Authors. Introduction. Experimental. Environmental Analysis

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1 Determination of Phenyl Urea and Triazine Herbicides in Potable and Groundwater by LC/MS Using API-ESI Selective Ion Monitoring and Direct Large-Volume Aqueous Injection Application Environmental Analysis Authors Neil Cullum Anglian Water Services Laboratories Huntingdon, UK Pete Stone Agilent Technologies Ltd Cheadle, UK Introduction The application note presents the analyses of a group of phenyl urea and triazine herbicides as a single analytical suite by large volume injection of an aqueous sample. Phenyl urea herbicides have found widespread use for weed control in crops, isoproturon being an example. The triazine herbicides have been extensively used as general weed control agents, atrazine being an example. Both phenyl urea and triazine herbicides have been detected in drinking water. The Prescribed Concentration or Value (PCV) for an individual pesticide in drinking water in the UK, as defined by the Water Supply (Water Quality) Regulations, is set at.1 µg/l; the method of analysis should be capable of detecting 25% of the PCV that is,.25 µg/l. Traditionally, these herbicides were analyzed by liquid chromatography/ultra violet (LC/UV) or diode array detector (DAD) with offline solid phase extraction (SPE) using typical sample volumes of between 5 ml and 1 L. With the introduction of liquid chromatography/mass spectrometry (LC/MS), sample volumes could be reduced down to between 25 5 ml due to the increased sensitivity of the detection system [1]. This application note details the analysis of these compounds by direct aqueous injection, negating the need for the normal sample preparation stages, and thus producing time and cost savings that would be attached to an offline SPE method. Experimental All analyses were performed using Agilent 11 series liquid chromatograph/mass selective detector (LC/MSD) quadrupole (G1956B) coupled to an Agilent 11 series LC system consisting of two binary pumps, vacuum degasser, thermostated wellplate autosampler, and thermostated column compartment with a 1-port valve installed. See Table 1 and Figure 1. The system also had a variable wavelength detector inline before the mass spectrometer for monitoring and trouble shooting purposes. The quadrupole mass spectrometer was fitted with an atmospheric pressure electrospray ionization (API-ESI) source operated in both positive and negative ion modes. See Table 2.

2 Table 1. Column HPLC Conditions ZORBAX Eclipse XDB-C8 5 mm 2.1 mm; C Flow rate.5 ml/min Precolumn ZORBAX SB C18 Rapid Resolution 3 mm 2.1 mm; C Injection volume 5 µl Mobile phase A =.1% Formic acid in water B = Methanol Pump program Pump 1 (Analytical column) Time Mobile phase Mobile phase Flow rate (min) A B (ml/min) Initial Pump 2 (Precolumn) Time Mobile phase Mobile phase Flow rate (min) A B (ml/min) Initial Column switching valve Time Valve Temp (L) Temp (R) (min) Position ( C) ( C) Initial Wellplate autosampler Additional 4-µL loop install to needle seat 5 1-µL injections: - Total 5 µl Draw speed = 2 µl/min Eject speed = 5 µl/min Flushport = 5. s (methanol) 2

3 Flow Path Diagram The flow path through the column compartment 1-port valve and the wellplate 6-port autosampler valve is shown in Figure 1. Right heat exchanger Analytical column Left heat exchanger Detector Column compartment Precolumn Waste Pump 1 Pump 2 Metering head Autosampler valve 4 5 Waste Needle seat + 4 µl loop Figure 1. Flow path diagram. During the sample injection stage, the switching valve in the column compartment is set at position 1 and pump 1 (the analytical gradient pump) equilibrates the analytical column. Pump 2 (the loading pump) is flowing through the autosampler, precolumn, and then on to waste isocratically. Both mobile phase compositions are at this point identical, set at the initial gradient point for the start of the separation. At the point where the total volume of sample injected is known to have passed through the sample precolumn (or trapping column), the switching valve is set to position 2 and flow from pump 1 (the analytical gradient pump) is then directed backwards through the precolumn and onto the analytical column before taking trapped analytes through with the method gradient to the mass spectrometer. Pump 2 (the sample loading pump) then goes straight to waste and its flow is reduced to save mobile phase useage. A 6- or 1-port, 2-position valve can be used for this purpose. 3

