Fast and Sensitive Analysis of Paraquat and Diquat in Drinking Water

Houssain El Aribi AB SCIEX Concord, Ontario (Canada)

Overview
This application note describes a fast and sensitive LC-MS/MS method for the determination of Paraquat and Diquat in drinking water. Using the Ultra Quat HPLC column and the AB SCIEX API 3200 LC/MS/MS System equipped with a Turbo V source, the limits of quantitation (LOQ) for this method in drinking water are 0.1 g/L and 5 g/L respectively for Diquat and Paraquat using a 10 L injection volume without sample preparation prior to analysis.

Introduction
Paraquat (1,1-dimethyl-4,4-bipyridylium dichloride, C12H14N2Cl2), and Diquat (1,1-ethylene-2,2-bipyridilium dibromide, C12H12N2Br2), are non-selective and nonsystematic contact herbicides widely used in agriculture to control broadleaf and grassy weeds. The use of these herbicides is very important because weeds compete vigorously with crops for water, light and other nutrients. As a result, if they are not suppressed they reduce crop yields by up to 80%. However both Parquat and Diaquat are toxic and ingestion of either compound can have serious effects as they can alter reduction-oxidation activities in biological systems. The analysis of these highly charged dual quaternary amines is complicated because of their ionic nature and therefore Paraquat and Diquat are difficult to retain by standard reversed phase HPLC. For drinking water the United States Environmental Protection Agency (EPA) has established 1 a maximum contaminant level of 20 g/L for Diquat. Paraquat is currently not regulated in drinking water to our knowledge. The EPA 549.2 method for the analysis of Paraquat and Diquat uses reversed phase/ion-pair extraction utilizing C8 SPE cartridges followed by ion-pair LC with ultraviolet (UV) and/or photodiode array (PDA) detection.2 This method is timeconsuming and requires large sample volume, and suffers from stability and reproducibility problems associated with ionexchange chromatography. Recently, various mass spectrometry (MS) methods have been developed for the analysis of these herbicides. Although these methods have lower limit of detection, 3-4 an extensive cleanup is generally required.

Experimental
Chemicals De-ionized (DI) water (Type I reagentgrade, 18 M-cm resistance) was produced in house. HPLC grade acetonitrile was purchased from J. T. Baker. Standards of Paraquat dichloride tetrahydrate and Diquat dibromide monohydrate were purchased from Supelco. The purity of these compounds was 99%. Internal standards (ISTD) D 8-Paraquat and D4-Diquat (both 99.6% isotope enriched) were obtained from CDN Isotopes. Heptafluorobutyric acid (HFBA), 99%, was obtained from SigmaAldrich. LC An Agilent 1100 series equipped with degasser, quaternary pump, and autosampler was used. HPLC separation was performed on a Restek Ultra Quat 3m (50x2.1mm) with guard column Ultra Quat 3 m (20x2.1 mm) and an isocratic mobile phase of 95% water + 5% acetonitrile + 10mM of HFBA at a flow rate of 500 L/min. The injection volume was set to 10 L. Low concentrations of HFBA effectively shield the positive charges of Paraquat and Diquat, increasing interaction with the Ultra Quat stationary phase, resulting in more retention required to separate analytes from matrix components.

p1

MS/MS An AB SCIEX API 3200 LC/MS/MS system equipped with a Turbo V source operating in Electrospray Ionization (ESI) mode and positive polarity was used. The following source and gas parameters were applied: TEM=700C; CUR=15 psi; GS1=70 psi; GS2=60 psi; IS=5500 V; and CAD=7. Compound dependent parameters, such as Declustering Potentials (DP), Collision Energies (CE), and Collision Cell Exit Potential (CXP) for each detected MRM transition are listed in Table 1. Two transitions a quantifier and a qualifier were monitored. A dwell time of 200 ms were used per transition.
Table 1. Detected MRM transitions for the analysis of Paraquat and Diquat Analyte Name
+ + Paraquat [M2 -H ]

Results and Discussion

At the analytical conditions used, Paraquat and Diquat preferentially form the singly charged [M2+-H+] ions as the MS + base peaks. However, the doubly charged [M2 ] ions at m/z 92 (Diquat) and m/z 93 (Paraquat) and the radical [M+.] cations at m/z 184 (Diquat) and m/z 186 (Paraquat) have also been formed at much lower relative intensities. The MS/MS spectrum of singly charged Diquat (m/z 183) is quiet simple compared to that of the singly charged Paraquat (m/z 185) as illustrated in Figure 1.

