Multiresidue screening of neutral pesticides in water samples by high performance liquid chromatography–electrospray mass spectrometry

Analytica Chimica Acta 505 (2004) 209–215

Multiresidue screening of neutral pesticides in water samples by high performance liquid chromatography–electrospray mass spectrometry J.M.F. Nogueira a,∗ , Tom Sandra b , Pat Sandra b a Departamento de Qu´ımica e Bioqu´ımica and Centro de Ciˆ encias Moleculares e Materiais, Faculdade de Ciˆencias da Universidade de Lisboa, Campo Grande Ed. C8, 1749-016 Lisboa, Portugal b Research Institute for Chromatography, Kennedypark 20, B-8500 Kortrijk, Belgium

Received 23 June 2003; received in revised form 13 October 2003; accepted 23 October 2003

Abstract A multiresidue method to screen ultra traces of neutral pesticides from water samples in compliance with European Union regulations is presented, using solid-phase extraction (SPE) followed by high performance liquid chromatography coupled to atmospheric pressure electrospray ionisation (ESI) mass spectrometry, SPE/LC–MS(ESI). Calibration conditions of LC–MS(ESI) in the SIM mode, showed excellent linear response for the 12 pesticides studied (carbaryl, carbofuran, methomyl, oxamyl, pirimicarb, chlortoluron, diuron, isoproturon, linuron, atrazine, propazine and simazine) in the range from 1 to 50 ␮g/l and precisions having a relative standard deviation (R.S.D.) below 4.9% (chlortoluron) was achieved. Instrumental limits of detection of 0.10 ␮g/l were found with the exception of chlortoluron, diuron and methomyl, for which 0.25 and 0.50 ␮g/l were measured. The SPE assays using octadecilsilica cartridges are relatively simple and highly reproducible, requiring low volume of water sample (50 ml). For laboratory-spiked water samples having 0.10 ␮g/l of individual pesticides, good average recoveries were obtained, within 65.0 and 97.8% with a relative standard deviation lower than 12.6% (methomyl). For real matrices, laboratory-spiked drinking and surface water samples at the 0.05 ␮g/l level, reasonable average recoveries were also achieved, ranging from 75.3 to 96.8% and 67.7 to 105.2%, respectively. The present work establishes a suitable protocol as a guideline for research and routine laboratories, to screen simultaneously carbamates, phenylureas and triazines from water samples. The methodology evidences very good reproducibility and is shown to be a suitable alternative to replace the currently dedicated analytical systems. The limits of detection reached (0.5–3.0 ng/l) clearly cover the maximum concentration admissible for pesticides in water samples, established by the European Union directive. © 2003 Elsevier B.V. All rights reserved. Keywords: LC–MS(ESI); Solid-phase extraction; Pesticides; Carbamates; Phenylureas; Triazines; Water analysis; Trace analysis; European Union guideline

1. Introduction N-Methylcarbamates, phenylureas and triazines are neutral pesticides extensively used as herbicides, insecticides, nematicides, molluscicides and acaricides, which are considered to have persistency and toxicological effects in the environment and more recently demonstrated to be potential endocrine disrupters [1,2]. Levels of their residues in drinking and surface waters is of public concern and according to the European Union directive on water quality (98/83/CE), the maximum concen-

∗ Corresponding author. Tel.: +351-217-500899; fax: +351-217-500088. E-mail address: [email protected] (J.M.F. Nogueira).

0003-2670/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2003.10.065

tration admissible for pesticides is 0.10 ␮g/l for individual and 0.50 ␮g/l for the sum of them [3]. Currently methods to screen neutral pesticides from environmental water matrices, such as carbamates, phenylureas and triazines, require an enrichment step, usually liquid–liquid extraction or solid-phase extraction (SPE), prior to analysis by high performance liquid chromatography or gas chromatography methods [4–8]. Since some classes of neutral pesticides are somewhat polar and thermally labile for the traditional gas chromatographic methods, as carbamates and phenylureas, liquid chromatography is the analytical method of choice. For detection, ultraviolet detectors used to be employed and for carbamates in particular, fluorescence detection following post column derivatization to enhanced sensitivity and selectivity are widely used [9–11].