4 Table 2. Mass Spectrometer Conditions MS Conditions Ionization mode: Positive/Negative API-ES Drying gas flow: 13. L/min Nebulizer pressure: 4 psig Drying gas temperature: 35 C V cap voltage: 3V (Positive); 25 (Negative) Selected Ion Monitoring (SIM) Table Parameters (Positive ion mode) Compound Time Group SIM ion Frag voltage Gain Metamitron. Group q* 7 Chloridazon q 1 Monuron 1.25 Group q 115 Simazine q 7 Carbetamide q 95 Cyanazine q 13 Isoproturon 13. Group q 14 Chlortoluron q 13 Atrazine q 135 Propazine Group Terbuthylazine 232. q 13 Trietazine Prometryn Terbutryn 243. q 13 SIM Table Parameters (Negative ion mode) Diuron. Group q 13 Linuron Group q 115 *q qualifier ion The selected ion monitoring (SIM) ions and fragmentor voltages listed in the SIM table parameters were all optimized using Flow Injection Analysis (FIA). Standard solutions of 1 mg/l each herbicide were injected using scan mode 15 4 amu and the fragmentor voltage was ramped from 7 to 15V in steps of 5V. 4

5 Results Figure 2, 3, and 4 show an extracted ion chromatogram from spiked tap water containing atrazine, isoproturon, and diuron respectively at a concentration of.1 µg/l. Figure 5 also shows an extracted ion chromatogram, again from a spiked tap water containing prometryn and terbutryn at a concentration of.1 µg/l (5 pg on-column). MSD1 216, EIC=215.7:216.7 (VAL3_4\PT3_426.D) API-ES, Pos, SIM, Frag: 7 (TT) MSD2 233, EIC=232.7:233.7 (VAL3_4\PT3_426.D) API-ES, Neg, SIM, Frag: 13 (TT) Diuron Atrazine Minute 15 Figure 4. Spiked tap water containing.1 µg/l of diuron. 1 5 Figure Minute Spiked tap water containing.1 µg/l of atrazine MSD1 242, EIC=241.7:242.7 (VAL3_4\PT3_424.D) API-ES, Pos, SIM, Frag: 7 (TT) Terbutryn Prometryn MSD1 27, EIC=26.7:27.7 (VAL3_4\PT3_426.D) API-ES, Pos, SIM, Frag: 7 (TT) Isoproturon Figure Minute Spiked tap water containing.1 µg/l of prometryn and terbutryn. 1 5 Figure Minute Spiked tap water containing.1 µg/l of isoproturon. Validation of the method was done on 11 sample batches. Standards were prepared at three levels.1,.1, and.4 µg/l. The borehole raw water was spiked at two levels:.1 and.1 µg/l. The potable tap water (which was derived from a surface water source) was also spiked at two levels:.1 and.1 µg/l. All samples were analyzed in duplicate in each batch in a random order. 5

6 The limit of detection (LOD) for each herbicide was calculated from the within-batch standard deviation of the standard spiked at.1 µg/l. Recovery for both borehole water and potable water samples was calculated from the.1 µg/l spike after subtraction of the.1 µg/l spiked value, hence the recovery value is based on.9 µg/l. See Table 3 for method performance data. Table 3. Method Performance Data Borehole raw water Potable treated water Compound %Recovery %RSD %Recovery %RSD LOD (µg/l) Metamitron Chloridazon Monuron Carbetamide Simazine Cyanazine Chlortoluron Atrazine Diuron Isoproturon Linuron Propazine Terbuthylazine Trietazine Prometryn Terbutryn

7 Calibration curves were produced using three calibration levels at.1,.3, and.5 µg/l. The calibration curves were all produced using a quadratic fit and forced through the origin. Typical correlation values are.999 or better for all the herbicides in the suite. A typical calibration curve is shown in Figure Simazine, MSD1 22 Area = *Amt^ *Amt + Conclusion The data shows that the method presented is capable of quantitative analysis for the 16 herbicides in a single analytical suite, using a direct aqueous injection of 5 µl, with no offline preconcentration step. Online trapping of analytes was undertaken by the precolumn, with the water matrix directly passing to waste as part of the method. The performance requirements set by the Drinking Water Inspectorate (DWI) for standard deviation, bias, recovery, and total error are all met. 12 Rel. Res%(1): References Validated method for the determination of Phenyl Urea and Triazine herbicides in potable and groundwater by LC/MS using selective ion monitoring. Agilent Technologies publication EN Area 6 2 For More Information 4 For more information on our products and services, visit our Web site at Correlation: Amount (µg/l) Figure 6. A typical calibration curve. 7

8 Agilent shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing, performance, or use of this material. Information, descriptions, and specifications in this publication are subject to change without notice. Agilent Technologies, Inc. 24 Printed in the USA March 29, EN