On Line SPE LC/MS/MS Applications
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1 On Line SPE LC/MS/MS Applications Dr. Sheher Bano Mohsin Mass Spectrometry Applications March 19, /21/2014
2 Introduction Why Online SPE? Sensitivity of the Mass Spectrometers Application Examples Water Samples Samples with dirty Matrices Introducing the Flexcube for Online SPE 2 3/21/2014
3 Solid Phase Extraction (SPE) Wouldn t it be great if you could automate SPE while reducing sample and solvent volumes? 3 3/21/2014
4 Agilent Online SPE System Switching valves Analytical column for separation Replace with Flexcube Quaternary pump MS/MS Autosampler Binary pump for gradient elution
5 Agilent 1200 Infinity Series Online SPE solution Solvent selection valve for up to three solvents Valves mounted in 1290 Infinity Flexible Cube Reciprocating singlepiston pump for flows up to 4 ml/min (60 bar) up to two Quick Change valves, according to application 5
6 Online SPE with the Flex Cube Column Compartment Autosampler with 900uL loop and extension seat capillary Flexcube Autosampler 1290 Autosampler 1290 Binary Pump 6460 QQQ
7 Flex Cube and Autosampler 6 port valve 10 port valve Single Piston Pump Autosampler 6 ml vials
8 Mass Hunter B with Flexcube Control
9 Flex cube pump Column SPE starter set Std. ALS 900 µl head SPE2 G4742A 1260 Bin Pump SPE1 Load System with alternating SPE loading: Load sample to SPE1 waste
10 Flex cube Column Mass Spec Load Std. ALS 900 µl head SPE Bin Pump SPE1 Elute waste System with alternating SPE loading: Elute sample from SPE1 to column / Load sample 2 on SPE2
11 Evaluation of On line Solid Phase Extraction and Direct Injection LC/MS/MS for the Analysis of Chemicals on U.S. EPA s Contaminant Candidate List 3 Sheher Bano Mohsin Michael Woodman
12 Chemicals for LC/MS/MS Evaluation * 4,4 methylenedianiline 3 hydroxycarbofuran bensulide clethodim fenamiphos fenamiphos sulfone fenamiphos sulfoxide methomyl quinoline tebuconazole tebufenozide thiodicarb 9 CCL3 chemicals 3 CCL3 degradates *Slide Taken From: Evaluation of On line Solid Phase Extraction LC/MS/MS for the Analysis of Chemicals on U.S. EPA s Contaminant Candidate List 3 Jody A. Shoemaker and Daniel R. Tettenhorst
13 Superimposed chromatograms Showing Reproducibility of Retention time as well as Area Counts for two runs Alternating Between two Different SPE Cartridges. The calibration standard used has a concentration of 10 ppt (ng/l) each for all 12 compounds with quantifier and qualifier ions. Each run alternates between two different SPE cartridges.
14 Mix at 1 ppt Quinoline Tebuconazole Carbofuran 3 Hydroxy Fenomiphos Fenomiphos Sulfoxide Tebufenozide Bensulide 4,4 diaminodiphenyl methane Methomyl Fenomiphos Sulfone Thiodicarb Clethodim
15 Mix at 0.1 ppt
16 1 ppt and 0.1 ppt 1 ppt 1ppt 1 ppt 0.1 ppt 0.1 ppt 1 ppt 0.1 ppt 1 ppt 0.1 ppt 0.1 ppt
17 Multi lab Verification Study of the first online SPE LC/MS EPA method EPA Method 543: Selected Organic Contaminants by Online SPE LC QQQ using the Agilent FlexCube and 6460 Tandem Quadrupole MS Don Noot, Ralph Hindle Vogon Laboratory Services Cochrane, AB, Canada
18 Goals Demonstrate acceptable method performance using Agilent online SPE (Flexible Cube) and LC QQQ Demonstrate acceptable performance of the Agilent Bond Elut SPE PLRP S cartridges Method verification requirements: Accuracy and Precision determination in reagent water and drinking water Accuracy: Recoveries between 70 and 130% Precision: RSDs 20% Determination of LCMRL, using calculator supplied by EPA 18
19 The results show that the Agilent online SPE LC QQQ system produces results (accuracy, precision and LCMRL) very similar to those produced by the US EPA. The Agilent Bond Elute SPE PLRP S cartridge produces results very similar to the Oasis HLB SPE cartridge, which is specified in the US EPA method. 19
