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 Method Results Summary 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 introduction

Anatune Girton, Cambridge (March 2012) VAR for Agilent Gerstel 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

Enrich trace level analytes in water 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%)

Correlation coefficient ITSP & LC/MS/MS Diurons in water Enrich 10 ml Elute in 0.8 ml Chromatogram Monuron (quantifier transition) in standard 0.40 μg/L after extraction Correlation coefficient Calibration after extraction Monuron 0.9989 Isoproturon 0.9995 Diuron 0.9997 Linuron 0.9984

NDMA and Metaldehyde very polar molecules Introduction NDMA and Metaldehyde very polar molecules Enrichment methods from water problematic Developed two separate automated methods Metaldehyde NDMA

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 better sensitivity but also selectivity!

Summary of talk Introduction Method Results Summary NDMA and Metaldehyde Method Automated Sample Preparation (focus on Gerstel instrumentation) GC/MS/MS method Results Summary introduction

Brand this set up - Multiflex Automated Sample Prep 2.5 ml HS Syringe 10 ul Syringe Brand this set up - Multiflex Consists of Dual Head MPS Thermal Desorption unit – Cold Inlet System - PTV

ITSP (Instrument Top Sample Preparation) Typically 15-25 mg sorbent Metaldehyde Biotage ENV (as in literature) NDMA – Coconut charcoal   Metaldehyde Results Metaldehyde_d16 (ISTD) Results Name Resp. Area Metaldehyde_DCM_Standard4 5202 6189 MetaldehydeSTD+IS_test_BiotageENV 4756 6115 6678 8082 5510 6795 MetaldehydeSTD+IS_test_DVBENV 959 857 926 919 880 779 MetaldehydeSTD+IS_test_C18 354 284 266 478 392. MetaldehydeSTD+IS_test_CoconutCharcoal 6392 7722 6650 7737 5910 7172

Right MPS (2.5 ml Headspace syringe) 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 Method Results Summary NDMA and Metaldehyde Method Automated Sample Preparation GC/MS/MS method Results Summary introduction

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 Metaldehyde Different method 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 Different method No reason in future to create one method WAX

Multiple Reaction Monitoring 74.1 44.1 Direct comparison at NDMA at 0.125 ng/ml

Optimised MRM Transitions for NDMA Compound Precursor Ion Product Ion CE (v) NDMA-d6 (Quant) 80.1 50.1 5 NDMA-d6 (Qual) 46.1 15 NDMA (Quant) 74.1 44.1 NDMA (Qual) 42.1 20

EI Ionisation Experiments Varying from 10 to 80 eV 70 eV

Varying Ion Source Temperature Varying between 150 & 350 °C

Optimised MRM Transitions for Metaldehyde Compound Precursor Ion Product Ion CE (v) Metaldehyde (Quant) 89.0 45.1 9 Metaldehyde (Qual) 43.1 42 Metaldehyde d16 (Quant) 98.1 50.1 12 Metaldehyde d16 (Qual) 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 Method Results - NDMA Summary NDMA and Metaldehyde Method Automated Sample Preparation GC/MS/MS method Results - NDMA Summary introduction

Extracted Water - NDMA Water spiked to build seven point calibration from 0.25 to 15 ng/l. Correlation co-efficient of 0.9995. 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 NDMA-d6

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 2.6 Ave. % 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

Developed two fully automated methods for NDMA and Metaldehyde 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