Simultaneous Determination of

Paraquat and Diquat in Environmental

Water Samples by HPLC-MS/MS

Richard Jack, Xiaodong Liu, Leo Wang and

Chris Pohl

Thermo Fisher Scientific

2 Proprietary & Confidential

Outline

• Introduction

• Stationary phase design

• Chromatographic evaluation

• LC-MS result

• Summary

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Paraquat (Pq) and Diquat (Dq)

• Non-selective, nonsystematic contact herbicides

• Environmental & safety concerns

• Toxic to humans through contact (e.g. oral, respiratory, dermal)

• Moderately hazardous: LD50 ~35 mg/kg for human

• Banned or restricted in several European countries and in Japan

• Regulation

• The U.S. EPA regulation: < 20 μg/L for Dq in drinking water

• European Union (EU)’s general rule for pesticides in drinking water (98/83/EC):

• < 0.1 μg/L of each individual pesticide

• < 0.5 μg/L for the total concentration

• Food safety concerns in developing countries

Paraquat (Pq) Diquat (Dq)

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Analytical Methods

• Colorimetric spectrophotometry

• Enzyme linked immunosorbent assay (ELISA)

• Liquid scintillation counting (LCS)

• HPLC – wide acceptance

• Ion exchange column – post-column derivatization – fluorescence

detection

• Reversed-phase column – UV, PDA, or MS

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U.S. EPA Method 549.2

• A HPLC method for the determination of diquat and paraquat in drinking water sources and finished drinking water

• Summary of the method

• Offline SPE

• RPLC/ion-pairing

• UV or PDA detection

• Detection limit: 0.72 μg/L for Dq; 0.68 μg/L for Pq

• Challenges

• Poor reproducibility

• Time consuming

• Complex mobile phase: water, phosphoric acid, acetonitrile, heptane or hexane sulfonic acid (ion pairing agent), and diethylamine (DEA)

• Separation and peak shape

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An Improved Method

• Specialty column – good separation and peak shape

• Special mobile phase additive

• Challenges

• Poor reproducibility

• Time consuming

• Complex mobile phase

• Incompatible with MS

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A Method Not Requiring Ion-Pairing Agent

• RP/AEX/CEX tri-mode column

• No ion-pairing agent required

• On-line SPE

• Challenges

• High concentration buffer

(not MS-friendly)

• Peak shape

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LC/MS – Greater Sensitivity w/o Sample Preparation

• Specialty RP column

• No sample enrichment

• LOQ: 0.1 μg/L for diquat and 5 μg/L for paraquat (10-µL )

• Challenges

• HFBA as ion-pairing agent – not desirable for MS

• High aqueous mobile phase (95%) – lower sensitivity

min

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UHPLC HILIC Column – Off-line SPE – LC/MS

• UHPLC HILIC column

• Off-line sample preparation (20x)

• LOQ: ~ 40 ppt for diquat and paraquat (10-µL )

• Challenges

• Dipuat and paraquat co-elution

• High buffer concentration – lower sensitivity

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Objective

To develop a method for determination of diquat and paraquat :

1. Retain k’ > 2

2. Resolve Rs > 2 (Dq/Pq)

3. Behave As < 1.5 (UV)

4. LC-MS compatible No ion-pairing agent

Solvent > 50% (v/v)

Buffer concentration < 50 mM

5. Simple Isocratic method

6. Fast < 5 min

7. Sensitive Better than reported

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Nano-polymer-Silica-Hybrid (NSH) Technology

Benefits:

• Versatile chemistry platform

• Cation-exchange and anion-exchange function simultaneously

• Distinctive spatial separation of the anion-exchange and cation-exchange regions,

which results in maximum flexibility in method development.

• Chromatography can be easily optimized by adjusting mobile phase buffer

concentration, pH, and solvent content, concurrently or independently.

• Ideal selectivity for simultaneous separation of basic, neutral, and acidic analytes

• Separation of hydrophilic ionic and ionizable analytes without ion-pairing reagent

Bare Silica Surface Modified Silica Nanopolymer Silica Hybrid

Surface Bonding Electrostatically Driven

Self-Assembly

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Stationary Phase Design

Bonded WAX phase for symmetrical peak shape

Nano-polymer bead (WCX) to retain and separate Dq and Pq

Retention mechanism:

WCX: Carboxylate

WAX: Tertiary amine

RP: Alkyl

Silica Substrate:

3 µm, high-purity, spherical, porous

Surface Area

100 m2/g

Pore Size

300 A

Operating Pressure limit

4000 psi

Operating flow rate range

0.3 – 0.9 mL/min for 3mm i.d.

0.15- 0.45 mL/min for 2.1 mm i.d.

pH range

2.5-7.0

Dq/Pq Phase

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Organic Solvent Effect

0 4 8 6 2

Minutes

mAU

0

150

Column: Dq/Pq prototype, 3 µm

Dimensions: 3.0 x 50 mm

Mobile Phase: MeCN/ 25 mM (total) NH4OAc, pH5

Temperature: 30 °C

Flow Rate: 0.60 mL/min

Inj. Volume: 2 µL

Detection: UV, 290 nm

Sample: Dq and Pq (0.1 mg/mL each)

