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Ultra trace determination of 31 pesticides in water samples by direct injection–rapid resolution liquid chromatography-electrospray tandem mass spectrometry
a n a l y t i c a c h i m i c a a c t a 6 2 4 ( 2 0 0 8 ) 90–96
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/aca
Ultra trace determination of 31 pesticides in water samples by direct injection–rapid resolution liquid chromatography-electrospray tandem mass spectrometry Laura Díaz ∗ , Julio Llorca-Pórcel, Ignacio Valor LABAQUA S.A. C/Dracma 16-18, Polígono industrial las Atalayas, 03114 Alicante, Spain
a r t i c l e
i n f o
a b s t r a c t
Article history:
A liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method for the
Received 23 April 2008
detection of pesticides in tap and treated wastewater was developed and validated according
Received in revised form
to the ISO/IEC 17025:1999. Key features of this method include direct injection of 100 L of
13 June 2008
sample, an 11 min separation by means of a rapid resolution liquid chromatography system
Accepted 13 June 2008
with a 4.6 mm × 50 mm, 1.8 m particle size reverse phase column and detection by electro-
Published on line 6 July 2008
spray ionization (ESI) MS–MS. The limits of detection were below 15 ng L−1 and correlation coefficients for the calibration curves in the range of 30–2000 ng L−1 were higher than 0.99.
Keywords:
Precision was always below 20% and accuracy was confirmed by external evaluation. The
Direct injection
main advantages of this method are direct injection of sample without preparative proce-
Water analysis
dures and low limits of detection that fulfill the requirements established by the current
Pesticides
European regulations governing pesticide detection.
Method validation
© 2008 Elsevier B.V. All rights reserved.
Rapid resolution Liquid chromatography-tandem mass spectrometry
1.
Introduction
Threats to the aqueous environment increase daily as the ever-expanding population density releases more chemical pollutants from manufacturing and agricultural processes into water sources. For this reason, the number of regulations designed to protect the quality of the environment has been growing exponentially over the last two decades. The industrial manufacturers of analytical equipment and the laboratories themselves have developed new analytical equipment and methodologies to adapt to the growing need for efficient, simple, and sensitive approaches to evaluating and regulating environmental contaminants. The increasing appearance of organic micro-contaminants in water sources
∗
Corresponding author. Tel.: +34 965 106070. E-mail address: [email protected] (L. Díaz). 0003-2670/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2008.06.053
has produced a need for the rapid development of new chromatography techniques. A clear example of this need is found in the 2000/60/EC Directive [1], which lists a set of priority compounds that must be monitored in aqueous environments at the community level. Of the total of 33 priority compounds, 29 are organic micro-contaminants present at low levels in aqueous environments and must be analysed by chromatographic techniques. Application of current chromatographic techniques for this purpose is hampered by low throughput, high detection limits, and tedious time-consuming sample extraction and concentration steps. In the last 20 years, laboratories have concentrated on the development of new extraction techniques to replace traditional liquid–liquid extraction, and the combination of
91
a n a l y t i c a c h i m i c a a c t a 6 2 4 ( 2 0 0 8 ) 90–96
these with gas chromatography and high-resolution mass spectrometry to improve sample processing efficiency [2–4]. More recently, throughput speed has been increased by the use of shorter columns between 1 and 5 m, thinner films of 0.1 m or less, and faster oven temperature programming up to 1200 ◦ C min−1 [5]. Gas chromatography–mass spectrometry (GC–MS) has been the preferred analytical method because liquid chromatography–mass spectrometry (LC–MS) was limited to the analysis of moderately high sample concentration levels [6]. However, new LC-MS/MS technology has improved the instrumental detection limits (IDLs) of LC–MS systems from nanogram to sub-picogram levels. These improvements make LC–MS an invaluable technique for the detection of polar contaminants and their transformation products in aqueous environments [7,8]. In the specific field of pesticide analysis in water samples many applications have been developed by combining a previous concentration step either off-line [9] or on-line [10–14] with an LC–MS or by directly injecting the sample in an LC-MS/MS [15]. The present paper describes a rapid and very sensitive LC-MS/MS method for the simultaneous direct injection–rapid resolution LC-MS/MS analysis of 31 pesticides in water samples. The list of pesticides includes some of the pesticides regulated among the 33 priority pollutants of the Water Framework Directive 2000/60/EC, simazine, atrazine, alachlor, chlorfenvinphos, chlorpyrifos and the phenylurea pesticides:
diuron and isoproturon which are difficult to be detected by GC–MS. The main pesticides which should be monitored in the water destined for human consumption according to Directive 98/83/EC [16] including some very polar pesticides such us 2-methyl-4-chlorophenoxyacetic acid (MCPA), ioxynil and triazine metabolites, were also included. The developed method was validated for tap and wastewater. For an external evaluation of the accuracy, the proposed method was tested by participating in an interlaboratory comparison test with real samples.
2.
Experimental
2.1.
Reagents, standards, and working solutions
Analytical standards were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany) and Riedel-de Häen (Seelze, Germany). The purity of all standards was greater than 97%. Individual stock solutions (around 1000 mg L−1 ) were prepared in pure acetonitrile or methanol, according to the solubility properties of each compound, and stored in the dark at −18 ◦ C. A working standard mixture of each pesticide (1 mg L−1 in methanol) was used for both spiking samples and calibration. Calibration standards were prepared daily in Milli-Q water (Millipore, Milford, MA, USA). Atrazine-D5 (Dr. Ehrenstorfer
Table 1 – Multiple reaction monitoring conditions for each of the compounds tested Compound Desisopropilatrazine Metamitron Dimethoate Desethyl atrazine Hydroxy atrazine Aldicarb Cyanazine Bromacil Simazine Carbofuran Metribuzine Atrazine Isoproturon Diuron Methyl-parathion Ametryn Propazine Linuron Terbuthylazine Malathion Prometryn Terbutryn Trietazine Alachlor Parathion Fenthion Diazinon Chlorfenvinphos Chlorpyrifos Ioxynil MCPA
RT (min) 2.57 3.04 3.05 3.45 3.68 3.80 4.08 4.35 4.36 4.38 4.38 4.39 5.05 5.10 5.40 5.50 5.64 5.68 5.70 5.77 5.90 5.94 5.96 6.00 6.10 6.25 6.30 6.57 7.08 3.69 3.75
Prec. ion 174.3 203.3 230.2 188.2 198.3 116.0 241.2 262.8 202.0 222.3 215.3 216.0 207.3 233.0 264.1 228.0 230.1 249.2 230.1 331.1 242.1 242.1 230.0 270.2 292.1 279.2 305.3 359.0 350.1 369.9 199/201
Prod. ion (P1 /P2 ) 104.0/68.3 104.1/175.3 125.0/198.9 146.1/104.1 156.2/86.2 70.0/89.0 214.1/132.0 206.8/189.4 132.0/124.0 123.1/165.1 84.2/187.3 174.0/96.0 165.2/72.1 188.0/160.0 232.0/125.1 185.9/95.8 146.1/188.0 182.1/160.1 104.2/174.4 127.1/99.1 200.2/158.1 68.1/186 98.9/131.7 238.1/162.3 236.0/140.1 169.0/247.0 169.2/153.1 155.0/99.1 198.0/97.1 126.9/214.7 141/143
Frag. (V) 110 50 60 110 110 70 110 100 110 70 130 110 130 90 110 70 110 110 110 90 110 90 70 70 110 110 110 70 110 110 90
Collision energy, E1 /E2 (V) 25/25 20/25 10/20 20/25 20/25 10/10 15/25 10/30 25/25 10/20 25/25 20/20 15/20 15/15 15/20 25/25 25/25 15/15 25/25 10/25 25/25 15/15 25/25 10/15 15/20 15/15 20/20 10/25 20/25 40/40 15
ESI mode Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Negative Negative
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a n a l y t i c a c h i m i c a a c t a 6 2 4 ( 2 0 0 8 ) 90–96
Fig. 1 – Signal to noise ratios obtained for different mobile phase flow rates.
