Quantification of trace-level herbicides in drinking water by online enrichment with the Agilent Infinity Series Online-SPE Solution and Triple Quadrupole MS Detection Application Note Environmental Author Terbutryn Edgar Naegele Agilent Technologies, Inc. Waldbronn, Germany Methabenzthiazuron Desmetryn Atrazine Diuron Monolinuron Terbuthylazine Chloroxuron Propazine Prometryn Trietazine Neburon Abstract This Application Note demonstrates the use of the Agilent Infinity Series Online-SPE solution combined with triple quadrupole mass spectrometric detection for the analysis of herbicides at trace levels down to ppt in drinking water. Performance data of the online-spe system for linearity, area and retention time precision, recovery and concentration precision, and accuracy in real samples is shown and discussed.
Introduction According to the requirements of the European Union drinking water directive 98/8/EC, pollutants such as neutral herbicides have to be monitored in drinking water. The current regulation demands a limit of detection (LOD) of ng/l ( ppt) for all pesticides. To achieve this limit of detection with an entry level or mid-range triple quadrupole mass spectrometer, a larger volume of the water sample (typically > ml) has to be enriched on a trapping column. Then, the compounds are eluted to the analytical HPLC column for separation. This Application Note describes the Agilent Infinity Series Online-SPE solution based on the Agilent 9 Infinity Flexible Cube for first, enriching different trace level herbicides and second, separating them from each other. The online-spe system comprises an Agilent Infinity Quaternary pump because commercially available SPE cartridges work satisfactorily at bar. The full functionality of the online-spe system is achieved using only one LC pump. The performance of the system is demonstrated by the mass spectrometric detection of a suite of 8 neutral herbicides down to a LOD of less than ppt. To enhance the throughput of the system, a valve solution for the parallel use of two alternating trapping columns is presented. Experimental Instrumentation Agilent Infinity Series Online-SPE solution system comprising: Agilent Infinity Quaternary Pump with internal degasser GC and LAN card G9C Agilent Infinity Standard Autosampler G9B with 9 µl head (G-), multidraw kit (G-8) and thermostat GB Agilent 9 Infinity Flexible Cube GA with -position/-port valve GB Agilent 9 Infinity Thermostatted Column Compartment GC Figure shows how the modules of the system are set up, and Table lists the capillaries and accessories needed to run the online-spe application. With the Agilent part number G-8 for the Online SPE Starter Set, all necessary parts such capillaries and the -position/-port valve head can be ordered in one package. MS-Detection Agilent Triple Quadrupole LC/MS with Agilent Jet Stream Technology Analytical Column Agilent ZORBAX Eclipse Plus C8,. mm,. µm (p/n 99-9) Trapping columns x Guard Column Hardware Kit (p/n 8999-9) Agilent PLRP-S Cartridges,.. mm, - µm (p/n 98-) Software Agilent MassHunter data acquisition for triple quadruple mass spectrometer, Version. Agilent MassHunter Optimizer software, Version. Agilent MassHunter Qualitative software, Version. Agilent MassHunter Quantitative software, Version. Column Compartment Flexible Cube MS Autosampler Quaternary Pump Thermostat Figure Setup of the Agilent Infinity Series Online-SPE solution with MS detector (the solvent bottles in the center are for SPE loading, rinsing and conditioning).