MRM transition 185/170 185/169

DP (V) 40 40 40 35 35 35

CE (V) 30 35 29 32 35 32

CXP (V) 3 3 3 3 3 3

D8-Paraquat [M -H ] Diquat [M2 -H ]

+ +

+ 2

193/178 183/157 183/168

D4-Diquat [M -D ]

+ 2

186/158

+ M S 2 3 .2 e 5 3 .0 e 5 2 .8 e 5 2 .6 e 5 2 .4 e 5 2 .2 e 5 2 .0 e 5 In t e n s it y , c p s In t e n s it y , c p s 1 .8 e 5 1 .6 e 5 1 .4 e 5 1 .2 e 5 1 .0 e 5 8 .0 e 4 6 .0 e 4 4 .0 e 4 2 .0 e 4

(1 8 5 . 0 0 ) C E

( 3 0 ): 2 0

M C A

s c a n s

fr o m

S a m p le 2

( C ID

o f 1 8 5 _ C E = 3 0 V _ Q 3

h ig h

R e s ) o f D iq u a t a n d P a r a q u a t . w i f f ( T u r b o S p r a y )

M a x . 3 . 2 e 5 1 8 5 .3

c p s .

Paraquat
a quantifier MRM 185/170 b qualifier MRM 185/169
1 7 0 .2

[M2+ - H+] a b

1 5 8 . 2 1 4 4 .3

1 1 8 .3 1 4 3 .2 1 0 7 .2 1 8 3 .2 1 4 2 . 1 9 2 .3 4 3 .2 2 0 3 0 4 0 ( 2 5 ): 2 0 M C A 5 5 . 2 5 0 s c a n s fr o m 5 7 .0 6 0 S a m p le 3 6 5 . 1 6 9 .2 7 0 ( C ID 7 7 . 1 8 3 .2 1 0 6 .1 1 1 5 .2 1 1 7 .3 1 2 8 .3 1 2 1 .3 1 3 1 .2 1 3 9 .2 1 4 5 .3 1 5 5 .0 1 5 3 . 2 1 5 0 1 6 8 .0 1 5 9 .2 1 8 2 .1 1 6 0 1 7 0 1 8 0 1 9 0 M a x . 2 . 8 e 6 1 5 6 .9 2 0 0 c p s .

+ M S 2

(1 8 3 . 0 0 ) C E

8 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 m /z , a m u o f 1 8 3 _ D iq u a t _ C E = 2 5 V ) o f F r a g m e n t a t i o n _ M a r c h 7 _ 0 6 . w i f f ( T u r b o S p r a y )

2 .8 e 6 2 .6 e 6

2 .4 e 6 2 .2 e 6

Diquat
a quantifier MRM 183/157 b qualifier MRM 183/168 a [M2+- H+]
1 8 2 .9

2 .0 e 6 1 .8 e 6

1 .6 e 6 1 .4 e 6 1 .2 e 6

1 .0 e 6 8 .0 e 5

6 .0 e 5 4 .0 e 5

b
4 3 .3 3 0 4 0 5 4 .8 5 0 5 6 .7 6 0 6 6 .8 7 0 7 8 . 1 7 9 .8 8 0 1 0 4 .2 8 2 . 7 9 2 .7 9 0 9 8 .8 1 0 0 1 3 0 .0 1 1 7 .2 1 2 2 . 9 1 1 4 .8 1 1 0 1 2 0 m /z , a m u 1 3 9 .7 1 3 0 1 4 2 .1 1 4 0 1 5 0 1 5 5 .3 1 6 5 .1 1 6 0 1 6 8 .1 1 8 2 .0 1 7 0 1 8 0 1 8 4 .2 1 9 0 2 0 0

2 .0 e 5

Figure 1. MS/MS spectra of Paraquat and Diquat

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XIC of +MRM (6 pairs): 183.0/157.0 amu from Sample 21 (P_D_Quats_500ng_mL_500ng_mL ISTD) of P_D_Quats_ISTDs_CAL_Marc... 100% 95% 90% 85% 80% 75% 70% 65% 60% 55% 50% 45% 40% 35% 30% 25% 20% 15% 10% 5% 0% 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 Time, min 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 5.03

Max. 3.1e5 cps.