GmbH) and ␣,␣,␣-(trifluoromethyl)phenol (Sigma–Aldrich, Schnelldorf, Germany) were used as internal standards for positive electrospray ionization (ESI) and negative ESI modes, respectively. HPLC-grade methanol and formic acid were purchased from Fluka (Seelze, Germany).
2.2.
Sample preparation
For the matrix effects experiments, water for human consumption (tap water) from the city of Alicante with 0.75 mg L−1 of total dissolved salts was used. Different treated wastewaters were analysed to check that none of the pesticides under study were present. Water from the outlet of the wastewater treatment plant of Monte Orgegia in the north of Alicante with a biochemical oxygen demand (BOD5 ) of 25 mg L−1 was finally
selected. Tap and wastewater samples were filtered through 0.22 m Millex® -GV syringe-driven filters (Millipore, Bedford, MA, USA). For tap water, 600 L L−1 of 3% Na2 S2 O3 ·5H2 O was added to eliminate chlorine prior to spiking with the target compounds. Samples were prepared daily using either MilliQ purified water for calibration or tap water and wastewater for the validation experiments by the addition of the corresponding working standard mixture containing all target compounds in methanol.
2.3.
Instrumentation
LC separations were performed with a 1200 Binary SL Rapid Resolution series pump (Agilent Technologies, Palo Alto, CA, USA). The so-called rapid resolution LC (RRLC) is able to work
Fig. 2 – Signal to noise ratios for different drying gas flow rates.
a n a l y t i c a c h i m i c a a c t a 6 2 4 ( 2 0 0 8 ) 90–96
93
Fig. 3 – Total ion chromatogram corresponding with a treated wastewater spiked with the target pesticides at 2 g L−1 (ESI+ ).
at pressures up to 9000 psi. An Agilent ZORBAX Eclipse® XDBC18 4.6 mm × 50 mm, 1.8 m particle size column was used held at 40 ◦ C with an Agilent 1200 series SL column compartment. A sample volume of 100 L was injected with an Agilent 1200 series SL autosampler using a binary mobile phase composed of solvent A (5 mM ammonium formate in 80% water/20% methanol) and solvent B (5 mM ammonium formate in 90% water/10% methanol) at a constant flow rate of 0.8 mL min−1 . The gradient was programmed to increase the amount of solvent B from 0 to 100% over 5.5 min, maintain 100% solvent B until 10 min, and return to the initial conditions after 11.5 min. Mass spectrometry was performed on an Agilent 6410 triple quadrupole mass spectrometer fitted with an ESI MS source and controlled by Mass Hunter (Version B.01.00) software. The ESI source conditions were established to obtain an average maximum intensity of the precursor ions. The nitrogen nebulizer pressure was set at 40 psi and the nitrogen drying gas was set at 350 ◦ C with a 9 L min−1 flow rate. The capillary voltage was set at 3500 V. A 1 unit resolution was set in both Q1 and Q2 with a dwell time of 40 ms. For both nebulizing and drying, a G4 Domnick Hunter (Dukesway, England) laboratory nitrogen generator was used. For MS–MS, 99.9995% N2 was used as a collision gas. To optimize the multiple reaction monitoring (MRM) transitions, direct injection of each individual pesticide at a concentration of 10 mg L−1 in methanol was used. Optimal conditions are summarized in Table 1.
2.4.
Validation studies
The performance characteristics of the optimized method were validated according to UNE-EN ISO/IEC 17025:1999 criteria [17]. The referred norm establishes the need to evaluate linearity, matrix effects, accuracy, repeatability, and calcula-
tion of uncertainties. The developed method was validated for both tap and wastewater. For an external evaluation of accuracy, the proposed method was tested in an interlaboratory comparison test.
3.
Results and discussion
3.1.
Mobile phase flow rate
When working with ESI-LC-MS interfaces, the mobile flow rate is generally sacrificed in favor of the optimal flow rates for ESI, approximately 200 L min−1 . This approach can limit the optimal flow rate in terms of efficiency of columns with 4.6 mm internal diameter (in which optimal flow rates are higher than 500 L min−1 ) and/or the throughput of columns with particle sizes smaller than 3 m capable to achieve great column efficiency at high flow rates (due to the flat shape of the Van Deenter equation for these columns), thus enabling good, quick separations. According to technical specifications, the orthogonal Agilent 6410 ESI source functions properly at mobile flow rates up to 1 mL min−1 due to its high desolvation efficiency, avoiding adduct formation and maintaining sensitivity. Fig. 1 shows the signal to noise (S/N) ratios obtained for two selected pesticides (dimethoate and carbofuran) at different flow rates of 0.6, 0.8, and 1.0 mL min−1 . The best S/N ratio was obtained at a mobile flow rate of 0.8 mL min−1 . The same result was observed for the remaining compounds. There was no significant difference between S/N ratios recorded with two nitrogen flow rates of 9 and 11 L min−1 (Fig. 2) that fell within the recommended range for the drying gas flow rate. According to the results, a 0.8 mL min−1 mobile flow rate and a 9 L min−1 drying gas flow rate were selected.
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3.2.
a n a l y t i c a c h i m i c a a c t a 6 2 4 ( 2 0 0 8 ) 90–96
MRM transition optimization
To optimize performance and sensitivity, the fragmentor voltage was selected to produce the highest signal intensity for the precursor ion. The collision energy was then adjusted to produce the highest intensity for the main fragment. To select the fragmentor voltage the flow injections of 10 mg L−1 for each individual standard solution were performed by varying the fragmentor voltages from 50 to 130 V in steps of 10 V. From the total ion chromatogram (TIC), the corresponding [M+H]+ for ESI+ and [M−H]+ for ESI− were used to obtain the extracted ion chromatogram (EIC) for the different fragmentor voltages; the optimum fragmentor voltages were then selected. With this optimized fragmentor voltage, an additional flow injection experiment was performed to optimize collision energy. Each time segment of the analysis was operated with increasing collision energy in steps of 5 V between 10 and 40 V. The optimum collision voltages were defined as generating the highest signal for the main fragment ion. The results of the optimized conditions for each MRM transition are summarized in Table 1. Two MRM transitions were chosen for each pesticide. The transition “precursor ion–P1 product ion” was used for quantification and the transition “precursor ion–P2 product ion” for confirmation (Table 1). The acquisition conditions for the positive ion mode were divided into four segments (Fig. 3). Even in segment two in which 27 transitions were monitored using a 40 ms dwell time for each transition, a 0.85 cycle s−1 was obtained for this segment, ensuring a minimum of 10 points per chromatographic peak.