Parts Qty Description Order No. -position/-port valve head Valve head to be mounted in Flexible Cube - Guard column hardware kit To insert SPE cartridge 8999-9 Online SPE capillary kit Contains required capillaries -8 Table a Parts of the Online SPE Starter Set GA which is required for the Agilent Infinity Series online SPE solution. Parts and Capillaries Qty Description Order No. mm,. mm id, SST Valve to guard column hardware and back to - valve; and one valve crossing BondElut online PLRP-S - µm.. mm Table b Capillaries and parts of the online SPE capillary kit (SST = stainless steel). SPE cartridges 98- mm,. mm id, SST Valve to column, valve to autosampler - Waste line m Valve to waste 89- mm,. mm id, SST Flexible Cube pump to autosampler - mm,. mm id, SST LC pump to valve -8 Finger tight fitting For waste line - HPLC Method Agilent Infinity Quaternary Pump: Solvent A: Water, mm ammonium formiate +.% formic acid Solvent B: ACN + % water, mm ammonium formiate +.% formic acid Flow rate:. ml/min Gradient: minutes % B, minutes % B, minutes 98% B. Stop time: minutes Post time: minutes Agilent 9 Infinity Thermostated Column Compartment: Column temperature: C Agilent 9 Infinity Flexible Cube: Right valve: -position/-port QuickChange valve head Pump:. ml/min Solvent: A: Water, B: ACN minutes Pump s, Solvent A minutes right valve change position minutes Pump 8 s, Solvent B minutes Pump s, Solvent A Agilent Infinity Standard Autosampler Injection volume:,8 µl (automated multidraw of times 9 µl) Needle wash in vial (MeOH) Draw and eject speed:, µl/min. Sample temperature: C. halftrays for ml vials G- -ml screw cap vials (glass, p/n 9-), screw caps (p/n 9-9), pre-slit septa for -ml screw cap vials (p/n 88-8)
In the setup of the online-spe LC system, the 9 Infinity Flexible Cube (Figure ) is hosting the -position/ port valve with two trapping columns next to the piston pump and the solvent selection valve for flushing the sample on the trapping columns and for the re-equilibration of those columns (Figures A and B). The piston pump inside the Flexible Cube is connected to the autosampler to flush the sample directly onto one trapping column (SPE ) while the other trapping column (SPE ) is eluted in front of the analytical column and connected to the LC pump (Figure A). Rail for additional valves Solvent selection valve (three wash solvents) Piston pump ( ml/min, bar) Quick-Change valves and Figure The Agilent 9 Infinity Flexible Cube is an additional module for the Agilent 9/ Infinity LC system, hosting up to two Agilent Infinity Series Quick-Change valves. Analytical column Agilent Triple Quadrupole MS Flexible cube SPE (Elute) Solvent selection valve 9 8 Piston pump SPE (Load) Standard Autosampler (9 µl Head) LC Pump Waste Figure A Valve positions for loading the sample on trapping column SPE while trapping column SPE is being eluted.
After loading the trapping column with sample, the -position/-port valve is switched and thus the positions of the trapping columns are exchanged (Figure B). Now, the LC pump delivers the gradient to elute the enriched analytes in backflush mode from the trapping column (SPE ) onto the analytical column. Simultaneously, the trapping column (SPE ) which had been loaded with sample in the previous run is cleaned and reconditioned by a purging procedure. This cleaning procedure is done by the piston pump with the cleaning solvents selected by the solvent selection valve. Table shows a summary of the LC method for the main modules. Flexible cube Solvent selection valve Piston pump Standard Autosampler (9 µl Head) SPE (Elute) Analytical column SPE (Load) 9 Agilent Triple Quadrupole MS 8 LC Pump Waste Figure B Valve positions for loading the sample on trapping column SPE while trapping column SPE is being eluted. Agilent Infinity Standard Autosampler Agilent Infinity Quaternary Pump multidraw 8 µl sample Inject % Solvent B Gradient % B to 98% B 98% Solvent B post-run Agilent 9 Infinity Flexible Cube Minutes Pump seconds Switch valve to next position pump 8 seconds pump seconds solvent 8 9 8 9 Table Summary of the LC method for the Agilent Infinity Standard Autosampler, the Agilent Infinity Quaternary Pump and the Agilent 9 Infinity Flexible Cube.
Triple Quadruple MS method Agilent Jet Stream thermal gradient focusing technology: Gas temperature: C Gas flow: 9 L/min Nebulizer: psi Sheath gas temperature: C Sheath gas flow: L Capillary:, Volt Nozzle: Volt The MRM and dynamic MRM triple quadrupole MS method was developed by means of the MassHunter optimizer software and direct injections of individual pesticide standards ( ng/µl) by flow injection into the mass spectrometer. The optimization was done to find the optimum fragmentor voltage for each individual compound and the optimum collision energies for the fragmentation to the quantifier and qualifier ions (Table ). Name RT Precursor Precursor ion [M+H]+ Fragmentor Fragment ion (quantifier) CE Fragment ion (qualifier) CE Atrazine desisopropyl... 9.. Carbendazim. 9. 9... Metamitron..... Fenuron.8.9. 8.. Atrazine desethyl.9 8. 88... 8 Chloridazon.9... 9. Carbetamide... 8. 8 9. Metoxuron. 8./. 9./... Monuron.9 98./. 99./. 9.. Simazine.8.8... Cyanazine..9... Methabenzthiazuron.8.. 9.. Chlorotoluron.8./../... Desmetryn.9... 8. Atrazine..9... 8 Isoproturon.8.... Diuron../../... Monolinuron... 8. 8. 8 Propazine. 9... 88. Linuron.8 8. 9. 9 9.9 8. Terbuthylazine.9 9.... Chloroxuron. 9.8/9.8 9./9... Irgarol... 98. 8. 8 Prometryn... 8.. Diflubenzuron... 9 8. 8. Terbutryn.8.. 8. 8. 8 Trietazine 8. 9.. 99.. Neburon 8... 88.. (Fragmentor = Voltage [V] RT = retention time [min] CE = collision energy [ev]) Table MRM and dynamic MRM MS method, showing the identified optimum fragmentor and collision energy values for the individual pesticides as well as for the quantifier and qualifier ions. The retention time was used to develop the dynamic MRM method with a window of ± times the peak width around the compound retention time. For some chlorinated compounds, the transition from both chlorine isotopes to the same fragment were used when other transitions were of lower abundance.
