Minimize Method Development Time for Pesticide Screening Using LC/MS Application Kit
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1 Minimize Method Development Time for Pesticide Screening Using LC/MS Application Kit Dr. Jerry Zweigenbaum Senior Applications Scientist Food Safety Page 1
2 Agilent 1260 LC SampliQ Extraction and Dispersive SPE Kits Agilent 6460 LC/QQQ Pesticide Application Kit with Dynamic MRM Database for the LC/MS/MS TRIPLE QUADRUPOLE Page 2
3 Methods for YOUR Pesticides!!!! Page 3
4 Copy YOUR list to the Dynamic MRM Database and Search Page 4
5 Search on the METHOD and the Names to get RT You will Use Page 5
6 Database is Directly Linked to OPTIMIZER Page 6
7 Many Columns and Info but may be Hidden Page 7
8 Test Mix Positive Ion Mode: Quick Start Verification Page 8
9 Test Mix Negative Ion Mode Page 9
10 Dynamic MRM Acquisition Parameters Customized Methods from Database Page 10
11 Dynamic MRM at the 10 pg level for 100 pesticides 1 min ΔR.T.- based acquisition window for each MRM transition Page 11
12 Dynamic MRM 11 Compounds (22 MRMs) eluting in about 1 min window Compound Name Precursor Ion Product Ion Retention Time Cinosulfuron Cinosulfuron (Q) Chlorotoluron Chlorotoluron (Q) Atrazine Atrazine (Q) Carbaryl Carbaryl (Q) Carboxin Carboxin (Q) Chlorsulfuron Chlorsulfuron (Q) Ethiofencarb Ethiofencarb (Q) Dodemorph Dodemorph (Q) Diuron Diuron (Q) Cyprodinil Cyprodinil (Q) Difenoxurone Difenoxurone (Q) Page 12
13 Typical results: 10 pg Atrazine on-column using Dynamic MRM 6460A QQQ with Jet Stream Technology Avg. Signal Height: 15,650 Avg. Signal Area: 50,966 RSD: 3.2 % Estimated LOQ: 100 fg or less 6-7 data points above FWHM 3 sec FWHM, 6 valley 20 data points baseline-tobaseline 3 sec FWHM 10% valley Page 13
14 Pesticides LC/MS method 1200 LC parameters Column Eclipse Plus-C18, 2.1 x 100mm, 1.8 µm Column temperature 35 ºC Injection volume 5.0 ml Autosampler temp 6 ºC Needle wash flushport (MeOH:H2O 75:25), 5 secs Mobile phase A = 0.01% formic acid in water B =5 mm ammonium formate 0.01% formic acid in 95:5 acetonitrile:water Flow rate 0.3 ml/min Gradient 0 min 6% B 15 min 95% B Stop time 20 min 6% B Post time 1 min Drying gas temperature 275 ºC Drying gas flow (nitrogen) 6 L/min Nebulizer gas pressure (nitrogen) 35 psig Capillary voltage 4000 V Sheath gas temperature 325 ºC Sheath gas flow 12 L/min Nozzle voltage Off Jet stream conditions 6460A QQQ settings MS1 and MS2 resolution Unit Time Filtering peak width = 0.03 min Dynamic MRM transitions up to 4000 Constant cycle time 373 ms Delta EMV 400 V Page 14
15 Dynamic MRM of 300 Pesticides two Transitions Each Run with 1200-SL LC 2.1 x 100 C18 Eclipse PLUS 1.8 u Page 15
16 Page 16
17 Pesticide Mix run with Agilent 1290 Infinity 1260 Bar LC: Benefit of HIGHER PEAK CAPACITY LC 2.1 x 150 C18 Eclipse PLUS HD 1.8 u, 0.6 ml/min, 800 bar Page 17
18 Column Agilent Poroshell 120 EC-C18, mm, 2.7 μm x Counts vs. Acquisition Time (min) IS MRM chromatograms of 50 ng/g fortified sample processed by EN method. Peak identification: 1. Acephate, 2. Pymetrozine, 3. Carbendazim, 4. Thiabendazole, 5. Imidacloprid, 6. Imazalil, 7. Propoxur, 8. Carbaryl, 9. Cyprodinil, 10. Ethoprophos, 11. Penconazole, 12. Kresoxim-methyl, IS: TPP. Page 18
19 Page 19
20 Using YOUR Chromatography DYNAMIC MRM CREATE MY OWN Page 20
21 Building a Dynamic MRM Acquisition Method Load tabular MRM from Database Compound names, ISTD (optional) MRM transitions, fragmentor voltages, collision energies Analyze medium level standard mixtures fixed dwell time for each MRM transition using one time segment MHW Quantitative Data Analysis gets retention times Create a Dynamic MRM method Import the results generated in the custom report into acquisition method Specify delta retention time window Page 21
22 Building a Dynamic MRM Method 4.00 Page 22
23 Select Datafile and Parameters Page 23
24 Acquisition is Automatically Populated with Retention Times and RT Windows Page 24
25 Next View the Method Page 25
26 Page 26
27 View Method Provides Important Information in Cohesive Format Page 27
28 Finally Set the Cycle Time Page 28
29 Typical results: 10 pg Atrazine on-column using Dynamic MRM 6460A QQQ with Jet Stream Technology Avg. Signal Height: 15,650 Avg. Signal Area: 50,966 RSD: 3.2 % Estimated LOQ: 100 fg or less 6-7 data points above FWHM 3 sec FWHM, 6 valley 20 data points baseline-tobaseline 3 sec FWHM 10% valley Page 29
30 Summary Dynamic MRM Database provides quick method setup with compound specific parameters and retention times Compounds not in method can be easily added New compounds can easily be added via optimizer to method and database Custom methods can be easily developed and Dynamic MRM readily created with Update Method Page 30
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