Determination of Chlorinated Acid Herbicides in Soil by LC/MS/MS Application Note

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1 Determination of Chlorinated Acid Herbicides in Soil by LC/MS/MS Application Note Environmental Author Chin-Kai Meng Agilent Technologies 85 Centerville Road Wilmington, DE USA Abstract Chlorinated acid herbicides were analyzed at the picogram level on column without any derivatization using liquid chromatography and tandem mass spectrometry (LC/MS/MS). Good linearity was observed for all the selected analytes, from low pg to low ng levels on column. Introduction Chlorinated acid herbicides are a popular class of broad-leaf weed killer in lawn and grain crops. Due to their widespread use, environmental contamination in water and soil from run-off is a serious concern. Traditional analytical methods based on gas chromatography (GC) and/or mass spectrometry (MS) require derivatization of the analytes. Combining LC and electrospray ionization (ESI) in negative ion mode, these herbicides can be analyzed without derivatization. The multiple reaction monitoring (MRM) mode in MS/MS operation provides low pg detection limits. This application note is based on standards and sample preparation procedures from the Montana Department of Agriculture in Bozeman, Montana. Experimental Standard and Sample Preparation A stock solution of each analyte at ppm is prepared in methanol. Intermediate mixed solutions for fortifying soil samples and making calibration standards are made by accurately mixing aliquots of each standard stock solution. The concentrations of each analyte in the intermediate solution used in this study are listed in Table. Table. Acid Herbicide Mixed Intermediate Standards in Methanol Clopyralid 59 (pg/µl),6-dichloro--pyridinecarboxylic acid Picloram 8 4-amino-,5,6-trichloropicolinic acid Dicamba 8,6-dichloro--methoxybenzoic acid,4-d 74,4-dichlorophenoxyacetic acid MCPA 548 -methyl-4-chlorophenoxyacetic acid Triclopyr 4 [(,5,6-trichloro--pyridinyl)oxy] acetic acid,4-dp 4,4-dichlorophenoxypropionic acid or dichloroprop MCPP 7 -(-methyl-4-chlorophenoxy) propionic acid,4-db 69,4-dichlorophenoxybutyric acid

2 Sample extraction and cleanup procedures are shown below. Sample Extraction Procedure. Weigh ±. g of soil.. Add 5 ml of.5n KOH in % KCl extracting solution to each sample. Mix thoroughly by shaking.. Place samples in boiling water bath for 5 minutes. 4. Place samples on horizontal shaker for 5 minutes. 5. Centrifuge samples at to 5 rpm for 5 minutes. 6. Transfer a.-ml aliquot into a -ml conical centrifuge tube and add 5 µl of N sulfuric aicd. 7. Vortex and confirm the ph is <.5. If not, add additional acid solution. Sample Cleanup Procedure. Add ml of chloroform to the acidified extract.. Vortex seconds and centrifuge at rpm for minutes.. Remove the lower chloroform layer into another centrifuge tube. Repeat these three steps two more times. 4. Evaporate the chloroform extract to just dryness. 5. Immediately add 4. ml HPLC-grade water, vortex briefly, sonicate 5 minutes, and briefly vortex again. Fill autosampler vial. See Reference for more detailed information on sample preparation. Instrumentation LC: LC Column: ZORBAX Extend-C8, RRHT,. mm mm,.8 µm Column temperature: 6 C Mobile phases: A:.4% Glacial acetic acid in water B: Acetonitrile (ACN) Flow rate:. ml/min Injection volume:. µl Gradient: Time, min. %B MS: G64A QQQ Ionization: ESI ( ) Mass range: to 4 amu Capillary: 5 V Nebulizer pressure: 4 psi Drying gas flow: 9 L/min Gas temperature: C Skimmer: 5 V MRM parameters are listed in Table. Table. Method Parameters for MRM Name RT MW Quant Qual Frag V Col cell Dwell Segment Clopyralid > 46 9 > Picloram > 95 4 > Dicamba 4. 9 > 75 9 > ,4-D 5. 9 > 6 > MCPA > 4 > 4 5 Triclopyr > > ,4-DP > 6 5 > MCPP > 4 5 > 4 5,4-DB > 6 49 > 6 8 5

3 Due to the concentration differences for these analytes in the intermediate solution (Table ), Dicamba s concentration was used as the concentration level shown in Table. Table. Concentration Levels (8 to pg/µl) Used in This Study Solution concentration level Clopyralid Picloram Dicamba ,4-D MCPA Triclopyr ,4-DP MCPP ,4-DB For example, concentration level 8 is the intermediate solution, and concentration level is a :4 dilution of the intermediate solution. Results and Discussion Figure shows the overlaid chromatograms of all nine herbicides from the MRM analysis. The run time was less than 6 minutes. Using a.8-µm particle size column, the peak widths of these analytes are in the range of. to. min. The narrower peak width helps to achieve a higher signal-tonoise ratio. The MRM results of all nine herbicides at.6 to. pg on column are shown in Figure. As shown in Table, the linearity range, incorporating,, 4, 8,, 4, 8, and 8 pg on column, mimics the concentration of Dicamba EIC (6. m/z) MRM (47. m/z); from mont_neg_8.d MCPA 4 MCPP Dicamba, 4-DB, 4-DP, 4-D Clopyralid Triclopyr Picloram Abundance vs. Acquisition Time (min) Figure. Overlaid MRM results from the nine selected herbicides.

