Quantitative Analysis of pesticides including …...©2013 Waters Corporation 1 Quantitative...
Transcript of Quantitative Analysis of pesticides including …...©2013 Waters Corporation 1 Quantitative...
©2013 Waters Corporation 1
Quantitative Analysis of pesticides including
Phenoxy-acids in surface and drinking
water matrices using UPLC-ON-LINE-SPE-MS/MS
Alexandre Paccou
Demo Centers and Application Support Manager, Southern Europe
Food & Environment Market Development Manager, Sothern Europe
Milan 12/03/2013
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Waters UPLC with On-line SPE Technology
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Off Line Sample Preparation Extraction Protocol
Barrels or Syringe
OFF-line SPE Extraction Manifold
Condition/Equilibrate 3 mL methanol/3 mL water
Load > 500 mL sample
Wash
Elute
Prepare Sample
For LC analysis, exchange to acetonitrile, then adjust to 1
mL
For GC analysis, dry extract over Na2SO4, then adjust to 1mL
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Off Line Sample Preparation Extraction Protocol
Volume
Availability 1 uL 50 uL 100 uL 1 mL 10 mL 100 mL 1 L 500 uL 5 mL 50 mL 500 mL
Final extract volume 1mL – inject 30 – 50 µL
10 x 100 x 1000 x
Target
LOQ 1 ng/L 10 ng/L 100 ng/L 1 ug/L 10 ug/L 100 ug/L 1 mg/L 10 mg/L 100 mg/L 1 g/L 10 g/L
10 x 100x
1000x
Instrument LOQ
Method LOQ
Elute
Evaporate and
Reconstitute
Condition/Equilibrate
Load
wash
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Analysis Collection Results Sample Preparation
Qualitative Quantitative
RSD’s > 15 % + LC/MS GC/MS
LC/MS/MS GC/MS/MS
Automated System
- Filtration - Extraction - Clean up - Concentration - Derivatization
- Manual Work - Hours to days - Consumable – Disposable - Sample storage - Lab space - Additional Equipment: - Glass ware - Manifold - Evaporator
1L to 20 L
- Shipping cost
Drinking Water Analysis How It’s Done Currently
- Sample container - Shipping container
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ON Line SPE/LC/MS/MS
ON-line analysis is a technique that reduces the
amount of manual labor to produce an analyzable
sample for LC/MS/MS. The sample is injected on an
extraction column using an automated injector.
Barrels or Syringe
OFF-line SPE Extraction Manifold
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Final extract volume is 0 (mL, L, nL …?)
Analytes are trapped on Extraction Column
Direct elution to HPLC Column
Volume
Availability 1 uL 50 uL 100 uL 1 mL 10 mL 100 mL 1 L 500 uL 5 mL 50 mL 500 mL
Elute
Condition/Equilibrate
Load
wash
500 mL
(starting volume)
1 mL final
(elution volume)
Off Line SPE Extraction
30 - 50 µL
(injection volume)
500:1 enrichment
LC/MS Analysis
30 µL – 1/33th of original volume – 15.0 mL
50 µL – 1/20th of original volume – 25.0 mL
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Analysis Collection Results Sample Preparation
Qualitative Quantitative
RSD’s < 15 %
Integrated System
- Fully Automated – no manual work - Results in minutes instead of hours/days - Reusable chemistries – longer cycle time - Reduction of sample storage space - Minimum lab space required - No additional equipment needed
- Reduction of analytical costs
20 mL
- Shipping cost
ON-line SPE/LC/MS/MS
- Sample container - Shipping container
Drinking Water Analysis Going Forward: ON-line SPE/LC/MS/MS System
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Analysis Collection Results Off-line Sample Preparation
Most time consuming steps
Traditional Off-Line Analytical Approach
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Complex and Labor Intensive Work Flow
– Large volumes of dilute samples
– Multiple Vessel Transfers
– Time Consuming Steps
– Separations are slow
– Rework can be high
– MS is required, skilled, expensive operators
– Competition between CRO’s is high and Costs must be reduced
Challenge
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Waters UPLC with Online SPE Technology
2777c or 2777
Column Heater/Cooler
BSM QSM Xevo TQD Xevo TQ-S
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Solid cap
Shipping Box
20 mL vial Autosampler
rack
Pre-slit Septum
Cap
Receiving and Preparing Samples
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Collection Results
Analysis
On-line Sample Preparation
New Analytical Approach For Analysis of Compounds of Concern in Water
20 ml
Sample concentration Elution
Analysis
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System Diagram
A: injection valve for online SPE UPLC B: switching valves for extraction columns C: injection valve for stand-alone UPLC
Parallel extraction columns increase throughput
Simple switch between online SPE system and stand alone
UPLC system
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Example events
