simultaneous determination of paraquat and diquat in...
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The world leader in serving science
Richard Jack, Xiaodong Liu, Leo Wang, and
Chris Pohl
OT70806_E 08/13S
Simultaneous Determination of Paraquat and Diquat in Environmental Water Samples by HPLC-MS/MS
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Outline
• Introduction
• Stationary Phase Design
• Chromatographic Evaluation
• Liquid Chromatography-Mass Spectrometry (LC-MS) Result
• Summary
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Paraquat (Pq) and Diquat (Dq)
• Nonselective, nonsystematic contact herbicides
• Environmental and safety concerns
• Toxic to humans through contact (e.g., oral, respiratory, dermal)
• Moderately hazardous: LD50 ~35 mg/kg for humans
• 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)
• High-performance liquid chromatography (HPLC)—wide
acceptance
• Ion-exchange column—postcolumn derivatization—fluorescence
detection
• Reversed-phase (RP) column—ultraviolet (UV), photodiode array (PDA), or
MS
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U.S. EPA Method 549.2
• An HPLC method for the determination of diquat and paraquat in drinking water sources and finished drinking water
• Summary of the method
• Off-line 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/anion-exchange/charge-
exchange/trimode 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
• Heptafluorobutyric acid (HFBA) as ion-pairing agent—not desirable for MS
• High aqueous mobile phase (95%)—lower sensitivity
Minutes
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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 coelution
• High buffer concentration—lower sensitivity
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Objective
To develop a method for determination of diquat and paraquat:
• Retain k’ > 2
• Resolve Rs > 2 (Dq/Pq)
• Behave As < 1.5 (UV)
• LC-MS-Compatible No ion-pairing agent
Solvent > 50% (v/v)
Buffer concentration <50 mM
• Simple Isocratic method
• Fast <5 min
• Sensitive Better than reported
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Nanopolymer 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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Bonded WAX phase for symmetrical peak shape
Nanopolymer bead (WCX) to retain and separate Dq and Pq
Dq/Pq Phase
Stationary Phase Design
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
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Organic Solvent Effect
Column: Dq/Pq prototype, 3 µm (3.0 50 mm)
Mobile Phase: MeCN/ 25 mM (total) NH4OAc, pH5
Flow Rate: 0.60 mL/min
Inj. Volume: 2 µL
Temp: 30 °C
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
0 4 8 6 2
Minutes
mAU
0
150 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
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
0 10 20 15 5 25
Minutes
mAU
0
150 Pq
Dq
25 mM
Pq
Dq
Pq
Dq
Pq Dq
20 mM
15 mM
10 mM
Column: Dq/Pq prototype, 3 µm (3.0 50 mm)
Mobile Phase: 75/25 v/v CH3CN/ various conc NH4OAc, pH5
Flow Rate: 0.60 mL/min
Inj. Volume: 2 µL
Temp: 30 °C
Detection: UV, 290 nm
Sample: Dq and Pq (0.1 mg/mL each)
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pH Effect
Pq/Dq pH 4 pH 5
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
0 4 8 6 2 10 Minutes
mAU
0
100 Pq
Dq
Pq
Dq
pH 5
pH 4
Column: Dq/Pq prototype, 3 µm (3.0 50 mm)
Mobile Phase: 75/25 v/v CH3CN/ 25 mM (total) NH4OAc, pH5
Flow Rate: 0.60 mL/min
Inj. Volume: 2 µL
Temp: 30 °C
Detection: UV, 290 nm
Sample: Dq and Pq (0.1 mg/mL each)
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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 Concn
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
* Selected Reaction Monitoring
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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
Minutes
100
100
100
100
Rela
tive
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: Thermo Scientific™ Dionex ™ UltiMate™ 3000
Rapid Separation (RS) UHPLC
Column: Dq/Pq
Mobile Phase: 25% Ammonium Acetate (100 mM, pH 5.0)
75% Acetonitrile
Flow Rate: 0.5 mL/min
Injection: 5 µL
Column Temp: Ambient
Mass Spectrometric Conditions
System: Thermo Scientific™ TSQ Quantum Access
MAX™ triple stage quadrupole mass
spectrometer
Interface: Heated electrospray ionization, Thermo
Scientific™ HESI-II probe
Spray Voltage: 1500 V
Vaporizer Temp: 400 ºC
Sheath Gas Pressure: 70
Aux Gas Pressure: 10
Capillary Temp: 350 ºC
Quantitation Mode: 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
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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
• 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
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Thank you for your attention!
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