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The United States Environmental Protection Agency methods using liquid chromatography for the analysis of neutral pesticides such as carbamates and phenylureas from water matrices (EPA 531.1, EPA 553, EPA 8318, EPA 8321 A and EPA 8325), requires high sample water volume and employs fluorescence detection, as well as particle beam and thermospray mass spectrometry interfaces. Nevertheless, the detection limits reported usually meet the Health Advisory Levels from EPA Office of Ground and Drinking Water and those methods as well as other researching works [12,13], have not been optimised to reach the European Union guideline for pesticides at the 0.10 ␮g/l level. Over recent years, mass spectrometry is becoming the detection system recommended for liquid chromatography and atmospheric pressure electrospray ionisation (ESI) source in particular, presents substantial advantages since the samples can be ionised directly in the liquid phase at quasi-ambient temperature, minimising the degradation of thermally labile compounds [14–16]. No derivatization procedures are required and the occurrence of interfering substances is also minimised, providing high selectivity and spectral evidence of individual solutes. Furthermore, the major attraction of ESI applied to environmental analysis is the low detection limits that could be reach in parts-per-trillion (ng/l) level, in particular when it is combined with SPE [17]. In last few years, several applications to monitor particular classes of pesticides from environmental matrices had been proposed in the literature using LC–MS analytical systems [18–29]. However, new multiresidue methodologies which could screen as far as possible several classes of pesticides from water samples in only one run presents all advantages, especially if optimised conditions could be establish in compliance with European Union regulations. The aim of the present contribution is to establish a suitable protocol as a guideline to research and routine laboratories, which combines an optimised SPE/LC–MS(ESI) procedure, to screen simultaneously 12 neutral pesticides (carbamates, phenylureas and triazines) from drinking and surface water samples according to the European Union directive on water quality.

2. Experimental 2.1. Reagents, standards and samples N-Methylcarbamates (carbaryl, carbofuran, methomyl, oxamyl and pirimicarb) were supplied from Riedel-de-Haën, having purity higher than 99.5%. Phenylureas (chlortoluron, diuron, isoproturon and linuron) were supplied from Labor Dr. Ehrenstorfer, having a purity of 99.9%. Triazines (atrazine, propazine and simazine) were supplied from Labor Dr. Ehrenstorfer, having purities of 97.2, 99.1 and 99.5%, respectively. Fig. 1 shows the chemical structures of the pesticides studied.

