EPA Method 538: Determination of Selected Organic Contaminants in Drinking Water with Direct Aqueous Injection LC/MS/MS

E. Michael Thurman and Imma Ferrer Center for Environmental Mass Spectrometry University of Colorado Boulder, CO, USA

Confidentiality Label 1 March 20, 2012 Abstract

EPA Method 538 is a new method from EPA for organophosphate pesticides in drinking water.

It uses direct aqueous injection; thus, no sample preparation is needed.

We use both UHPLC (Agilent 1290) and MS/MS (Agilent 6460) analysis for rapid analysis and sensitive detection with ng/L limits of detection.

A second MRM is added for more reliable identification.

Confidentiality Label 2 March 20, 2012 Hypothesis

Direct injection of organophosphate pesticides (EPA Method 538) will work by UHPLC (Agilent Model 1290) and LC/MS/MS with Jetstream (Agilent Model 6460) with trace level detection at ng/L concentrations.

Confidentiality Label 3 March 20, 2012 1. Introduction-Summary

1.1 EPA Method 538 (published in November 2009 by Shoemaker) deals with Organophosphate pesticides in drinking water (1) and one other contaminant, quinoline.

1.2 The method consists of 10 compounds: acephate, aldicarb, aldicarb sulfoxide, dicrotophos, diisopropylmethylphosphonate (DIMP), fenamiphos sulfone, fenamiphos sulfoxide, methamidophos, oxydemeton methyl, quinoline, and thiofanox with 5 labeled internal standards.

1.3 Direct aqueous injection is used with a large volume sample of 100 microliters; thus, no sample preparation is needed.

1.4 Because solid phase extraction (i.e. concentration of the sample is not carried out) suppression is mimimized in the analysis.

1.5 Part-per-Trillion Detection Limits.

Confidentiality Label 4 March 20, 2012 Introduction 1.1:

EPA Method 538: Determination of Selected Organic Contaminants in Drinking Water by Direct Aqueous Injection

by Jody Shoemaker, EPA Cincinnati, OH [email protected] 513-569-7298

Confidentiality Label 5 March 20, 2012 Introduction: 1.2. Ten Organophosphates and Quinoline

Confidentiality Label 6 March 20, 2012 Introduction:1.3. Organophosphate Structures

Aldicarb Sulfoxide Acephate Aldicarb O O O O O O N N P NH NH H3C N OCH3 H S S O SCH3

+ + + C4H11NO3PS C7H15N2O2S C7H15N2O3S Exact Mass: 184.0192 Exact Mass: 191.0849 Exact Mass: 207.0798

Fenamiphos Sulfone Dicro.tophos Diisopropyl methylphosphonate (DIMP)

H3C O N O O H O P O N CH3 H3C O P P O O O O O CH O 3 . S C H NO P+ 8 17 5 C H O P+ Exact Mass: 238.0839 7 18 3 . Exact Mass: 181.0988 O + C13H23NO5PS Exact Mass: 336.1029

Confidentiality Label 7 March 20, 2012 Introduction 1.3. Organophosphate Structures

Fenamiphos Sulfoxide Methamidophos Oxydemeton-methyl

H3C O

O H S O N CH P H C O 3 O S P 3 H N P 2 S O O O O CH3 + O C2H9NO2PS S + Exact Mass: 142.0086 C6H16O4PS2 Exact Mass: 247.0222 + Thiofanox C13H23NO4PS Exact Mass: 320.108

Quinoline

N O N S

HN

O C H N+ 9 8 C H N O S+ Exact Mass: 130.0651 9 19 2 2 Exact Mass: 219.1162

Confidentiality Label 8 March 20, 2012 Introduction 1.4. Five Deuterated Standards

Confidentiality Label 9 March 20, 2012 Introduction 1.4. Deuterated Internal Standards

Diisopropylmethylphosphonate-d14 (DIMP-d14) Acephate-d6 Methamidophos-d6

O O O O

O P O CD3 P P O H3C N OCD3 D C CD H2N CD3 H 3 3 S SCD3 D CD D C D 3 3 + + C2H3D6NO2PS C4H5D6NO3PS + Exact Mass: 148.0463 Exact Mass: 190.0568 C7H4D14O3P Exact Mass: 195.1867 Quinoline-d7

Oxydemeton-methyl-d6 D

D N D S O

S P CD3 CD O O 3 O D D C H D O PS + 6 10 6 4 2 D D Exact Mass: 253.0599 + C9HD7N Exact Mass: 137.1091

Confidentiality Label 10 March 20, 2012 Introduction 1.5 No solid-phase extraction!

