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!