Stock solutions of each pesticide having a concentration of 100 mg/l were prepared in methanol and stored at 4 ◦ C. Six calibration standard solutions of mixing pesticides, ranging from 1 to 50 ␮g/l each, were prepared in ultra-pure water by appropriate dilution of aliquots of the stock solutions. Laboratory-spiked water samples were prepared in 50 ml of ultra-pure, drinking and surface waters by appropriate dilution of aliquots of the stock solutions to a concentration ranging from 0.03 to 0.30 ␮g/l of each pesticide. For the SPE assays, a vacuum manifold (Agilent Technologies) and three types of cartridges were used: LC-18 (Supelco; 3 ml, 250 mg), Oasis HLB (Waters; 6 ml, 200 mg) and Bond Elut (Varian-Envi; 3 ml; 500 mg). HPLC-grade methanol, acetonitrile, ammonium acetate and ultra-pure water were obtained from Merck. Drinking and surface water samples were obtained from Kortrijk (Belgium). 2.2. Sample preparation For SPE assays, each cartridge was conditioned with 3 ml of methanol/acetonitrile (50/50, v/v), 3 ml of methanol and two times of 3 ml of ultra-pure water and slowly aspirated (−0.2 bar). After loaded with the water samples (−0.4 bar), the column was wash twice with 3 ml of ultra-pure water, followed by vacuum dry for 2 min. Subsequently, the elution took place with three times of 1 ml of methanol/acetonitrile (50/50, v/v), followed by evaporation to dryness under a gentle stream of nitrogen. The dry residues obtained from the SPE assays were redissolved in 0.2 ml of ultra-pure water and after agitation by vortex were analysed by LC–MS(ESI). For blank assays the same procedure as above was performed using water samples without spiking. 2.3. Instrumentation The analyses were carried out on a benchtop Agilent 1100 series LC–MS SL single quadrupole instrument (Agilent Technologies). An Inertsil ODS3 column, 100 mm×2.1 mm, 5 ␮m particle size (Zorbax Eclips XDB C18, Agilent Technologies) was used. The mobile phase consisted of 10% (v/v) ammonium acetate (10 mM) aqueous solution in methanol (solvent A) and 10% (v/v) methanol in ammonium acetate (10 mM) aqueous solution (solvent B), and the gradient applied was: 0–10 min: 10–90% B, 10–20 min: 90% B, isocratic. The flow rate was 0.25 ml min−1 , the analyses were performed at 25 ◦ C, and the injection volume was 50 ␮l, having a draw speed of 200 ␮l/min. Atmospheric pressure electrospray ionisation was carried out in the positive mode. In the full-scan mode the parameters were as follows: mass range from 100 to 500 u; fragmentor voltage 20–100 V; gain 1; N2 drying gas flow rate: 12 l min−1 at 350 ◦ C; nebulizer pressure: 35 psig; quadrupole temperature: 100 ◦ C; capillary voltage: 4000 V. Flow injection analyses (10 ␮l) were performed for individual pesticide solutions (10 mg/l) in order

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Fig. 1. Structures and common names of the neutral pesticides studied.

to obtained the mass spectral data, from which ions were careful chosen for analysis in the selected ion monitoring (SIM) mode, using the parameter conditions as above.

3. Results and discussion 3.1. Optimisation of the LC–MS(ESI) instrumentation Twelve neutral pesticides constituted by five carbamates (carbaryl, carbofuran, methomyl, oxamyl and pirimicarb), four phenylureas (chlortoluron, diuron, isoproturon and linuron) and three triazines (atrazine, propazine and simazine), were selected as model compounds for the present study.

To evaluate the best mass spectral data of each pesticide, flow injection analysis of individual standards, at fragmentor voltages of 20, 40, 60, 80 and 100 V were performed, to find the maximum response under the optimum ESI conditions. In the positive mode, the cone voltage at 60 V provided molecular mass information through the base peak, where the sensitivity was highest for almost all compounds studied. Under such experimental conditions, the full-scan mode provides mainly protonated, ammoniated and sodiated species in the mass spectra of almost all pesticides studied, according to previous works [13–15,28]. Protonated ions ([M + H]+ ) were the base peaks for carbofuran, methomyl, pirimicarb, chlortoluron, diuron, isoproturon, linuron, atrazine, propazine and simazine at m/z 222, 163, 239, 213, 233, 207, 249, 216, 230 and 202, respectively. For carbaryl