1.Sorption on C-18 or 100-200 mL sample polymer.

2. Washing and cleanup. Condition cartridge Elute with Methanol with Methanol and (5 mL) to remove 50% of the WaterTime and Effortpesticides or 3. Elution and AnalysisRemoved. from Analysispharmaceuticals  

Cartridges: - C18, polymeric - Oasis HLB, OPT

Effluent to waste

Confidentiality Label 11 March 20, 2012 Introduction 1.6 Minimizing Compound Suppression

•SPE increases background organic matrix by 200 to 500 fold OH •Salts, such as sodium and metal ions, are also concentrated along with the background organic matrix

ClFeOOC •These salts suppress the ionization of target analytes. OH HO OH OH O OH

O

NaOOC O O

NaOOC

Confidentiality Label 12 March 20, 2012 Introduction 1.6 Direct Aqueous Injection with the 1290 Infinity—UHPLC and 6460 LC/MS/MS

Confidentiality Label 13 March 20, 2012 2.0 Experimental Methods: Summary

1. Collect 40 mL sample and preserve with sodium omadine and ammonium acetate.

2. Aliquot 950 µL of sample and add 50 µL of internal standard.

3. Analyze by UHPLC/MS/MS with the Agilent 1290 LC and the Agilent Model 6460 LC/MS/MS.

Confidentiality Label 14 March 20, 2012 Experimental Methods 2.1 MRM Transitions for QqQ Method

Compound Name Precursor Ion Product Ion Dwell Fragmentor Collision Energy Polarity Acephate 206 165 10 90 5 Positive Acephate 184 143 10 50 0 Positive Aldicarb 213 116 10 90 5 Positive Aldicarb 213 89 10 90 15 Positive Aldicarb-Sulfoxide 229 166 10 70 5 Positive Aldicarb-Sulfoxide 229 109 10 70 10 Positive Dicrotophos 238 193 10 70 0 Positive Dicrotophos 238 112 10 70 5 Positive DIMP 181 139 10 70 0 Positive DIMP 181 97 10 70 5 Positive Fenamiphos-Sulfone 336 308 10 110 10 Positive Fenamiphos-Sulfone 336 266 10 110 15 Positive Fenamiphos-Sulfoxide 320 292 10 110 10 Positive Fenamiphos-Sulfoxide 320 233 10 110 20 Positive Methamidophos 142 125 10 70 10 Positive Methamidophos 142 94 10 70 10 Positive Oxydemeton-methyl 269 191 10 110 5 Positive Oxydemeton-methyl 247 169 10 70 10 Positive Quinoline 130 103 10 110 25 Positive Quinoline 130 77 10 110 35 Positive Thiofanox 241 184 10 90 5 Positive Thiofanox 241 57 10 90 15 Positive

Confidentiality Label 15 March 20, 2012 Experimental Methods 2.2 Transitions for Deuterated Standards

Compound Transition Frag. CE

Acephate-d5 190149 50 0

DIMP –d14 19599 70 5

Methamidophos-d6 14897 70 10

Oxydemeton-methyl-d6 253175 70 10

Quinoline-d7 13781 110 35

Confidentiality Label 16 March 20, 2012 Experimental Methods 2.3 QqQ Experimental Source Parameters

Confidentiality Label 17 March 20, 2012 3.0 Results and Discussion

1. EPA Method 538: Organophosphate Pesticides in Drinking Water.

2. Direct Aqueous Injection; Thus, No Sample Preparation Needed.

3. UHPLC/MS/MS Analysis with Minimal Suppression.

4. Part-per-Trillion Detection Limits.

Confidentiality Label 18 March 20, 2012 3.1 Multiple Reaction Monitoring

Quad Mass Filter (Q1) Quad Mass Filter (Q3)

Ions

Collision Cell

Spectrum with Q1 lets only Collision cell Q3 monitors only background target ion 210 breaks ion 210 characteristic ions (from ESI) pass through apart fragments 158 and 191 from ion 210 210 210 for quant and

222 qual.