The developed MRM method was applied to a ng/l ( ppt) mixture of all standards to identify the retention time of the individual compounds in the final SPE LC method. From the resulting data file, the dynamic MRM method was developed with a retention time window of ± times the measured peak width around the retention time of each compound (Figure ). Chemicals All solvents were LC/MS grade. acetonitrile was purchased from J.T. Baker, Germany. Fresh ultrapure water was obtained from a Milli-Q Integral system equipped with LC-Pak Polisher and a.-µm membrane point-of-use cartridge (Millipak). All pesticide standards were purchased from Dr. Ehrenstorfer GmbH, Germany at a concentration of mg/l in acetonitrile. Calibration standards A stock solution containing all pesticides was prepared by dilution of the purchased standards to ng/l ( ppt) each in water. The dilution series for determination of the LOD, LOQ, and the calibration curve was,,,,,,, and. ppt. Samples Water samples were taken directly from the Rhine river, from tap water, and from a spring in the region of Karlsruhe, Germany. The water samples were spiked to a final concentration of ppt with a concentrated pesticide solution containing all 8 pesticides, vortexed, filtered with a syringe filter (. µm) and injected without further sample prep. 9 8 8 9 Figure MRM chromatograms for a calibration standard with a concentration of ppt (ng/l) each for all 8 pesticides measured by the final SPE LC dynamic MRM method with quantifier and qualifier ion. Results and Discussion Calibration curves for each individual compound were obtained by diluting the stock solution containing all 8 pesticides at a concentration of ng/l ( ppt) in a dilution series down to. ng/l (. ppt). The pesticides were measured with the developed online-spe LC method using dynamic MRM. Each calibration standard was injected four times with a volume of,8 µl and enriched on the SPE trapping column. The value at a signal-to-noise (S/N) ratio of was used for the LOD and the value at a S/N ratio of was used as LOQ. The calibration curve was calculated from LOQ up to ng/l. Figures A and B show the quantifier transition (m/z. & m/z.) of isoproturon for the concentration level of ppt to ppt (Figure A) and for ppt to the LOQ of ppt (Figure B) at a retention.8 ppt ppt ppt ppt ppt....8.9........8.9 Figure A Dynamic MRM chromatograms for the quantifier transition m/z. &. of Isoproturon, at a concentration of to ppt.