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and oxamyl, the base peaks were ammoniated adducts ([M + NH4 ]+ ) from ions at m/z 219 and 237, respectively. The separation of the neutral pesticides was carried out on a reverse phase column using a standard methanol– water gradient, showing suitable analytical time (<15 min) for which ammonium acetate was added to the mobile phase (10 mM) in order to stabilise the cationic species formation. In the full-scan mode, oxamyl/methomyl pair had sufficient baseline resolution, but other peak groups such as simazine/carbofuran, pirimicarb/chlortoluron/atrazine/isoproturon/diuron and propazine/linuron were not completely resolved under such conditions. Nevertheless, the low peak resolution did not constitute a difficulty since enough selectivity could be easily achieved for the identification and quantification in the SIM mode. From the abundance of the base peaks observed in the mass spectra of individual pesticides, calibration plots in the SIM mode were performed, using external standardisation. For carbaryl and oxamyl calibration plots, better linearity was achieved with [M + H]+ and [M + Na + 2 × H2 O]+ species [14] from ions at m/z 202 and 278, respectively, having relative abundances above 80%. Although the same target ion (m/z 202) was selected for simazine and carbaryl, it did not affect their quantification since both pesticides present different retention times. Good linear responses were achieved for three replicates, using calibration standard mixtures having 1, 2, 5, 10, 25 and 50 ␮g/l of each pesticide, presenting excellent correlation coefficients. Table 1 shows the molecular mass (Mr ) of each pesticide studied, the target ions selected at 60 V, the assignment of each species, as well as the corresponding correlation coefficients obtained from the regression plots. Furthermore, the standard solutions of pesticides mixtures were stable over one week without significant degradation.

In order to evaluate the instrumental precision, six replicates of a standard mixture at 10 ␮g/l level of each pesticide were carried out under optimum experimental conditions. The data obtained from the repeatability studies allowed a relative standard deviation (R.S.D.) below 4.9% (chlortoluron). The instrumental limit of detection (LOD) was studied through the injection of standard mixtures of pesticides in the SIM mode and calculated with a signal-to-noise (S/N) ratio of three. A LOD of 0.10 ␮g/l was achieved for all pesticides studied with the exception of methomyl, chlortoluron and diuron, for which 0.50 and 0.25 ␮g/l were measured, respectively (Table 1). Thus, a sensitivity ranging from 5 to 25 pg on-column was found for the 12 pesticides studied, under the optimised experimental conditions. 3.2. Multiresidue analysis of laboratory-spiked water samples SPE is currently the sample preparation method of choice for enrichment of pollutants from water samples. In our study, laboratory-spiked water samples of mixed pesticides having individual concentrations at 0.03 and 0.30 ␮g/l were used to survey the SPE assays. In a first approach, cartridges having solid phases with different polarities, such as octadecilsilica (Supelco LC-18), N-vinylpyrrolidane divinylbenzene (Waters Oasis HLB) and polystyrene-divinylbenzene (Varian-Envi Bond Elut), were tested to evaluate the best recoveries, under the experimental conditions used. Preliminary assays indicated acceptable average recoveries for the pesticides studied in those cartridges, for which octadecilsilica shows better effective results (76–111%) than N-vinylpyrrolidane divinylbenzene (30–123%) and polystyrene-divinylbenzene (17–125%), as shown in Table 2.

Table 1 Molecular mass (Mr ), assignment of the target ions used in the calibration plots by LC–MS(ESI) in the SIM mode, correlation coefficients and instrumental LODs achieved for the 12 pesticides studied r2 (1–50 ␮g/l)c

LOD (␮g/l)d

+ H]+ + H]+ + H]+ + Na + 2 × H2 O]+ + H]+

0.9998 0.9993 0.9989 0.9999 0.9989

0.10 0.10 0.50 0.10 0.10

+ H]+ + H]+ + H]+ + H]+

0.9948 0.9989 0.9988 0.9976

0.25 0.25 0.10 0.10

[M + H]+ [M + H]+ [M + H]+

0.9989 0.9994 0.9994

0.10 0.10 0.10

Class

Pesticide

Mr

Ion (m/z)

Assignation (species)

Carbamates

Carbaryl Carbofuran Methomyl Oxamyl Pirimicarb

201 221 162 219 238

202b 222a 163a 278b 239a

[M [M [M [M [M

Phenylureas

Chlortoluron Diuron Isoproturon Linuron

212 232 206 248

213a 233a 207a 249a

[M [M [M [M

Triazines

Atrazine Propazine Simazine

215 229 201

216a 230a 202a

a b c d

Base peak. Relative abundance higher than 80%. SIM mode, n = 3. S/N=3.