158 268 280 158 165 191 210 191

170 210 250 290 190 210 150 170 190 210 160 190 no chemical background

Confidentiality Label 19 March 20, 2012 +ESI Product Ion:1 (0.400-0.546 min, 54 Scans) Frag=70.0V [email protected] (229.0 -> **) Aldicarb Sulfoxide_PI Scan Positive 229.d 3.2 Example: x10 4 8

CE=10V 166.0

Fragmentation 6 109.0

Study of Aldicarb 4

2 O O 91.9

0 4 +ESI Product Ion:2 (0.401-0.546 min, 54 Scans) Frag=70.0V [email protected] (229.0 -> **) Aldicarb Sulfoxide_PI Scan Positive 229.d N x10

NH 5 Na CE=15V 109.0

S O 4

3

2 + 166.0

[M+Na] = 229 1 91.9

0 +ESI Product Ion:3 (0.402-0.547 min, 54 Scans) Frag=70.0V [email protected] (229.0 -> **) Aldicarb Sulfoxide_PI Scan Positive 229.d O x10 5 O 1 N CE=5V 166.0

1 NH Na 0 0

0 109.0

m/z = 166 0 0 +ESI Product Ion:4 (0.402-0.548 min, 54 Scans) Frag=70.0V (229.0 -> **) Aldicarb Sulfoxide_PI Scan Positive 229.d x10 5 HO 1 N 1 CE=0V 1

Na 0 166.0

0 m/z = 109 0 0 108.9

0 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 Counts vs. Mass-to-Charge (m/z) Confidentiality Label 20 March 20, 2012 3.3 Extracted Ion Chromatograms of Compounds

Column: C18 Eclipse Plus 2.1mm x 50mm, 1.8 um Mobile phase: A=ACN, B=H2O (0.1% Acetic Acid) Flow-rate: 0.4 mL/min Gradient: t=0min. 10% A/90% B t=1.7min. 10% A/90% B t=10 min. 100% B Injection volume: 40uL

x10 5

1 1

3

2.

2.

2. Thiofanox

2. methyl 2 DIMP Sulfoxide Aldicarb 1.

1.

1.

1.

- Oxydemeton 1 Aldicarb 0. Dicrotophos

0. Sulfone Fenamiphos Quinoline

0. Sulfoxide Fenamiphos Methamidophos Acephate 0.

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 7.4 7.6 7.8 8 8.2 8.4 8.6 8.8 9 9.2 9.4 9.6 9.8 10 10.2 Counts vs. Acquisition Time (min) Confidentiality Label 21 March 20, 2012 3.4 Examples of Standard Curves

Fenamiphos Sulfone

Quinoline

Confidentiality Label 22 March 20, 2012 3.5 EPA Method 538: Limits of Detection

Compound LOD (ppt, ng/L) Acephate 1000 Aldicarb 10 Aldicarb Sulfoxide 5 DIMP 50 Dicrotophos 50 Fenamiphos Sulfone 50 Fenamiphos Sulfoxide 50 Methamidophos 500 Oxydemeton-methyl 20 Quinoline 50 Thiofanox 10 Confidentiality Label 23 March 20, 2012 4.0 Conclusions 1. EPA Method 538 works well on the Agilent Model 1290 UHPLC coupled to the Agilent Model 6460 LC/MS/MS.

2. 10 Organophosphates and quinoline are analyzed by direct aqueous injection and UHPLC/MS/MS.

3. A second MRM transition is added for QA/QC.

4. Detection Limits are in the ng/L range.

5. Method is robust and has low suppresion.

6. Agilent has the competitive advantage in Direct Aqueous injection with UHPLC/MS/MS analysis.

Confidentiality Label 24 March 20, 2012 References

1. Shoemaker, EPA Method 538: Determination of selected organic contaminants in drinking water by direct aqueous injection-liquid chromatography/Tandem Mass Spectrometry (DAI-LC/MS/MS): EPA/600/R- 09/149.

Confidentiality Label 25 March 20, 2012 References 2. EPA Method 538:Determination of selected Organic Contaminants in Drinking Water by Direct Aqueous Injection with the Agilent 6460 Triple Quadrupole LC/MS System. Michael Thurman and Imma Ferrer. Agilent Application Note#5990-9670EN

Confidentiality Label 26 March 20, 2012