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Online SPE Method Fundamentals
Loading Eluting
Basic
Neutral
Acidic
100 % aqueous + 2 % NH4OH pH 10
High K’ - basic functionality neutral- highest trapping efficiency
100 % aqueous + no additive
100 % aqueous + 2 % FA pH 3
High K’ ’ - acidic functionality neutral- highest trapping efficiency
Organic gradient – 1, 3 or 5 min + 0.5 % acidic additive (pH 3- 5)
FA, AA, NH4Formate or NH4Acetate
Low K’ - basic functionality ionized
Organic gradient – 1, 3 or 5 min + 0.5 % basic additive (pH 10)
NH4OH or NH4HCO3
Low K’ - acidic functionality ionized
Organic gradient – 1, 3 or 5 min with or without additive
(ionisation may improve with additive)
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ESI+ MS/MS
Load High K’
100% Aqueous
Waste
UPLC
Elute Low K’
Backflush Elution
SPE Sorbents: • Oasis HLB 20m pH 1-14 • XBridge C18 10mpH 1-12 • XBridge C8 10mpH 1-12
Loading and Eluting Conditions
SPE
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Recommended Columns
UPLC packed online SPE direct connect columns:
BEH columns are recommended for elution
– pH tolerance +++
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Analysis of Pesticides in ESI +
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Analysis of Pesticides in ESI +
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Analysis of Phenoxy-acids pesticides in ESI -
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Analysis of Phenoxy-acids pesticides in ESI -
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Linearity Example 10 to 150 ppt
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Linearity Example 10 to 150 ppt
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Sensitivity ESI+ 10ppt
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Sensitivity ESI- 10ppt
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LOD/LOQ
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Repeatability
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Time8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50
%
0
8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50
%
0
8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50
%
0
MRM of 15 Channels ES+284.2 > 252.2
6.59e59.84
MRM of 15 Channels ES+284.2 > 252.2
4.55e59.83
MRM of 15 Channels ES+284.2 > 252.2
4.02e59.81
Time8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50
%
0
8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50
%
0
Time8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50
%
0
8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50
%
0
8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50
%
0
MRM of 15 Channels ES+284.2 > 252.2
6.59e5
8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50
%
0
MRM of 15 Channels ES+284.2 > 252.2
6.59e59.84
MRM of 15 Channels ES+284.2 > 252.2
4.55e5
9.84
MRM of 15 Channels ES+284.2 > 252.2
4.55e59.83
MRM of 15 Channels ES+284.2 > 252.2
4.02e5
9.83
MRM of 15 Channels ES+284.2 > 252.2
4.02e59.81
Metolachlor
500th injection
250th injection
1st injection
Lifetime
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Results Off-line Sample
Preparation
UPLC with On-line SPE Technology for Complex Matrices
Sample concentration Elution
Analysis
On-line Sample Concentration
Analysis
20 ml
Collection
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UPLC with On-line SPE Technology
– Sensitivity
o Range of solutions addressing ppm to low ppt analyte levels
– Robustness
o Column lifetime in excess of 500 injections for Drinking Water
– Flexibility
o Operate as On-line SPE and Standalone
o Multiple Detectors
– Reproducibility
o RSDs always less than 15%
– Transferability/Ease of Use
o Advance software suite
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A UPLC-on-line-SPE-MS/MS method for the analysis of pesticides in Surface
and Drinking Water samples at ppt level was developed
On-line SPE and UPLC parameters were optimized in order to be able to
analyze the widest range possible of pesticides
Only a single 5mL Water Sample injection was required for this analysis
A throughput of 3 to 4 samples per hours including preparation was typically
achieved
Apart from an eventual filtration, no sample pre-treatment is needed,
An excellent linearity was observed across the studied range of 10 to 150ppt
with coefficient of determination higher than 0.995 for all studied compounds
Standard deviation from the calibration curve always stayed below 10% even
at 10ppt
An excellent sensitivity was observed for all compounds
Calculated detection limits ranged from below 0.05 to 3.695 ppt
Repeatability studies revealed Rsds typically lower than 10% for all compounds
even at low level
Excellent column life-time > 500 injections
Conclusion
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Thank You!