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Table 2 Average recoveries of two assays using several SPE cartridges followed by LC/MS(ESI) in SIM mode for the 12 neutral pesticides from laboratory-spiked ultra-pure water samples at the 0.03 and 0.30 ␮g/l levels Class; n = 2

Pesticide

Recovery (%) Varian-Envi (Bond Elut)

Supelco (LC-18)

Waters (Oasis HLB)

Carbamates

Carbaryl Carbofuran Methomyl Oxamyl Pirimicarb

82.0 108.8 120.0 90.3 32.9

(48.4) (51.2) (87.3) (89.6) (15.8)

111.2 93.7 84.1 82.8 99.1

(94.8) (84.9) (85.6) (76.3) (97.6)

53.0 72.5 80.4 66.4 65.0

(30.1) (60.8) (85.4) (78.5) (72.8)

Phenylureas

Chlortoluron Diuron Isoproturon Linuron

74.4 75.2 125.0 72.1

(35.9) 35.7) (35.3) (30.9)

81.5 91.6 103.0 80.0

(84.3) (106.3) (108.4) (100.7)

93.6 80.0 85.0 92.4

(75.2) (95.3) (94.9) (99.8)

Triazines

Atrazine Propazine Simazine

54.1 (30.1) 27.1 (17.2) 66.9 (47.1)

86.8 (91.7) 89.8 (87.9) 84.7 (89.1)

84.3 (82.5) 123.2 (82.9) 92.5 (83.6)

Values in brackets are obtained at the 0.30 ␮g/l level.

The optimised SPE procedure proved to be very simple, sensitive and not much volume of water sample is required (50 ml), evidencing to be a suitable routine alternative in relation to other sample preparation methods established to screen ultra-traces of pesticides. Because the enrichment factor used is 250 (from 50 to 0.2 ml), LODs in water samples drop easily to the low ng/l level, reaching the European Union guideline for pesticides on water samples at the 0.10 ␮g/l level. To evaluate the accuracy of the present multiresidue method, three replicates of laboratory-spiked water samples, having a concentration for individual pesticides at the 0.10 ␮g/l level were analyzed. Screening assays using octadecilsilica cartridges under the optimised experimental conditions showed good robustness, allowed average recoveries between 65.0 and 97.8% with a relative standard deviation lower than 12.6% (methomyl) for the 12 neutral pesticides studied (Table 3). Octadecilsilica cartridges promoted substantial precision for ultra-trace enrichment of those neutral pesticides from water samples in compliance with European Union regulations. Fig. 2 provides an example of a typical mass fragmentograms for the 12 pesticides studied by SPE/LC–MS(ESI) in the SIM mode, from a laboratory-spiked ultra-pure water sample at the 0.10 ␮g/l level under the conditions used. The present methodology showed substantial selectivity for ultra-trace screening of the neutral pesticides in water samples, where 12 discrete fragmentograms of individual compounds could be observed at the 0.10 ␮g/l level. Moreover, blank assays showed no relevant interference substances, in particular residues from the cartridges tested. In order to evaluate the present multiresidue method in real matrices, assays with laboratory-spiked drinking and surface water samples having the mixture of individual pesticides at the 0.05 ␮g/l level, were carried out

Table 3 Average recoveries and relative standard deviation (R.S.D.) of three assays using SPE enrichment (Supelco LC-18) followed by LC–MS(ESI) in the SIM mode, for the 12 neutral pesticides from laboratory-spiked ultra-pure water samples at the 0.10 ␮g/l level Class; n = 3

Pesticide

Recovery (% ± R.S.D.)