3.3.
Limits of detection and linearity
The limits of detection and quantification have been established for a signal to noise ratio of 3 and 10, respectively, from the chromatogram corresponding to the treated water fortified at 0.03 g L−1 level. As shown in Table 2, the limits of detection for all compounds were below 15 ng L−1 , ranging from 2 to 15 ng L−1 . Taking into account the volume injected (100 L) this allows to estimated an instrumental detection limit from 0.2 to 1.5 pg depending on the compound. In a recent work published by Mezcua [8] an LC-MS/MS method for simazine, atrazine, diuron and isoproturon among others was developed. The limits of detection with the referred method were 0.8 and 0.6 ng L−1 for simazine and atrazine, respectively and 0.1 ng L−1 for both, diuron and isoproturon. However, a much larger (300 mL) sample was required for the solid phase extraction. Taking into account the extraction/concentration step an IDL could be estimated for the UPLC-MS/MS method ranging from 0.2 to 2.3 pg which are in the same range than the ones obtained for the same compounds (0.2–0.8 pg) with our rapid resolution LC-MS/MS instrument. In a previous paper by Ingelse [14] a direct injection of aqueous samples into and atmospheric pressure chemical ionization (APCI) mass spectrometer was developed for some very polar organophosphorus pesticides such as acephate, metamidophos, monocrotophos and others. Limits of detection were in the range of 10–30 ng L−1 by the injection of 10× the volume of sample used in our method. The limits of detection achieved by the developed method are appropriate for assessing compliance with the European
Table 2 – Limits of detection (LOD in g L−1 ) and correlation coefficients (r2 ) for calibration curves Compound Desisopropilatrazine Metamitron Dimethoate Desethyl atrazine Hydroxy atrazine Aldicarb Cyanazine Bromacil Simazine Carbofuran Metribuzine Atrazine Isoproturon Diuron Methyl-parathion Ametryn Propazine Linuron Terbuthylazine Malathion Prometryn Terbutryn Trietazine Alachlor Parathion Fenthion Diazinon Chlorfenvinphos Chlorpyrifos Ioxynil MCPA
r2
LOD
0.996 0.996 0.999 0.999 0.996 0.998 0.999 0.993 0.997 0.999 0.996 0.998 0.999 0.995 0.998 0.998 0.998 0.999 0.997 0.999 0.998 0.998 0.999 0.998 0.998 0.999 0.998 0.994 0.999 0.999 0.999
5 6 2 4 5 6 4 4 4 4 6 2 3 8 15 3 2 4 5 6 4 5 4 6 4 7 4 4 7 9 6
limits for pesticides in water destined for human consumption (100 ng L−1 ) and for the more restrictive values of the Water Framework Directive 2000/60/CE (as low as 30 ng L−1 ). The linearity of the method was studied in the range from 30 to 2000 ng L−1 for compounds having a LOD < 5 ng L−1 (consequently a LOQ < 17) and from 100 to 2000 ng L−1 for compounds having a LOD ≥ 5 ng L−1 at five concentration levels (30 or 100, 250, 500, 1000 and 2000 ng L−1 ). As can be seen (Table 2), the linearity across the studied range was excellent, with correlation coefficients higher than 0.99 for all the studied compounds.
3.4.
Precision and overall uncertainties
Precision, intermediate precision and uncertainties were calculated for tap water and treated wastewater at two concentration levels (low and high level) according to current requirements of the EN/ISO 17025. These levels were 30 and 2000 ng L−1 for compounds having a LOD < 5 ng L−1 (consequently a LOQ < 17) and from 100 to 2000 ng L−1 for compounds having a LOD ≥ 5 ng L−1 . For precision six replicates were analysed in the same day by the same analyst. In the case of intermediate precision six replicates were analysed in different days by different analysts. Table 3 shows the results obtained for the low level expressed as relative standard deviation (R.S.D.%). A good precision was observed for all the investigated compounds presenting R.S.D.% values below 20%
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for both tap and wastewater matrices. Random differences between intermediate precision and precision were observed but, on average, similar R.S.D.% values were obtained, demonstrating that precision is not significantly affected by variances in either test day or analyst. Due to difficulties in the calculations of the individual uncertainty contributions following a “bottom up” procedure, as proposed by the ISO Guide to the Expression of Uncertainty in Measurement (GUM) [18], the different contributions were grouped as recommended by the EURACHEM/CITAC guide [19]. The contributions in the direct injection–RRLC-MS-MS method can be grouped into three terms, permitting the calculation of the overall uncertainty according to the following equation:
U=k
SR u2CRM + w √ nR
2
+
tolerance 2 √ 3
The first term (uCRM ) represents the uncertainty from the certified reference material used for calibration and the subsequent uncertainties introduced during weighing and diluting √ the sample. The second term (w(SR / nR )) represents the contribution of the intermediate precision of the method in which
w is a statistical coefficient for a desirable level of confidence and SR is the standard deviation for n experiments performed under intermediate precision conditions (nR ). The third term represents the tolerance that each laboratory establishes for their internal quality controls. Finally, k is the coverage factor to expand the uncertainty to the desired level of confidence. The second and third terms generally contribute the most to overall uncertainty. In the present work, the overall uncertainties were calculated for both the low and high concentration levels. uCRM was calculated by taking into account all the dilution steps, the uncertainties from the CRM, and all the volumetric material and balances used to prepare the calibration standards. SR was calculated from the nR = 6 results from the precision experiments (then w = 1.3 for a 95% level of confidence). The third term was calculated with a 10% tolerance which is the standard value in our laboratory for the verification of the daily calibration curve. Finally, a coverage factor of k = 2 was used for a confidence interval of 95%. As shown in Table 3, the uncertainties calculated for the low concentration level (0.03 and 0.1 g L−1 ) are in the order of 20% which is a typical value for a chromatographic method in a routine
Table 3 – Precision and intermediate precision (expressed as R.S.D.%) and overall uncertainties expressed as % (k = 2) calculated for the lowest calibration level Compound
Tap water Precision
Intermediate precision
0.03 g L Dimethoate Desethyl atrazine Cyanazine Bromacil Simazine Carbofuran Atrazine Isoproturon Ametryn Propazine Linuron Prometryn Trietazine Diazinon Chlorfenvinphos Ioxynil
2 13 11 3 16 5 8 14 6 12 13 10 8 15 10 10
10 11 10 5 15 5 6 10 11 12 11 8 14 5 7 13
0.1 g L−1 Desisopropilatrazine Metamitron Hydroxy atrazine Aldicarb Metribuzine Diuron Methyl-parathion Terbuthylazine Malathion Terbutryn Alachlor Parathion Fenthion Chlorpyrifos MCPA
9 11 8 8 14 15 13 3 5 6 2 9 10 8 15
9 7 8 10 10 7 7 13 6 15 10 12 3 11 6
Wastewater Uk=2
precision
Intermediate precision
Uk=2
19 20 20 15 27 15 16 20 20 20 20 17 23 15 17 12
4 10 7 15 12 7 9 8 7 6 7 10 13 7 16 16
20 9 14 7 10 15 11 6 7 14 15 3 14 8 14 12
27 17 23 15 17 34 20 15 15 23 20 14 20 17 20 12
18 15 16 17 18 17 15 21 15 20 18 20 14 20 17
8 7 5 7 6 13 9 2 13 3 6 14 12 8 10
3 6 4 7 9 23 8 10 3 8 3 9 7 9 6
14 15 14 17 17 28 13 14 14 16 13 18 15 16 17
−1
n = 6.