4 -EIC (46. m/z) MRM (9. m/z); from mont_neg_.d Smooth ().5 Clopyralid, 7.4 pg EIC (95. m/z) MRM (9. m/z); from mont_neg_.d Smooth ().7 Picloram,. pg -EIC (75. m/z) MRM (9. m/z); from mont_neg_.d Smooth () 4.79 Dicamba,. pg -EIC (6. m/z) MRM (9. m/z); from mont_neg_.d Smooth (),4 -D,. pg EIC (4. m/z) MRM (99. m/z); from mont_neg_.d Smooth () MCPA, 6.9 pg EIC (96. m/z) MRM (54. m/z); from mont_neg_.d Smooth () 5.7 Triclopyr,.6 pg 6 -EIC (6. m/z) MRM (. m/z); from mont_neg_.d Smooth (),4 -DP,.8 pg EIC (4. m/z) MRM (. m/z); from mont_neg_.d Smooth () MCPP,.4 pg.5 -EIC (6. m/z) MRM (47. m/z); from mont_neg_.d Smooth () 5.67,4 -DB, 8.6 pg Abundance vs. Acquisition Time (min) Figure. MRM results. Therefore, the corresponding on column amounts for Triclopyr are:.6,., 6.,.4,, 6, 4, and 4 pg. The calibration model used was a linear model that included origin with no weighting. All analytes showed excellent linearity. In the repeatability study, seven -µl injections of the level 4 solution (Table 4) were analyzed to calculate the RSD. At this low concentration, all RSDs were <5%, with the majority in the single digits. The matrix effect from three different matrices was evaluated. Water and soil extracts were spiked with the herbicide standards (5 µl of the level 4 standard were added to 95 µl of water, silt, clay, or sandy extracts). The resulting concentration of the analytes is equivalent to the level solution (Table ). Table 4. Linearity ( to 8 pg/µl) and Repeatability Compound Clopyralid Picloram Dicamba,4-D MCPA Triclopyr,4-DP MCPP,4-DB R (linear fit, include origin) Repeatability, %RSD (n=7) Amount on column 9.7 pg Table 5 shows the RSD of eight -µl injections of the level solutions, that is, < pg of each ana- 4

5 lyte on column, in three different matrices. As expected, analytes with lower absolute responses showed higher RSD value. Table 5. RSDs from Eight Injections of < pg of Each Analyte in Three Matrices Clay Sandy Silt Triclopyr 7 MCPP MCPA 6 5 Clopyralid 4 5,4-DP 9 8,4-DB 9 9,4-D 4 On column. pg The repeatabilities of responses in three matrices for all analytes were <5% except for Triclopyr, which was >%. The responses of ~ pg analytes among water and matrices are compared in Figure. The listed response for each matrix is the average of responses from eight injections. The RSDs for water and the three matrices are shown in Figure and are comparable. In general, the variation of the responses among water and different matrices is less than 5% for all analytes except for,4-d, which has an RSD close to % due to the higher responses from the silt matrix. This shows that the method described in this application note is free from matrix interferences from clay, sand, and silt. The RSDs of < pg analytes in the selected matrices were comparable to that in water (RSDs ~5%), except for,4-d, due to the higher response from the silt. Acknowledgement The author would like to thank Ms. Heidi Hickes and Ms. Angela Schaner from the Montana Department of Agriculture for valuable discussions, sample preparation procedures, and the samples used in this study. Reference. Determination of Chlorinated Acid Herbicides in Soils by Liquid Chromatography- Electrospray/Mass Spectrometry/Mass Spectrometry, by Angela Schaner and Laura Luckey, Revision, April, 4. Montana Department of Agriculture, Laboratory Bureau, McCall Hall, Montana State University, Bozeman, MT For More Information For more information on our products and services, visit our website at Conclusion Using LC/MS/MS, chlorinated acid herbicides were analyzed at pg levels on column without any derivatization. At <4 pg on column, the repeatability of seven -µl injections showed RSD <5%. Good linearity was observed for all analytes from low pg to low ng on column Triclopyr MCPP MCPA Clopyralid,4-DP,4-DB,4-D Level in water Level in clay Level in sandy Level in silt Figure. Analyte RSDs from water and soil extracts are comparable. 5

6 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. 6 Printed in the USA July 5, EN

Determination of Pharmaceuticals in Water by SPE and LC/MS/MS in Both Positive and Negative Ion Modes Application

Determination of Pharmaceuticals in Water by SPE and LC/MS/MS in Both Positive and Negative Ion Modes Application Environmental Authors Chin-Kai Meng Agilent Technologies, Inc. 85 Centerville Road Wilmington,