Pq/Dq 30% MeCN 40% MeCN 50% MeCN 60% MeCN 70% MeCN 75% MeCN

Rs 2.45 3.69 5.5 7.5 8.0 8.8

As 1.88/1.18 1.17/1.35 1.15/1.07 1.03/0.98 0.93/0.96 1.08/0.96

Efficiency 1900/2175 3060/3370 4090/4600 5550/5560 6000/4840 6230/5670 Pq

Dq

75% MeCN

10

Pq Dq

70% MeCN

60% MeCN

50% MeCN

40% MeCN

30% MeCN

Pq Dq

Pq Dq

Pq Dq

Pq Dq

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Buffer Concentration Effect

0 10 20 15 5 25

Minutes

mAU

0

150

Column: Dq/Pq prototype, 3 µm

Dimensions: 3.0 x 50 mm

Mobile Phase: 75/25 v/v CH3CN/ various conc. NH4OAc, pH5

Temperature: 30 °C

Flow Rate: 0.60 mL/min

Inj. Volume: 2 µL

Detection: UV, 290 nm

Sample: Dq and Pq (0.1 mg/mL each)

Pq/Dq 10 mM 15 mM 20 mM 25 mM

Rs 10.7 10.3 9.5 8.8

k 26.4/46.8 11.8/21.0 6.9/12.2 4.5/7.9

As 1.02/0.96 1.02/0.93 1.03/0.97 1.08/0.96

Efficiency 5900/6160 5860/6170 5760/5770 6230/5670

Pq

Dq

25 mM

Pq

Dq

Pq

Dq

Pq Dq

20 mM

15 mM

10 mM

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pH Effect

0 4 8 6 2 10 Minutes

mAU

0

100 Column: Dq/Pq prototype, 3 µm

Dimensions: 3.0 x 50 mm

Mobile Phase: 75/25 v/v CH3CN/ 25 mM (total) NH4OAc, pH5

Temperature: 30 °C

Flow Rate: 0.60 mL/min

Inj. Volume: 2 µL

Detection: UV, 290 nm

Sample: Dq and Pq (0.1 mg/mL each)

Pq/Dq pH4 pH5

Resolution (Rs) 5.1 8.8

Retention (k) 4.7/6.8 4.5/7.9

Asymmetry (As) 1.31/1.18 1.08/0.96

Efficiency 3900/4800 6200/5600

Pq

Dq

Pq

Dq

pH5

pH4

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LC/MS configuration

Inject / Cleanup Separation

Reconditioning

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MS and Test Matrix parameters

SRM Scan Events Precursor Quantitative

SRM (CID)

Confirmative

SRM (CID)

Paraquat 185 169 (27) 170 (17)

Paraquat-d8 193 178 (17)

Diquat 183 157 (22) 130 (31)

Diquat-d3 186 158 (22)

Matrix Conc.

Na+ and K+ > 5000 mg/L

NH4+ 1000 mg/L

NO3- 200 mg/L

HCO3- 1500 mg/L

SO42- 2500 mg/L

Cl- 3500 mg/L

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LC-MS-MS: Paraquat and Diquat at 10 ppb

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Time (min)

100

100

100

100

Re

lati

ve

Ab

un

da

nc

e

100

100 NL: 1.19E5

NL: 5.46E4

NL: 5.47E4

NL: 9.67E5

NL: 3.11E5

NL: 9.98E5

185 169

185 170

193 178

183 157

183 130

186 158

Paraquat: Q-SRM

Paraquat: C-SRM

Paraquat-IS

Diquat: Q-SRM

Diquat: C-SRM

Diquat-IS

Chromatographic Conditions

System: UltiMate 3000 RS UHPLC System

Column: Dq/Pq

Column Temp.: Ambient

Mobile Phase: 25% Ammonium Acetate (100 mM, pH 5.0)

75% Acetonitrile

Flow Rate: 0.5 mL/min

Injection: 5 µL

Mass Spectrometric Conditions

System: Quantum TSQ Access MAX Triple Quad

Interface: Heated Electrospary Ionization

with HESI II probe

Spray Voltage: 1500 V

Vaporizer Temperature.: 400 ºC

Sheath Gas Pressure: 70

Aux Gas Pressure: 10

Capillary Temperature: 350 ºC

Quantitation Mode: Selected Reaction Monitoring (SRM)

Scan Events Precursor

Quantitative SRM

(CID)

Confirmative SRM

(CID)

Paraquat 185 169 (27) 170 (17)

Paraquat-d6 193 178 (17)

Diquat 183 157 (22) 130 (31)

DiQuat-d3 186 158 (22)

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Quantitation: Diquat from 0.1 to 100 ng/mL

Diquat

Y = 0.0224505+0.152363*X

R2 = 0.9995 W: 1/X

0.1 to 100 ng/mL

0 20 40 60 80 100

0

16

Are

a R

ati

o

Concentration (ng/mL)

0 1 2 3 4 5 6 7 8 9 10 0.0

1.6

LOQ – 100 ppt (10-µL) or 1 pg

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Spike Recoveries

ng/mL Replicates Paraquat Diquat

Observed %Recovery %RSD Observed %Recovery %RSD

Creek Water

0.50 (n=3) 0.39 78.0 1.73 0.44 88.0 3.14

5.0 (n=2) 5.12 102 3.17 5.37 107 1.30

50 (n=3) 47.2 94.4 0.52 52.1 104 1.04

Heavy Matrix 10 (n=30) 10.5 105 3.48 9.43 94.3 1.09

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Summary

• Nanopolymer Silica Hybrid (NSH) technology allows for tailoring column

chemistry to meet specific needs.

• A column for diquat and paraquat analysis has been developed

• Adequate retention k’ > 2

• Excellent resolution Rs > 4 (Dq/Pq)

• Good peak shape As < 1.5

• MS-compatible No ion-pairing agent

Acetonitrile > 50% (v/v)

Buffer concentration < 25 mM

• Simple Isocratic method

• Fast 5 min

• Sensitive 1 pg (or 0.1 ppb with 10-µL injection)

• Robust Recoveries in real and synthetic matrices