96
a n a l y t i c a c h i m i c a a c t a 6 2 4 ( 2 0 0 8 ) 90–96
Table 4 – Results obtained (ng L−1 ) in the interlaboratory comparison test Mean value by LC/MS
Target value
z-Score
Sample A Atrazine Prometryne Propazine Simazine Terbuthylazine Terbutryn
0.123 ± 0.025 0.079 ± 0.017 0.205 ± 0.049 0.215 ± 0.064 0.178 ± 0.045 0.289 ± 0.069
0.119 ± 0.02 0.065 ± 0.007 0.190 ± 0.03 0.206 ± 0.035 0.166 ± 0.022 0.270 ± 0.038
−0.45 −0.38 −0.83 −0.79 −0.98 −0.65
Sample B Atrazine Prometryne Propazine Simazine Terbuthylazine Terbutryn
<0.03 0.145 ± 0.032 0.125 ± 0.030 0.156 ± 0.047 <0.1 0.149 ± 0.036
<0.01 0.131 ± 0.016 0.101 ± 0.016 0.145 ± 0.025 <0.03 0.139 ± 0.023
− −0.87 −0.79 −0.96 – −0.83
z-Score = (mean value − target)/Std of accepted labs.
quality control laboratory. The average of the uncertainties for tap water is 17.9% and 17.7% for treated wastewater, demonstrating that this type of matrix does not contribute to the uncertainties of the method.
3.5.
Accuracy and proficiency testing with real samples
Accuracy was tested for the two investigated matrices at both low and high concentration levels by evaluating the six results of the intermediate precision experiment with the Student’s t-test. A method is defined as traceable when the value for the Student’s t-test calculated from the experimental results, tcal , is less than or equal to the Student’s t-test tabulated value (two-tailed). A confidence interval of 95% was considered with (n − 1) = 5 degrees of freedom, thus ttab = 2.015. For all the compounds under study in both matrices at both concentration levels, the values calculated for the Student’s t-test were below 2.015. For an external evaluation of accuracy, the proposed method was tested by an interlaboratory comparison test with real samples. The interlaboratory test was organized by IFA Tulln (Center for Analytical Chemistry of the Institute for Agrobiotechnology Tulln, Austria). Two samples encoded A and B, containing different levels of herbicides (simazine, cyanazine, atrazine, propazine, terbuthylazine, and prometryn) in real matrices (surface waters), were distributed to 12 different laboratories. The mean value obtained with the proposed direct injection RRLC/MS/MS method and the target value proposed by the organization with their corresponding uncertainties are presented in Table 4. The z-scores for both samples were less than −2, indicating that the results from all laboratories produced equivalent results using this method.
4.
Conclusions
The developed method allows the analysis of 31 pesticides in both tap water and wastewater in 11 min, making possible the analysis of the selected pesticides of up to 80 water samples per day, by the direct injection of 0.1 mL sample with LODs less than 10 ng L−1 for the majority of compounds. The low limits of detection, reliable linearity, precision, and accuracy of the developed method exceed the requirements for assessing adherence to limits established by the current European regulations governing pesticide contamination of aqueous environments. The increased efficiency and ease of sample processivity make this method a key tool for the routine quality control of priority pesticides in water.
references
[1] Directive 2000/60/EC of 23 October 2000 of the European Parliament and of the Council Establishing a Framework for the Community Action in the Field of Water Policy. [2] I. Valor, C. Cortada, D. Apraiz, Quim. Anal. 4 (1995) 228. [3] I. Valor, J.C. Moltó, D. Apraiz, G. Font, J. Chromatogr. A 767 (1997) 195. [4] J. Llorca-Porcel, G. Martínez-Sánchez, B. Álvarez, M.A. Cobollo, I. Valor, Anal. Chim. Acta 569 (2006) 113. [5] X. Yang, L. Ma, P.W. Car, J. Chromatogr. A 1079 (2005) 213. [6] D. Barceló, M. Petrovic, Trends Anal. Chem. 26 (2007) 2. [7] C. Zwiener, F.H. Frimmel, Anal. Bioanal. Chem. 378 (2004) 851. [8] S.D. Richardson, Anal. Chem. 78 (2006) 4021. [9] M. Mezcua, A. Agüera, J.Ll. Lliberia, M.A. Cortés, B. Bagó, A.R. Fernández-Alba, J. Chromatogr. A 1109 (2006) 222. [10] F. Hernández, J.V. Sancho, O. Pozo, A. Lara, E. Pitarch, J. Chromatogr. A 939 (2001) 1. [11] J.M. Marín, J.V. Sancho, O.J. Pozo, F.J. López, F. Hernández, J. Chromatogr. A 1133 (2006) 204. [12] H. Sabik, R. Jeannot, B. Rondeau, J. Chromatogr. A 885 (2000) 217. [13] R.J. Vreeken, P. Speksnijder, I. Bobeldijk-Pastorova, Th.H.M. Noij, J. Chromatogr. A 794 (1998) 187. [14] K. Stoob, H.P. Singer, C.W. Goetz, M. Ruff, S.R. Mueller, J. Chromatogr. A 1097 (2005) 138. [15] B.A. Ingelse, R.C.J. van Dam, R.J. Vreeken, H.G.J. Mol, O.M. Steijger, J. Chromatogr. A 1918 (2001) 67. [16] Council Directive 98/83/EC of 3 November 1998 On the Quality of Water Intended for Human Consumption, OJL 330, p. 32. [17] UNE-EN ISO/IEC 17025:1999: General Requirements for the Competence of Testing and Calibration Laboratories, CEN/CENELEC, Bruxelles, 2000. [18] ISO Guide to the Expression of Uncertainty in Measurement (GUM). BIPM, IEC, IFCC, ISO, IUPAC, IUPAP, OIML, Geneva, 1993. [19] EURACHEM, Quantifying Uncertainty in Analytical Measurements. EURACHEM Secretariat, Teddington, Middlesex, UK, 1995.