Carbamates

Carbaryl Carbofuran Methomyl Oxamyl Pirimicarb

89.5 76.3 68.3 65.0 91.7

± ± ± ± ±

2.2 8.1 12.6 11.8 2.1

Phenylureas

Chlortoluron Diuron Isoproturon Linuron

89.9 87.4 97.8 70.2

± ± ± ±

6.8 6.4 6.3 7.0

Triazines

Atrazine Propazine Simazine

85.0 ± 6.1 77.9 ± 3.6 81.5 ± 4.1

using octadecilsilica cartridges (Supelco LC-18) under the optimised experimental conditions. Control assays using laboratory-spiked ultra-pure water samples were simultaneously performed. Average recoveries were good, ranging from 64.7 to 98.2%, 75.3 to 96.8% and 67.7 to 113.9% for laboratoryspiked ultra-pure, drinking and surface water samples, respectively (Table 4). In summary, the present methodology [SPE/LC–MS(ESI)] could be established as a suitable protocol for the simultaneous screening of different classes of neutral pesticides such as carbamates, phenylureas and triazines from real water samples. Good reproducibility and high sensitivity could be obtained with LODs between 0.5 and 3.0 ng/l, which more than adequately covers the maximum concentration admissible for pesticides in water samples according to European Union directive.

214 J.M.F. Nogueira et al. / Analytica Chimica Acta 505 (2004) 209–215 Fig. 2. LC–MS(ESI) in the SIM mode showing the mass fragmentograms of the 12 neutral pesticides from a laboratory-spiked ultra-pure water sample at the 0.10 ␮g/l level, after SPE enrichment (Supelco LC-18).

J.M.F. Nogueira et al. / Analytica Chimica Acta 505 (2004) 209–215 Table 4 Average recoveries of two assays using SPE enrichment (Supelco LC-18) followed by LC–MS(ESI) in the SIM mode, for the 12 neutral pesticides from laboratory-spiked ultra-pure, drinking and surface water samples at the 0.05 ␮g/l level Class; n = 2

Pesticide

Recovery (%) Ultra-pure water

Drinking water

Surface water

Carbamates

Carbaryl Carbofuran Methomyl Oxamyl Pirimicarb

94.6 90.5 72.6 64.7 95.1

85.3 83.3 91.0 75.3 96.4

78.8 78.9 82.9 67.7 92.5

Phenylureas

Chlortoluron Diuron Isoproturon Linuron

90.0 83.1 98.2 74.7

82.3 78.7 96.8 94.2

89.3 113.9 105.2 94.5

Triazines

Atrazine Propazine Simazine

84.8 80.4 91.5

90.9 78.7 89.9

84.8 80.0 82.2

4. Conclusion An optimised multiresidue method to screen ultra-trace levels of 12 neutral pesticides (carbamates, phenylureas and triazines) from water samples was developed using SPE/LC–MS(ESI). Instrumental conditions of LC–MS(ESI) in the SIM mode, showed excellent linear responses for the pesticides studied (1–50 ␮g/l) and precisions with a relative standard deviation below 4.9% (chlortoluron). An instrumental LOD of 0.10 ␮g/l was found with the exception of chlortoluron, diuron and methomyl, for which 0.25 and 0.50 ␮g/l were measured. The SPE assays were easy, sensitive, and did not require much volume of water sample (50 ml). Good recoveries (65.0–97.8%) were obtained for laboratory-spiked water samples at the 0.10 ␮g/l level. For laboratory-spiked drinking and surface water samples containing concentrations of the individual pesticides at the 0.05 ␮g/l level, average recoveries ranging from 75.3 to 96.8% and 67.7–113.9% were measured, respectively. The present methodology proved to be very reproducible and a suitable alternative to the conventional methods used to screen different classes of neutral pesticides in water samples, reaching LODs in the range of 0.5–3.0 ng/l in compliance with the European Union directive on water quality.

Acknowledgements We thank Agilent Technologies for supporting our LC–MS research and ICCTI-OTAN for a grant.

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