time of.8 minutes. The calibration curve of the seven levels from the LOQ of ppt to ppt was calculated including all 8 injections and resulted in a linear coefficient of.998 (Figure )..8 ppt ppt ppt ppt....8.9........8.9 Figure B Dynamic MRM chromatograms for the quantifier transition m/z. &. of isoproturon at a concentration of to ppt. R =.998 Responses 8 8 9 9 Concentration (ng/l) Figure Calibration curve of Isoproturon at a concentration of ppt ppt (seven levels, seven levels used, 8 points, 8 points used), linear coefficient.998, LOQ ppt. 8
Table outlines the complete set of data for all 8 pesticides. Typically, the LOQs were between ppt and ppt and the respective LODs were between ppt and. ppt. Linear coefficients were good for all compounds, with values typically better than.99. The relative standard deviation (RSD) of the retention timed was excellent with values typically below.%. The RSD of the peak areas was typically between.% and.%. The recovery of the SPE trapping process was determined by comparing the peak areas of an injection onto the SPE column to a direct injection of the same concentration level and volume (9 µl of ppt standard) onto the analytical column. The recoveries of compounds were > 9%, the other compounds were between 8 and 9% (Table ). Name r.t. LOQ (ng/l) (S/N=) R LOD (ng/l) (S/N=) Area RSD (%) r.t. RSD (%) Recovery (%) Atrazine desisopropyl..999... 8. Carbendazim..99..8. 88.8 Metamitron..9988... 8.8 Fenuron.8.998... 9. Atrazine desethyl.9.99... 9. Chloridazon.9.99..9. 9.8 Carbetamide..998..9. 98. Metoxuron..998... 9.8 Monuron.9.998..8. 9. Simazine.8.998... 9.9 Cyanazine..99... 9. Methabenzthiazuron.8.998... 9. Chlorotoluron.8.998... 9.9 Desmetryn.9.998... 9. Atrazine..998... 9.9 Isoproturon.8.998..8. 98. Diuron..998...8 8. Monolinuron..99..8. 9. Propazine..998..8. 9. Linuron.8.998...8 8. Terbuthylazine.9.99....9 Chloroxuron..998.... Irgarol..99. 8.. 89.8 Pormetryn..9988... 9. Diflubenzuron..99... 8. Terbutryn.8.9988...8 9. Trietazine 8..998..9. 9. Table Performance data for all pesticide compounds present in the study. (R = linear coefficient, RSD = Relative standard deviation, r.t. = retention time, RSD (%) and recovery (%)). 9
The carryover was determined for three of the most intense compounds isoproturon (Figure A), terbutryn (Figure B) and metoxuron (Figure C). The carryover from a ppt injection of isoproturon to a subsequent blank injection was determined to be at.%. This was approximately % of the LOQ (Figure A). The carryover from a ppt injection of terbutryn to a subsequent blank injection was determined to be.8%, which was approximately % of the LOQ (Figure B). The carryover from a ppt injection of Metoxuron to a subsequent blank injection was below the LOD (Figure C).....,.... ppt Test injection ppt LOQ Carryover from ppt Figure A Carryover from a ppt injection of isoproturon to a following blank injection was determined to be.%. This was approximately % of the LOQ. 8 8,8 ppt Test injection. 88 ppt LOQ 9 8 Carryover from ppt.. 8 Figure B Carryover from a ppt injection of terbutryn to a following blank injection was determined to be.8%. This was approximately % of the LOQ.
Finally, water samples from the Rhine river, tap water, and spring water were spiked with all 8 pesticides to a final concentration of ppt. Analysis of all samples yielded comparable intensities for a large number of the spiked herbicides independent from the source of the water sample (Figure 8). This indicates that residual salt contaminations from the water samples or other contaminants with high ion strength which might cause ion suppression were effectively flushed out of the SPE column. The spiked tap water and river water samples were rich in calcium hydrogen carbonate. The measured concentrations of all pesticides shown in Figure 8 were averaged dependent on the source of water. The calculated concentration precision was between.% and.8%. The concentration accuracy was always above 9%. Conclusion This Application Note demonstrates the use of the Agilent Infinity Series Online-SPE solution for enrichment, separation, and detection in trace level analysis of pesticide residues in water samples by HPLC with triple quadrupole MS detection. It was demonstrated that lowest LOD of. ppt and LOQ as low as ppt could be achieved. The methodology shows a high sample-to-sample reproducibility with area deviation of less than %. The efficient online-spe trapping process allows pesticide detection in real drinking water samples well below the regulatory limits with high precision and accuracy. Reference. European Union Drinking Water Directive 98/8/EC http://ec.europa. eu/environment/water/water-drink/ index_en.html...., ppt Test injection ppt LOQ Carryover from ppt. Figure C Carryover from a ppt injection of Metoxuron to a following blank injection could not be determined.... Rhine river water Tap water Spring water Average concentration (n=) [ng/l]..8.88 SD..8.9 Precision RSD [%]...8 Accuracy [%] 9. 98. 9. Methabenzthiazuron Terbutryn. Chloroxuron Terbuthylazine Prometryn Desmetryn. Propazine Atrazine Diuron Trietazine Neburon Monolinuron.... 8 8. 9 Figure 8 Water samples from a tap, Rhine river, and a spring spiked with ppt of all 8 pesticides. The retention time window from. minutes to 9. minutes is shown. The table shows the average measured concentrations of all pesticides (n=) within the retention time window dependent on the sources of water together with precision RSD and accuracy.
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