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/aca
Ultra trace determination of 31 pesticides in water samples by direct injection–rapid resolution liquid chromatography-electrospray tandem mass spectrometry Laura Díaz ∗ , Julio Llorca-Pórcel, Ignacio Valor LABAQUA S.A. C/Dracma 16-18, Polígono industrial las Atalayas, 03114 Alicante, Spain
a r t i c l e
i n f o
a b s t r a c t
Article history:
A liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method for the
Received 23 April 2008
detection of pesticides in tap and treated wastewater was developed and validated according
Received in revised form
to the ISO/IEC 17025:1999. Key features of this method include direct injection of 100 L of
13 June 2008
sample, an 11 min separation by means of a rapid resolution liquid chromatography system
Accepted 13 June 2008
with a 4.6 mm × 50 mm, 1.8 m particle size reverse phase column and detection by electro-
Published on line 6 July 2008
spray ionization (ESI) MS–MS. The limits of detection were below 15 ng L−1 and correlation coefficients for the calibration curves in the range of 30–2000 ng L−1 were higher than 0.99.
Keywords:
Precision was always below 20% and accuracy was confirmed by external evaluation. The
Direct injection
main advantages of this method are direct injection of sample without preparative proce-
Water analysis
dures and low limits of detection that fulfill the requirements established by the current
Pesticides
European regulations governing pesticide detection.
Method validation
© 2008 Elsevier B.V. All rights reserved.
Rapid resolution Liquid chromatography-tandem mass spectrometry
1.
Introduction
Threats to the aqueous environment increase daily as the ever-expanding population density releases more chemical pollutants from manufacturing and agricultural processes into water sources. For this reason, the number of regulations designed to protect the quality of the environment has been growing exponentially over the last two decades. The industrial manufacturers of analytical equipment and the laboratories themselves have developed new analytical equipment and methodologies to adapt to the growing need for efficient, simple, and sensitive approaches to evaluating and regulating environmental contaminants. The increasing appearance of organic micro-contaminants in water sources
∗
Corresponding author. Tel.: +34 965 106070. E-mail address: [email protected] (L. Díaz). 0003-2670/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2008.06.053
has produced a need for the rapid development of new chromatography techniques. A clear example of this need is found in the 2000/60/EC Directive [1], which lists a set of priority compounds that must be monitored in aqueous environments at the community level. Of the total of 33 priority compounds, 29 are organic micro-contaminants present at low levels in aqueous environments and must be analysed by chromatographic techniques. Application of current chromatographic techniques for this purpose is hampered by low throughput, high detection limits, and tedious time-consuming sample extraction and concentration steps. In the last 20 years, laboratories have concentrated on the development of new extraction techniques to replace traditional liquid–liquid extraction, and the combination of
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these with gas chromatography and high-resolution mass spectrometry to improve sample processing efficiency [2–4]. More recently, throughput speed has been increased by the use of shorter columns between 1 and 5 m, thinner films of 0.1 m or less, and faster oven temperature programming up to 1200 ◦ C min−1 [5]. Gas chromatography–mass spectrometry (GC–MS) has been the preferred analytical method because liquid chromatography–mass spectrometry (LC–MS) was limited to the analysis of moderately high sample concentration levels [6]. However, new LC-MS/MS technology has improved the instrumental detection limits (IDLs) of LC–MS systems from nanogram to sub-picogram levels. These improvements make LC–MS an invaluable technique for the detection of polar contaminants and their transformation products in aqueous environments [7,8]. In the specific field of pesticide analysis in water samples many applications have been developed by combining a previous concentration step either off-line [9] or on-line [10–14] with an LC–MS or by directly injecting the sample in an LC-MS/MS [15]. The present paper describes a rapid and very sensitive LC-MS/MS method for the simultaneous direct injection–rapid resolution LC-MS/MS analysis of 31 pesticides in water samples. The list of pesticides includes some of the pesticides regulated among the 33 priority pollutants of the Water Framework Directive 2000/60/EC, simazine, atrazine, alachlor, chlorfenvinphos, chlorpyrifos and the phenylurea pesticides:
diuron and isoproturon which are difficult to be detected by GC–MS. The main pesticides which should be monitored in the water destined for human consumption according to Directive 98/83/EC [16] including some very polar pesticides such us 2-methyl-4-chlorophenoxyacetic acid (MCPA), ioxynil and triazine metabolites, were also included. The developed method was validated for tap and wastewater. For an external evaluation of the accuracy, the proposed method was tested by participating in an interlaboratory comparison test with real samples.
2.
Experimental
2.1.
Reagents, standards, and working solutions
Analytical standards were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany) and Riedel-de Häen (Seelze, Germany). The purity of all standards was greater than 97%. Individual stock solutions (around 1000 mg L−1 ) were prepared in pure acetonitrile or methanol, according to the solubility properties of each compound, and stored in the dark at −18 ◦ C. A working standard mixture of each pesticide (1 mg L−1 in methanol) was used for both spiking samples and calibration. Calibration standards were prepared daily in Milli-Q water (Millipore, Milford, MA, USA). Atrazine-D5 (Dr. Ehrenstorfer
Table 1 – Multiple reaction monitoring conditions for each of the compounds tested Compound Desisopropilatrazine Metamitron Dimethoate Desethyl atrazine Hydroxy atrazine Aldicarb Cyanazine Bromacil Simazine Carbofuran Metribuzine Atrazine Isoproturon Diuron Methyl-parathion Ametryn Propazine Linuron Terbuthylazine Malathion Prometryn Terbutryn Trietazine Alachlor Parathion Fenthion Diazinon Chlorfenvinphos Chlorpyrifos Ioxynil MCPA
RT (min) 2.57 3.04 3.05 3.45 3.68 3.80 4.08 4.35 4.36 4.38 4.38 4.39 5.05 5.10 5.40 5.50 5.64 5.68 5.70 5.77 5.90 5.94 5.96 6.00 6.10 6.25 6.30 6.57 7.08 3.69 3.75
Prec. ion 174.3 203.3 230.2 188.2 198.3 116.0 241.2 262.8 202.0 222.3 215.3 216.0 207.3 233.0 264.1 228.0 230.1 249.2 230.1 331.1 242.1 242.1 230.0 270.2 292.1 279.2 305.3 359.0 350.1 369.9 199/201
Prod. ion (P1 /P2 ) 104.0/68.3 104.1/175.3 125.0/198.9 146.1/104.1 156.2/86.2 70.0/89.0 214.1/132.0 206.8/189.4 132.0/124.0 123.1/165.1 84.2/187.3 174.0/96.0 165.2/72.1 188.0/160.0 232.0/125.1 185.9/95.8 146.1/188.0 182.1/160.1 104.2/174.4 127.1/99.1 200.2/158.1 68.1/186 98.9/131.7 238.1/162.3 236.0/140.1 169.0/247.0 169.2/153.1 155.0/99.1 198.0/97.1 126.9/214.7 141/143
Frag. (V) 110 50 60 110 110 70 110 100 110 70 130 110 130 90 110 70 110 110 110 90 110 90 70 70 110 110 110 70 110 110 90
Collision energy, E1 /E2 (V) 25/25 20/25 10/20 20/25 20/25 10/10 15/25 10/30 25/25 10/20 25/25 20/20 15/20 15/15 15/20 25/25 25/25 15/15 25/25 10/25 25/25 15/15 25/25 10/15 15/20 15/15 20/20 10/25 20/25 40/40 15
ESI mode Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Negative Negative
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a n a l y t i c a c h i m i c a a c t a 6 2 4 ( 2 0 0 8 ) 90–96
Fig. 1 – Signal to noise ratios obtained for different mobile phase flow rates.
