Automated SPE for NDMA and Metaldehyde in water

using GC-QqQ

Dan Carrier, Applications Chemist

www.anatune.co.uk

Purpose of this presentation

“show how methods NDMA & Metaldehyde fully automated using Gerstel and Agilent instrumentation”

Summary of talk• Introduction

– Background NDMA and Metaldehyde– Why trace level method for both analytes

• Method– Automated Sample Preparation– Chromatographic method using GC/MS/MS

• Results– ~1 ng/l NDMA in extracted water– ~2 ng/l Metaldehyde in extracted water

• Summary– Good overview of system

Anatune

• Girton, Cambridge (March 2012)

• VAR for Agilent– GC and LC products– MSD, QqQ, QTOF

• Gerstel– MPS – DHS, Twister, ITSP

• Focus - Sell and Support Solutions– Wide number of industries - Environmental, Food and

Flavours, Petrochem, Pharmaceutical, Forensic, and Clinical

• Dual Head MPS Solution with LC/UV – Formaldehyde and acetaldehyde in

air

Twister SBSE

• Enrich trace level analytes in water– Twisters made PDMS (ethylene glycol)– Adsorption based on lipophibicity– Log K o/w

Twister SBSE – PAH solution

• 100 ml water samples (2 hours)– Dried and placed in TDU tubes– SIM 16 PAH (0.02 ug/l to 1 ug/l)– Acenaphthene 0.999 (1-2%)

ITSP & LC/MS/MS

• Diurons in water– Enrich 10 ml– Elute in 0.8 ml

Correlation coefficient

Calibrationafter extraction

Monuron 0.9989

Isoproturon 0.9995

Diuron 0.9997

Linuron 0.9984

Chromatogram Monuron (quantifier transition) in standard 0.40 μg/L after extraction

Introduction

• NDMA and Metaldehyde very polar molecules – Enrichment methods from water

problematic– Developed two separate automated

methods

NDMAMetaldehyde

Harm - NDMA

• Industrial by-product many processes

• IARC (International Agency for Research on Cancer) NDMA is probably carcinogenic to humans

• WHO Guideline limit NDMA of 100 ng/l in drinking water.

Metaldehyde

• Widespread Pesticide

• Regulatory limit 100 ng/L in drinking water

NDMA

• Initial method on MSD 5975C (before QqQ)

– Method Detection limit 25 ng/L– AS 119– Needed to get bettersensitivity but also selectivity!

Summary of talk

• Introduction– NDMA and Metaldehyde

• Method– Automated Sample Preparation (focus on

Gerstel instrumentation)– GC/MS/MS method

• Results

• Summary

Automated Sample Prep

• Brand this set up - Multiflex – Consists of Dual Head MPS– Thermal Desorption unit – Cold Inlet System

- PTV

2.5 ml HS Syringe10 ul Syringe

ITSP (Instrument Top Sample Preparation)

• Typically 15-25 mg sorbent• Metaldehyde

– Biotage ENV (as in literature)– NDMA – Coconut charcoal

  Metaldehyde ResultsMetaldehyde_

d16 (ISTD) Results

Name Resp. AreaMetaldehyde_DCM_Standard4 5202 6189MetaldehydeSTD+IS_test_BiotageENV 4756 6115MetaldehydeSTD+IS_test_BiotageENV 6678 8082MetaldehydeSTD+IS_test_BiotageENV 5510 6795MetaldehydeSTD+IS_test_DVBENV 959 857MetaldehydeSTD+IS_test_DVBENV 926 919MetaldehydeSTD+IS_test_DVBENV 880 779MetaldehydeSTD+IS_test_C18 354 284MetaldehydeSTD+IS_test_C18 354 266MetaldehydeSTD+IS_test_C18 478 392.MetaldehydeSTD+IS_test_CoconutCharcoal

6392 7722

MetaldehydeSTD+IS_test_CoconutCharcoal

6650 7737

MetaldehydeSTD+IS_test_CoconutCharcoal

5910 7172

Coconut Charcoal ITSP cartridges (NDMA)ENV (Metaldehyde)

Right MPS (2.5 ml Headspace syringe)

Conditioned 750 µl dichloromethane

1000 µl of methanol

Equilibrated 2000 µl of HPLC grade water

Load 10 ml of sample (in water)

Dried 15 minutes

Eluted 400 ul dichloromethane

Left MPS (10 ul) Large Volume injection

X 25 concentration

Large Volume Injection– removing DCM boiling point 40 °C, Metaldehyde and NDMA both exceed 100 °C

Inlet kept at 10 °C (peltier cooled)

Slow injection speed at 0.5 ul/s (to remove DCM) ramped to 250 °C (12 °C /s)NDMA (similar for Metaldehyde)

- Glass beads liner gave best results

Summary of talk

• Introduction– NDMA and Metaldehyde

• Method– Automated Sample Preparation– GC/MS/MS method

• Results

• Summary

Agilent GC/QqQ

• Agilent 7890A GC• Agilent 7000 GC/MS triple quad

– Increased Sensitivity and Selectivity

Direct comparison at NDMA at 0.125 ng/ml (without extraction)

Single Ion Monitoring Multiple Reaction monitoring

GC Methods• NDMA

– DB-WAX 30 m x 250 μm x 0.5 μm– Thermal gradient from 35 deg C to 240 deg C over 10 minutes

• Metaldehyde– DB5 30m x 250 μm x 0.25 μm– Thermal gradient from 35 deg C to 250 deg C over 10 minutes