20 Calibration Compounds showed best fit using quadratic, 1/X weighting Fenamiphos example shown (typical), from 0.02 to 20 ng/l 20
21 Calibration Range Compounds were analysed at different concentrations due to differences in sensitivity Calibration standards were prepared and down to levels where the compounds were not detected The range of concentrations used for the calibration curves for each compound is shown in the table Compound Range (ng/l) 3 Hydroxycarbofuran Bensulide Fenamiphos Fenamiphos sulfone Fenamiphos sulfoxide Tebuconazole Tebufenozide
22 Accuracy and Precision Preserved tap water (surface water source) was fortified at a mid level compared to the calibration curve range Seven fortified replicates were analysed to determine Accuracy and Precision Accuracy is presented as the average recovery of all seven replicates Precision is presented as the % Relative Standard Deviation (%RSD) 22
23 Accuracy LFB Accuracy (%) HLB PLRP S EPA* Accuracy LFSM Accuracy (%) HLB PLRP S EPA* * EPA data from fortifications at a similar concentration level, and from a surface water for LFSM 23
24 Precision LFB Precision (%) HLB PLRP S EPA* 1.6% 1.7% % 2.7% % 3.4% % 3.7% % 2.0% % 4.0% % 3.4% 2.9 Precision LFSM Precision (%) HLB PLRP S EPA* 4.8% 4.2% % 1.7% % 1.7% % 3.8% % 5.1% % 3.9% % 2.4% 12 * EPA data from fortifications at a similar concentration level, and from a surface water for LFSM 24
25 LCMRL Calculations Calculations are determined by entering values in EPA supplied LCMRL Calculator 3 LCMRL is defined as the lowest spiking concentration at which recovery of between 50 and 150% is expected 99% of the time by a single analyst Requires minimum of 4 replicates at each of 7 fortification levels, plus 4 LRBs The LCMRL Calculator constructs mean and variance models of measurement as a function of spiking level This procedure takes into account both precision and accuracy When the LCMRL value is not bracketed by the fortified sample levels, the calculator gives a warning that an additional set of replicates will be needed Next table shows calculated values and also US EPA values 1. Results are similar to EPA values, and PLRP S values are similar to HLB. 25
26 LCMRL Results LCMRL (ng/l) Compound Oasis HLB Agilent PLRP S EPA (Oasis HLB) 3 Hydroxycarbofuran Fenamiphos sulfoxide Fenamiphos sulfone Fenamiphos Tebuconazole Tebufenozide Bensulide Note: the EPA method specifies use of Ammonium Acetate as the mobile phase. Better sensitivity can be achieved by using Ammonium Formate (especially for 3 Hydroxycarbofuran) 26
27 LCMRL Example Plots Fenamiphos sulfoxide Fenamiphos 27
28 Discussion and Conclusions An Agilent on line SPE /6460 LC QQQ system is suitable for the analysis of pesticides and metabolites in drinking water for EPA Method 543. The Agilent Flexible Cube incorporates seamlessly into the LC system and is controlled by MassHunter software. Online SPE provides many benefits for the analytical lab. Agilent Bond Elut SPE PLRP S cartridges perform similarly to the Oasis HLB SPE cartridges for US EPA method 543. LCMRLs are near or below EPA levels. Accuracy and Precision values were similar to US EPA values. The Agilent Poroshell PhenylHexyl column provides excellent retention and separation of the target compounds. The analysis method time (injection to injection) of less than 17 minutes (including online SPE), allows many samples to be automatically processed each day. 28
29 Acknowledgements Many Thanks to Trisa Robarge of Agilent Technologies for supplying the Bond Elute SPE PLRP S cartridges Sheher Mosin, Bettina Schuhn and Jean Francois Roy of Agilent Technologies for advice on setting up the Flexible Cube 29
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