GmbH) and ␣,␣,␣-(trifluoromethyl)phenol (Sigma–Aldrich, Schnelldorf, Germany) were used as internal standards for positive electrospray ionization (ESI) and negative ESI modes, respectively. HPLC-grade methanol and formic acid were purchased from Fluka (Seelze, Germany).
2.2.
Sample preparation
For the matrix effects experiments, water for human consumption (tap water) from the city of Alicante with 0.75 mg L−1 of total dissolved salts was used. Different treated wastewaters were analysed to check that none of the pesticides under study were present. Water from the outlet of the wastewater treatment plant of Monte Orgegia in the north of Alicante with a biochemical oxygen demand (BOD5 ) of 25 mg L−1 was finally
selected. Tap and wastewater samples were filtered through 0.22 m Millex® -GV syringe-driven filters (Millipore, Bedford, MA, USA). For tap water, 600 L L−1 of 3% Na2 S2 O3 ·5H2 O was added to eliminate chlorine prior to spiking with the target compounds. Samples were prepared daily using either MilliQ purified water for calibration or tap water and wastewater for the validation experiments by the addition of the corresponding working standard mixture containing all target compounds in methanol.
2.3.
Instrumentation
LC separations were performed with a 1200 Binary SL Rapid Resolution series pump (Agilent Technologies, Palo Alto, CA, USA). The so-called rapid resolution LC (RRLC) is able to work
Fig. 2 – Signal to noise ratios for different drying gas flow rates.
a n a l y t i c a c h i m i c a a c t a 6 2 4 ( 2 0 0 8 ) 90–96
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Fig. 3 – Total ion chromatogram corresponding with a treated wastewater spiked with the target pesticides at 2 g L−1 (ESI+ ).
at pressures up to 9000 psi. An Agilent ZORBAX Eclipse® XDBC18 4.6 mm × 50 mm, 1.8 m particle size column was used held at 40 ◦ C with an Agilent 1200 series SL column compartment. A sample volume of 100 L was injected with an Agilent 1200 series SL autosampler using a binary mobile phase composed of solvent A (5 mM ammonium formate in 80% water/20% methanol) and solvent B (5 mM ammonium formate in 90% water/10% methanol) at a constant flow rate of 0.8 mL min−1 . The gradient was programmed to increase the amount of solvent B from 0 to 100% over 5.5 min, maintain 100% solvent B until 10 min, and return to the initial conditions after 11.5 min. Mass spectrometry was performed on an Agilent 6410 triple quadrupole mass spectrometer fitted with an ESI MS source and controlled by Mass Hunter (Version B.01.00) software. The ESI source conditions were established to obtain an average maximum intensity of the precursor ions. The nitrogen nebulizer pressure was set at 40 psi and the nitrogen drying gas was set at 350 ◦ C with a 9 L min−1 flow rate. The capillary voltage was set at 3500 V. A 1 unit resolution was set in both Q1 and Q2 with a dwell time of 40 ms. For both nebulizing and drying, a G4 Domnick Hunter (Dukesway, England) laboratory nitrogen generator was used. For MS–MS, 99.9995% N2 was used as a collision gas. To optimize the multiple reaction monitoring (MRM) transitions, direct injection of each individual pesticide at a concentration of 10 mg L−1 in methanol was used. Optimal conditions are summarized in Table 1.
2.4.
Validation studies
The performance characteristics of the optimized method were validated according to UNE-EN ISO/IEC 17025:1999 criteria [17]. The referred norm establishes the need to evaluate linearity, matrix effects, accuracy, repeatability, and calcula-
tion of uncertainties. The developed method was validated for both tap and wastewater. For an external evaluation of accuracy, the proposed method was tested in an interlaboratory comparison test.
3.
Results and discussion
3.1.
Mobile phase flow rate
When working with ESI-LC-MS interfaces, the mobile flow rate is generally sacrificed in favor of the optimal flow rates for ESI, approximately 200 L min−1 . This approach can limit the optimal flow rate in terms of efficiency of columns with 4.6 mm internal diameter (in which optimal flow rates are higher than 500 L min−1 ) and/or the throughput of columns with particle sizes smaller than 3 m capable to achieve great column efficiency at high flow rates (due to the flat shape of the Van Deenter equation for these columns), thus enabling good, quick separations. According to technical specifications, the orthogonal Agilent 6410 ESI source functions properly at mobile flow rates up to 1 mL min−1 due to its high desolvation efficiency, avoiding adduct formation and maintaining sensitivity. Fig. 1 shows the signal to noise (S/N) ratios obtained for two selected pesticides (dimethoate and carbofuran) at different flow rates of 0.6, 0.8, and 1.0 mL min−1 . The best S/N ratio was obtained at a mobile flow rate of 0.8 mL min−1 . The same result was observed for the remaining compounds. There was no significant difference between S/N ratios recorded with two nitrogen flow rates of 9 and 11 L min−1 (Fig. 2) that fell within the recommended range for the drying gas flow rate. According to the results, a 0.8 mL min−1 mobile flow rate and a 9 L min−1 drying gas flow rate were selected.
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3.2.
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MRM transition optimization
To optimize performance and sensitivity, the fragmentor voltage was selected to produce the highest signal intensity for the precursor ion. The collision energy was then adjusted to produce the highest intensity for the main fragment. To select the fragmentor voltage the flow injections of 10 mg L−1 for each individual standard solution were performed by varying the fragmentor voltages from 50 to 130 V in steps of 10 V. From the total ion chromatogram (TIC), the corresponding [M+H]+ for ESI+ and [M−H]+ for ESI− were used to obtain the extracted ion chromatogram (EIC) for the different fragmentor voltages; the optimum fragmentor voltages were then selected. With this optimized fragmentor voltage, an additional flow injection experiment was performed to optimize collision energy. Each time segment of the analysis was operated with increasing collision energy in steps of 5 V between 10 and 40 V. The optimum collision voltages were defined as generating the highest signal for the main fragment ion. The results of the optimized conditions for each MRM transition are summarized in Table 1. Two MRM transitions were chosen for each pesticide. The transition “precursor ion–P1 product ion” was used for quantification and the transition “precursor ion–P2 product ion” for confirmation (Table 1). The acquisition conditions for the positive ion mode were divided into four segments (Fig. 3). Even in segment two in which 27 transitions were monitored using a 40 ms dwell time for each transition, a 0.85 cycle s−1 was obtained for this segment, ensuring a minimum of 10 points per chromatographic peak.
