LC Technical Note

Analysis of Paraquat and Diquat in Urine

LT104
GL Sciences Inc.

In this note, a determination method for paraquat anddiquat using HPLC is described. Paraquat and diquat, which
are quaternary ammonium herbicide widely used in theworld, are also toxic to human being. Their concentrationin urine or serum after ingestion is determined foraccurate prognosis.
To determine the concentration in physiological sample,
purification by solid-phase extraction is required before
HPLC analysis. However, it is difficult to obtain good
recovery with C18 cartridge because alkaline solution, in
which paraquat and diquat is unstable, should be used toretain the compounds to the solid-phase.

Strong cation- exchange type is also not desirable because
solution containing high concentration of salt has to be
used for elution from the solid-phase. If the eluted sample
is injected into an HPLC system without any dilution ordesalting, peak shape often gets worse owing to the salt.
In this note, MonoSpin PBA, which has carboxylate groupfor weak cation-exchange mode, was used. As a result,excellent recoveries were obtained from urinary sample
with simple procedures.

(S. Ota)

A Chromatogram Obtained from Standard Solution

1. Paraquat dichloride
2. Diquat dibromide
3. Ethylparaquat diiodide

1 µg/mL
1 µg/mL
1 µg/mLConditions
Column10EluentmAU

1

Flow rate
Temp.
Detection
Inj. Vol.

2
3: Inertsil ODS-3(5 μm, 150 x 4.6 mm I.D.): A) Phosphate buffer *B) CH3CN
A/B = 89/11, v/v: 1.0 mL/min: 40 ℃: UV 290 nm: 50 μL* Phosphate buffer ；0Phosphoric acid (200 mM), diethylamine(100 mM), and sodium 1octanesulfonate (7.5 mM) wasdissolved in water.

02468Time [min]101214

Chemical Structures
ClH3CClN+

N+ CH3

Paraquat

Br-BrN+N+Diquat
Structures are created using Chemistry 4-D Draw which is provided by ChemInnovation Software, Inc.

GL Sciences LC Technical Note
What is MonoSpin ?
MonoSpin is a series of spin columns for solid phase extraction (SPE). Owing to the high permeability of monolithic silica disk
packed into the spin column, the procedures, such as conditioning, sample loading, washing, and elution can be carried out onlyby centrifuging the column. It is also the advantage that the elution volume is only 200 µL.

An example of procedures

Centrifuge

Centrifuge

Conditioning Adsorption

Centrifuge

Rinsing

Centrifuge

ElutionMonolithic silica disk

MonoSpin
Enlarged view of monolithic silica

Background of pretreatment using MonoSpin CBAMonoSpin CBA has carboxylate group (COOH). This functional group is a typical weak acid, and its ratio of COOanion changes depending on the pH of solvent passing through. When cationic paraquat and diquat are loaded
onto MonoSpin PBA, basic solvent should be passed through to dissociate carboxylate group. In contrast,strong acid solvent is used for elution to inhibit dissociation of COOH group.
In case of MonoSpin SCX, its functional group is almost all dissociated even at pH 1. Therefore, elution of
paraquat and diquat from MonoSpin CBA is much easier than that from MonoSpin SCX.

MonoSpin CBA (pKa ≒ 4.8)

< 1 % dissociated

50 % dissociatedElutionpH 1.02.8> 99 % dissociatedAdsorption
4.8
6.8 (Rinsing)
MonoSpin SCX（pKa ＜1.0）

14.0> 99 % dissociatedpH 1.014.0

GL Sciences LC Technical Note
Chromatograms obtained from urinary sample (1 g/mL each standard addition)

0mAU

10

＜Before purification with MonoSpin CBA＞

02468Time [min]10121410＜After purification with MonoSpin CBA＞

2mAU

1030268Time (min)4101214Procedures for purification of paraquat and diquat
Preparation of buffer；
Buffer A：
Buffer B：

10 mM Phosphate buffer (pH 7.0) Hydrochloric
acid：Methanol：Water＝1：30：69

Attach the spin column to tube for waste fluid↓ ＋ Buffer A
200 μLCentrifuge*↓ ＋ Buffer A↓ ＋ SampleCentrifuge↓ ＋ Buffer A

Centrifuge↓（to top right）

400 μL200 μL200 μL(from bottom left)↓
Put the spin column into collection tube
↓ ＋ Buffer B
200 μLCentrifuge↓

Collected solution was injected into HPLC system* Centrifugation was carried out at 10,000 g for 30
seconds.

GL Sciences LC Technical Note
Validation of this method
Excellent recovery was obtained because strong basic solution,
under which the compounds are unstable, was not used. MonoSpin
series is also suitable for pretreatment of basic compound
adsorptive to glass because each tube used is made of plastic.
Concentration(µg/mL)Intra-day (n=5)
Recovery (%)

RSD (%)

Inter-day (n=3)
Recovery (%)

RSD (%)

Paraquat

1.0
0.1

97.5
99.1

6.5
3.5

95.4
95.4

2.6
3.3

Diquat

1.0
0.1

94.3
98.7

8.9
3.6

97.4
97.7

3.0
3.1

Ethylparaquat

1.0
0.1

88.0
101.6

5.9
3.2

89.8
97.3

6.0
4.0

The spin column used in this note;
MonoSpin CBA

50/pk

Cat.No. 5010-21729

MonoSpin CBA

100/pkCat.No. 5010-21730
Detailed instructions for using MonoSpin spin columns can be viewed in the following link.
http://www.youtube.com/watch?v=uVh0Bw8QiGg

The HPLC column used in this note; Inertsil ODS-3 (5 µm, 4.6 x 150 mm)
Cat.No. 5020-01731

GL Sciences disclaims any and all responsibility for any injury or damage which may be caused by this data directly or indirectly. We reserve theright to amend this information or data at any time and without any prior announcement.

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