• Different method– No reason in future to create one method– WAX

Multiple Reaction Monitoring

Direct comparison at NDMA at 0.125 ng/ml

74.1 44.1

Optimised MRM Transitions for NDMA

CompoundPrecursor

IonProduct Ion CE (v)

NDMA-d6 (Quant) 80.1 50.1 5NDMA-d6 (Qual) 80.1 46.1 15NDMA (Quant) 74.1 44.1 5NDMA (Qual) 74.1 42.1 20

6x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

2

2.1

2.2

2.3

2.4

2.5

2.6

2.7

2.8

2.9

3

3.1

+EI TIC MRM CID@** (** -> **) DC160113_20.d

Counts vs. Acquisition Time (min)11.88 11.9 11.92 11.94 11.96 11.98 12 12.02 12.04 12.06 12.08 12.1 12.12 12.14 12.16 12.18 12.2 12.22 12.24 12.26 12.28 12.3 12.32 12.34 12.36 12.38

EI Ionisation Experiments

Varying from 10 to 80 eV 70 eV

Varying Ion Source Temperature

3x10

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

3

3.2

3.4

3.6

3.8

4

4.2

4.4

4.6

4.8

5

5.2

5.4

5.6

5.8

6

6.2

6.4

6.6

6.8

7

7.2

+EI MRM CID@5.0 (74.1 -> 44.1) PR01072013_11.D

Counts vs. Acquisition Time (min)5.68 5.685 5.69 5.695 5.7 5.705 5.71 5.715 5.72 5.725 5.73 5.735 5.74 5.745 5.75 5.755 5.76 5.765 5.77 5.775 5.78 5.785 5.79 5.795 5.8 5.805 5.81 5.815 5.82 5.825 5.83 5.835 5.84 5.845 5.85 5.855 5.86

Varying between 150 &350 °C

Optimised MRM Transitions for Metaldehyde

CompoundPrecursor

IonProduct Ion CE (v)

Metaldehyde (Quant)

89.0 45.1 9

Metaldehyde (Qual)

89.0 43.1 42

Metaldehyde d16 (Quant)

98.1 50.1 12

Metaldehyde d16 (Qual)

98.1 46.1 39

Summary of Method development• Metaldehyde method developed

within 2 weeks

• NDMA over 3 months– Contamination issues

– Exposing LC/MS grade water to light!

Summary of talk

• Introduction– NDMA and Metaldehyde

• Method– Automated Sample Preparation– GC/MS/MS method

• Results - NDMA

• Summary

Extracted Water - NDMA

Water spiked to build seven point calibration from 0.25 to 15 ng/l. Correlation co-efficient of 0.9995.

NDMA - 7 Levels, 7 Levels Used, 7 Points, 7 Points Used, 0 QCs

Concentration (ng/L)-1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Re

lati

ve

Re

spo

nse

s -1x10

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

3

y = 0.019033 * x + 0.007651R^2 = 0.99955163Type:Linear, Origin:Include, Weight:1/x

X 25 concentration

NDMA comparison with blank

1 ng/l NDMA in LC/MS grade water with blank (extracted LC/MS grade water) - around detection limit

Standard Extracted Water 10 ng/l

NDMA-d6

NDMA

Absolute Recovery

Absolute recovery of NDMA established at 1 ng/l & 10 ng/l spikes

1 ng/L NDMA – Absolute recovery 55 % (% RSD 9.2)10 ng/l NDMA – Absolute recovery 61 % (% RSD 2.6)

40 ng/l NDMA-d6 – Absolute recovery 56 % (% RSD 7.9)

Recovery and precision of water extractions

Calculated accuracy of all calibration points 86.5 – 107.1 %Accuracy and precision of 1 ng/l & 10 ng/l standards (n=3)

NDMA 1 ng/l 10 ng/l

 

Mean ng/l result 0.997 10.329

SD 0.005 0.266

% RSD 0.5 2.6Ave. % Corrected Recovery 100.2 102.7

40 ng/l Metaldehyde extracted standard comparison with

blank

Detection limit approx 2 ng/l (based on signal to noise from this standard)

Extracted Water - Metaldehyde

Water spiked to build seven point calibration from 40 to 800 ng/l. Correlation co-efficient of 0.9993.

X 25 concentration

Recovery and precision of water extractions

(Metaldehyde)

Amount spiked (μg/L) 0.06080 0.70400

Amount detected (μg/L) 0.05734 0.63858

0.05721 0.71908

0.06000 0.70449

0.05628 0.72256

0.05641 0.72204

Mean 0.05745 0.70135

SD 0.0015 0.035856

% RSD 2.61 5.11

% Recovery 94.49 99.62

Summary

• Developed two fully automated methods for NDMA and Metaldehyde

• Good linearity and recovery for NDMA and Metaldehyde

• Detection limit (after extraction)– NDMA between 0.5 to 1 ng/l– Metaldehyde approx 2 ng/l

Acknowledgements

• Anatune– Paul Roberts– Anais Maury– Matthew Carson

• Rick Youngblood• Ken Brady

Any Questions?

Additional Work on MRM method• Work was completed to assess the impact of running

MS1 & MS2 in unit, wide and widest resolution modes.• Increased sensitivity as resolution decreased, but

possibility of interferences increases• Unit 0.7 amu at half height• Wide 1.2 amu at half height• Widest 2.5 amu at half height

• Default QQQ Collision Cell conditions for Collision Cell and Quench Gas