3.3.
Limits of detection and linearity
The limits of detection and quantification have been established for a signal to noise ratio of 3 and 10, respectively, from the chromatogram corresponding to the treated water fortified at 0.03 g L−1 level. As shown in Table 2, the limits of detection for all compounds were below 15 ng L−1 , ranging from 2 to 15 ng L−1 . Taking into account the volume injected (100 L) this allows to estimated an instrumental detection limit from 0.2 to 1.5 pg depending on the compound. In a recent work published by Mezcua [8] an LC-MS/MS method for simazine, atrazine, diuron and isoproturon among others was developed. The limits of detection with the referred method were 0.8 and 0.6 ng L−1 for simazine and atrazine, respectively and 0.1 ng L−1 for both, diuron and isoproturon. However, a much larger (300 mL) sample was required for the solid phase extraction. Taking into account the extraction/concentration step an IDL could be estimated for the UPLC-MS/MS method ranging from 0.2 to 2.3 pg which are in the same range than the ones obtained for the same compounds (0.2–0.8 pg) with our rapid resolution LC-MS/MS instrument. In a previous paper by Ingelse [14] a direct injection of aqueous samples into and atmospheric pressure chemical ionization (APCI) mass spectrometer was developed for some very polar organophosphorus pesticides such as acephate, metamidophos, monocrotophos and others. Limits of detection were in the range of 10–30 ng L−1 by the injection of 10× the volume of sample used in our method. The limits of detection achieved by the developed method are appropriate for assessing compliance with the European
Table 2 – Limits of detection (LOD in g L−1 ) and correlation coefficients (r2 ) for calibration curves Compound Desisopropilatrazine Metamitron Dimethoate Desethyl atrazine Hydroxy atrazine Aldicarb Cyanazine Bromacil Simazine Carbofuran Metribuzine Atrazine Isoproturon Diuron Methyl-parathion Ametryn Propazine Linuron Terbuthylazine Malathion Prometryn Terbutryn Trietazine Alachlor Parathion Fenthion Diazinon Chlorfenvinphos Chlorpyrifos Ioxynil MCPA
r2
LOD
0.996 0.996 0.999 0.999 0.996 0.998 0.999 0.993 0.997 0.999 0.996 0.998 0.999 0.995 0.998 0.998 0.998 0.999 0.997 0.999 0.998 0.998 0.999 0.998 0.998 0.999 0.998 0.994 0.999 0.999 0.999
5 6 2 4 5 6 4 4 4 4 6 2 3 8 15 3 2 4 5 6 4 5 4 6 4 7 4 4 7 9 6
limits for pesticides in water destined for human consumption (100 ng L−1 ) and for the more restrictive values of the Water Framework Directive 2000/60/CE (as low as 30 ng L−1 ). The linearity of the method was studied in the range from 30 to 2000 ng L−1 for compounds having a LOD < 5 ng L−1 (consequently a LOQ < 17) and from 100 to 2000 ng L−1 for compounds having a LOD ≥ 5 ng L−1 at five concentration levels (30 or 100, 250, 500, 1000 and 2000 ng L−1 ). As can be seen (Table 2), the linearity across the studied range was excellent, with correlation coefficients higher than 0.99 for all the studied compounds.
3.4.
Precision and overall uncertainties
Precision, intermediate precision and uncertainties were calculated for tap water and treated wastewater at two concentration levels (low and high level) according to current requirements of the EN/ISO 17025. These levels were 30 and 2000 ng L−1 for compounds having a LOD < 5 ng L−1 (consequently a LOQ < 17) and from 100 to 2000 ng L−1 for compounds having a LOD ≥ 5 ng L−1 . For precision six replicates were analysed in the same day by the same analyst. In the case of intermediate precision six replicates were analysed in different days by different analysts. Table 3 shows the results obtained for the low level expressed as relative standard deviation (R.S.D.%). A good precision was observed for all the investigated compounds presenting R.S.D.% values below 20%
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for both tap and wastewater matrices. Random differences between intermediate precision and precision were observed but, on average, similar R.S.D.% values were obtained, demonstrating that precision is not significantly affected by variances in either test day or analyst. Due to difficulties in the calculations of the individual uncertainty contributions following a “bottom up” procedure, as proposed by the ISO Guide to the Expression of Uncertainty in Measurement (GUM) [18], the different contributions were grouped as recommended by the EURACHEM/CITAC guide [19]. The contributions in the direct injection–RRLC-MS-MS method can be grouped into three terms, permitting the calculation of the overall uncertainty according to the following equation:
U=k
SR u2CRM + w √ nR
2
+
tolerance 2 √ 3
The first term (uCRM ) represents the uncertainty from the certified reference material used for calibration and the subsequent uncertainties introduced during weighing and diluting √ the sample. The second term (w(SR / nR )) represents the contribution of the intermediate precision of the method in which
w is a statistical coefficient for a desirable level of confidence and SR is the standard deviation for n experiments performed under intermediate precision conditions (nR ). The third term represents the tolerance that each laboratory establishes for their internal quality controls. Finally, k is the coverage factor to expand the uncertainty to the desired level of confidence. The second and third terms generally contribute the most to overall uncertainty. In the present work, the overall uncertainties were calculated for both the low and high concentration levels. uCRM was calculated by taking into account all the dilution steps, the uncertainties from the CRM, and all the volumetric material and balances used to prepare the calibration standards. SR was calculated from the nR = 6 results from the precision experiments (then w = 1.3 for a 95% level of confidence). The third term was calculated with a 10% tolerance which is the standard value in our laboratory for the verification of the daily calibration curve. Finally, a coverage factor of k = 2 was used for a confidence interval of 95%. As shown in Table 3, the uncertainties calculated for the low concentration level (0.03 and 0.1 g L−1 ) are in the order of 20% which is a typical value for a chromatographic method in a routine
Table 3 – Precision and intermediate precision (expressed as R.S.D.%) and overall uncertainties expressed as % (k = 2) calculated for the lowest calibration level Compound
Tap water Precision
Intermediate precision
0.03 g L Dimethoate Desethyl atrazine Cyanazine Bromacil Simazine Carbofuran Atrazine Isoproturon Ametryn Propazine Linuron Prometryn Trietazine Diazinon Chlorfenvinphos Ioxynil
2 13 11 3 16 5 8 14 6 12 13 10 8 15 10 10
10 11 10 5 15 5 6 10 11 12 11 8 14 5 7 13
0.1 g L−1 Desisopropilatrazine Metamitron Hydroxy atrazine Aldicarb Metribuzine Diuron Methyl-parathion Terbuthylazine Malathion Terbutryn Alachlor Parathion Fenthion Chlorpyrifos MCPA
9 11 8 8 14 15 13 3 5 6 2 9 10 8 15
9 7 8 10 10 7 7 13 6 15 10 12 3 11 6
Wastewater Uk=2
precision
Intermediate precision
Uk=2
19 20 20 15 27 15 16 20 20 20 20 17 23 15 17 12
4 10 7 15 12 7 9 8 7 6 7 10 13 7 16 16
20 9 14 7 10 15 11 6 7 14 15 3 14 8 14 12
27 17 23 15 17 34 20 15 15 23 20 14 20 17 20 12
18 15 16 17 18 17 15 21 15 20 18 20 14 20 17
8 7 5 7 6 13 9 2 13 3 6 14 12 8 10
3 6 4 7 9 23 8 10 3 8 3 9 7 9 6
14 15 14 17 17 28 13 14 14 16 13 18 15 16 17
−1
n = 6.