Diquat
MRM 183/157 MRM 183/168 MRM 186/158 (ISTD)

Paraquat
MRM 185/170 MRM 185/169 MRM 193/178 (ISTD)

9.5

Figure 2. Separation and detection Diquat and Paraquat in drinking water (500 g/L) by LC-MS/MS

For the highest sensitivity and selectivity used Multiple Reaction Monitoring (MRM) was used to quantify Paraquat and Diquat in DI water and drinking water. Figure 2 shows the analysis of drinking water spiked with 500 g/L of Paraquat, D8-Paraquat, Diquat, and D4-Diquat. Two MRM transitions were selected for each analyte. Internal standards were used to improve the accuracy of quantitation, to compensate for matrix effects, and to correct for random and systematic errors in separation and detection. Triplicate injections of 9 concentrations of analytes in DI water and in drinking water, from 0.1 to 500 g/L for Diquat and from 0.5 to 500 g/L for Paraquat were used to investigate the performance of the developed method.
Table 2. Summary of statistical parameters Analyte Name Diquat Paraquat MRM transition 183/157 185/170

Correlation coefficients for calibration curves were >0.999, using a linear fit and 1/x weighting factor. These results indicate that quantification can be performed with good linearity and sensitivity. Figure 3 and 4 show the calibration curve for Diquat (MRM 183/157) and Paraquat (185/170) in the case of drinking water. Table 2 summarizes the statistical parameters for the analysis of Diquat and Paraquat in drinking water. The limits of quantitation (LOQ) of the analytes were calculated from the chromatograms, at a signal-to-noise-ratio of >10.

LOQ (g/L) 0.1 0.5

Linear range ( g/L) 0.1 5000 0.5 5000

0.9996 0.9999

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Di_Para _Quats_ISTD_Ma rch 9_06 .rdb (183 .0 / 157.0 ): "Linear" Regression ("1 / x" weighting): y = 1.01 x + 0.000817 (r = 0.9996)

Summary
The use of the Restek Ultra Quat 3 m HPLC column with an eluent containing Heptafluorobutyric acid allows sufficient separation of Paraquat and Diquat. Coupled to an API 3200 LC/MS/MS systems enough sensitivity of detection is provided to inject water samples directly without any time-consuming sample preparation prior to analysis. The method was found to be robust, selective and sensitive.

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

Diquat
MRM 183/157 R2 = 0.9996

References
1
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 Analyte Conc. / IS Conc. 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0

Figure 3. Calibration curve for Diquat (183/157) in drinking water from 0.1 g/L to 5000 g/L

US EPA, Drinking Water Health Advisory: Pesticides, US Environmental Protection Agency, Lewis, Chelsea, MI, 1989 J. W. Munch, W. J. Bashe, US EPA 549.2, US Environmental Protection Agency, Cincinnati, OH, 1997 R. Castro, E. Moyano, M. T. Galceran J. Chromatogr. A 2001, 914, 111-121 L. Grey, B. Nguyen, P. Yang J. Chromatogr. A 2002, 958, 2533

Di_Para_Quats_ISTD_March 9_06.rdb (185 .0 / 170.0): "L inear" Regression ("1 / x" we ighting): y = 1.28 x + -0.00099 (r = 0.9999)

13 13 12 12 11 11 10 10 9 9 8 8 7 7 6 6 5 5 4 4 3 3 2 2 1 1 0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 Analyte Conc. / IS Conc. 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0

Paraquat
MRM 185/170 R2 = 0.9999
4

Figure 4. Calibration curve for Paraquat (185/170) in drinking water from 0.5 g/L to 5000 g/L

For Research Use Only. Not for use in diagnostic procedures. 2010 AB SCIEX. The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners. AB SCIEX is being used under license. Publication number: 1281110-01

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