi-molecular ion and the [Mÿ72] ion were used; the latter can be attributed to the losses of the propionic carboxylic acid moiety. The linear dynamic range and the absolute sensitivity of the ES-MS detector were estimated by injecting in triplicate a known, variable amount of ArPP and measuring the peak area ratios of the chromatograms relative to m/z 288, 360 (haloxyfop), 254, 326 (¯uazifop) and 254, 326 (diclofop). The response was linear for all compounds in the concentration range 0.2±40 ng mlÿ1 and the relative slopes and R2 values (n5) were respectively, 1.57 and 0.998 for haloxyfop, 2.57 and 0.999 for ¯uazifop, and 1.97 and 0.998 for diclofop. The variability of triplicates ranged from 6.2% to 9.8%. The signal-to-noise ratio (S/N) from the lowest injected amount of ArPP suggests the absolute limits of detection (S/N3) of the ES-MS detector
were about 0.1 ng for ¯uazifop, and 0.2 ng for haloxifop and diclofop. 3.5. Soil analysis Soil samples from three different sources were spiked with the appropriate standard solution to give forti®ed samples at 10 ng gÿ1. A typical chromatogram depicting full scan conditions for the Pomaia soil is shown in Fig. 2. Quanti®cation was performed by external calibration under SIM conditions. Considering the sample preparation procedure involved in the method, the limit of detection (LOD) was calculated using a signal-to-noise ratio of 3 (the ratio of the peak intensity under SIM conditions and the intensity of the noise was used) and varied from 0.1 to 0.3 ng gÿ1 for the three herbicides. A chromatogram using SIM
Fig. 2. Chromatogram obtained from Pomaia soil sample spiked with the ArPP herbicides at the level of 10 ng gÿ1 each under full-scan conditions. (1) fluazifop, (2) haloxyfop and (3) diclofop. For the list of ions monitored, see Table 2. For other experimental conditions, see Section 2.
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Fig. 3. Chromatogram obtained from Pomaia soil sample spiked with the ArPP herbicides at the level of 1 ng gÿ1 each using time scheduled SIM conditions under NI mode. (1) fluazifop, (2) haloxyfop and (3) diclofop. For the experimental conditions, see Section 2.
under NI mode for the Pomaia soil sample (Fig. 3) spiked at 1 ng gÿ1 is close to the detection limit. The sensitivity is improved over previous methods since no satisfactory analytical method has been available for the determination of ArPPs at concentration levels as low as 0.1 ng gÿ1 in soils. 4. Conclusions A simple isolation and enrichment procedure and LC/ES-MS method has been developed to routinely quantify ArPP at low ng gÿ1 concentrations. The method gave reproducible and accurate results for three different soil samples. The sensitivity and selectivity of SCE followed by LC/ES-MS for ArPP analysis greatly reduces the amount of extract that must be processed through clean-up procedures. As a consequence, the complexity of the analytical method and the sample preparation time are reduced. Use of SCE makes the sample preparation method ef®cient and economical.
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