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Table 4 – Results obtained (ng L−1 ) in the interlaboratory comparison test Mean value by LC/MS
Target value
z-Score
Sample A Atrazine Prometryne Propazine Simazine Terbuthylazine Terbutryn
0.123 ± 0.025 0.079 ± 0.017 0.205 ± 0.049 0.215 ± 0.064 0.178 ± 0.045 0.289 ± 0.069
0.119 ± 0.02 0.065 ± 0.007 0.190 ± 0.03 0.206 ± 0.035 0.166 ± 0.022 0.270 ± 0.038
−0.45 −0.38 −0.83 −0.79 −0.98 −0.65
Sample B Atrazine Prometryne Propazine Simazine Terbuthylazine Terbutryn
<0.03 0.145 ± 0.032 0.125 ± 0.030 0.156 ± 0.047 <0.1 0.149 ± 0.036
<0.01 0.131 ± 0.016 0.101 ± 0.016 0.145 ± 0.025 <0.03 0.139 ± 0.023
− −0.87 −0.79 −0.96 – −0.83
z-Score = (mean value − target)/Std of accepted labs.
quality control laboratory. The average of the uncertainties for tap water is 17.9% and 17.7% for treated wastewater, demonstrating that this type of matrix does not contribute to the uncertainties of the method.
3.5.
Accuracy and proficiency testing with real samples
Accuracy was tested for the two investigated matrices at both low and high concentration levels by evaluating the six results of the intermediate precision experiment with the Student’s t-test. A method is defined as traceable when the value for the Student’s t-test calculated from the experimental results, tcal , is less than or equal to the Student’s t-test tabulated value (two-tailed). A confidence interval of 95% was considered with (n − 1) = 5 degrees of freedom, thus ttab = 2.015. For all the compounds under study in both matrices at both concentration levels, the values calculated for the Student’s t-test were below 2.015. For an external evaluation of accuracy, the proposed method was tested by an interlaboratory comparison test with real samples. The interlaboratory test was organized by IFA Tulln (Center for Analytical Chemistry of the Institute for Agrobiotechnology Tulln, Austria). Two samples encoded A and B, containing different levels of herbicides (simazine, cyanazine, atrazine, propazine, terbuthylazine, and prometryn) in real matrices (surface waters), were distributed to 12 different laboratories. The mean value obtained with the proposed direct injection RRLC/MS/MS method and the target value proposed by the organization with their corresponding uncertainties are presented in Table 4. The z-scores for both samples were less than −2, indicating that the results from all laboratories produced equivalent results using this method.
4.
Conclusions
The developed method allows the analysis of 31 pesticides in both tap water and wastewater in 11 min, making possible the analysis of the selected pesticides of up to 80 water samples per day, by the direct injection of 0.1 mL sample with LODs less than 10 ng L−1 for the majority of compounds. The low limits of detection, reliable linearity, precision, and accuracy of the developed method exceed the requirements for assessing adherence to limits established by the current European regulations governing pesticide contamination of aqueous environments. The increased efficiency and ease of sample processivity make this method a key tool for the routine quality control of priority pesticides in water.
references
[1] Directive 2000/60/EC of 23 October 2000 of the European Parliament and of the Council Establishing a Framework for the Community Action in the Field of Water Policy. [2] I. Valor, C. Cortada, D. Apraiz, Quim. Anal. 4 (1995) 228. [3] I. Valor, J.C. Moltó, D. Apraiz, G. Font, J. Chromatogr. A 767 (1997) 195. [4] J. Llorca-Porcel, G. Martínez-Sánchez, B. Álvarez, M.A. Cobollo, I. Valor, Anal. Chim. Acta 569 (2006) 113. [5] X. Yang, L. Ma, P.W. Car, J. Chromatogr. A 1079 (2005) 213. [6] D. Barceló, M. Petrovic, Trends Anal. Chem. 26 (2007) 2. [7] C. Zwiener, F.H. Frimmel, Anal. Bioanal. Chem. 378 (2004) 851. [8] S.D. Richardson, Anal. Chem. 78 (2006) 4021. [9] M. Mezcua, A. Agüera, J.Ll. Lliberia, M.A. Cortés, B. Bagó, A.R. Fernández-Alba, J. Chromatogr. A 1109 (2006) 222. [10] F. Hernández, J.V. Sancho, O. Pozo, A. Lara, E. Pitarch, J. Chromatogr. A 939 (2001) 1. [11] J.M. Marín, J.V. Sancho, O.J. Pozo, F.J. López, F. Hernández, J. Chromatogr. A 1133 (2006) 204. [12] H. Sabik, R. Jeannot, B. Rondeau, J. Chromatogr. A 885 (2000) 217. [13] R.J. Vreeken, P. Speksnijder, I. Bobeldijk-Pastorova, Th.H.M. Noij, J. Chromatogr. A 794 (1998) 187. [14] K. Stoob, H.P. Singer, C.W. Goetz, M. Ruff, S.R. Mueller, J. Chromatogr. A 1097 (2005) 138. [15] B.A. Ingelse, R.C.J. van Dam, R.J. Vreeken, H.G.J. Mol, O.M. Steijger, J. Chromatogr. A 1918 (2001) 67. [16] Council Directive 98/83/EC of 3 November 1998 On the Quality of Water Intended for Human Consumption, OJL 330, p. 32. [17] UNE-EN ISO/IEC 17025:1999: General Requirements for the Competence of Testing and Calibration Laboratories, CEN/CENELEC, Bruxelles, 2000. [18] ISO Guide to the Expression of Uncertainty in Measurement (GUM). BIPM, IEC, IFCC, ISO, IUPAC, IUPAP, OIML, Geneva, 1993. [19] EURACHEM, Quantifying Uncertainty in Analytical Measurements. EURACHEM Secretariat, Teddington, Middlesex, UK, 1995.