Pestic. Sci. 1997, 51, 102È107

Communication to the Editor

Determination of N-(3-Ethylsulfonyl-2-pyridinyl)-4,6-dimethoxy-2-pyridineamine in Soil afterTreatment with RimsulfuronCesare Marucchini1* & Riccardo Luigetti21 Istituto di Chimica Agraria, Universita degli Studi di Perugia, Borgo XX Giugno 72, 06121-Perugia, Italy2 Centro di Studio sulla Chimica e Biochimica dei Fitofarmaci del CNR, Borgo XX Giugno 72,06121-Perugia, Italy

(Received 9 December 1996 ; revised version received 26 February 1997 ; accepted 3 April 1997)

Abstract : An analytical procedure to detect N-(3-ethylsulfonyl-2-pyridinyl)-4,6-dimethoxy-2-pyridineamine, a degradation product deriving from the hydrolysisof rimsulfuron in soil, has been developed. The analytical standard was preparedby basic hydrolysis of rimsulfuron at pH 9 and puriÐcation on a silica gel chro-matographic column. The compound obtained was stable at high temperature,thus enabling determination by gas chromatographic analysis. Soil samples wereextracted with acetonitrile, puriÐed with a SPE C18 cartridge and analysed usingboth nitrogen phosphorus (NPD) and mass spectrometer detectors. The analyti-cal procedure described proved to be sensitive and reproducible. Recoveriesvaried from 84 to 90%. The limit of sensitivity was 0É001 mg kg~1.

Pestic Sci., 51, 102È107, 1997No. of Figures : 4. No. of Tables : 2. No. of Refs : 8

Key words : rimsulfuron, herbicide, sulfonylurea, analytical procedure

1 INTRODUCTION

Rimsulfuron, 1-(4,6-dimethoxypyrimidin-2-yl)-3-(3-ethylsulfonyl-2-pyridylsulfonyl)urea, is widely used as aselective post-emergence herbicide in maize crops. Themode of action of rimsulfuron, like other sulfonylureas,is the inhibition of acetolactate synthase (ALS) andbranched-chain aminoacid biosynthesis.1h3 The speciÐcand high activity of rimsulfuron allows treatments atrates as low as 10È30 g ha~1.

Degradation pathways of rimsulfuron in soil and inhydrolytic solutions under laboratory conditions haveshown to produce Ðve degradation products derivedfrom cleavage or contraction of the sulfonylureabridge.4 The hydrolysis rate was pH-dependent and was

* To whom correspondence should be addressed.

also inÑuenced by temperature. Under Ðeld conditionsrimsulfuron degraded rapidly with a half-life of 5É6 daysand the major metabolite was formed by a contractionof the sulfonylurea bridge forming N-(3-ethylsulfonyl-2-pyridinyl)-4,6-dimethoxy-2-pyridineamine.

The very low dosages and rapid dissipation of somesulfonylureas, including rimsulfuron, in soil1,4,5 compli-cate residue analysis and although trace-level methodsfor sulfonylurea herbicides have been proposed,5,8 it isstill not easy to conduct routine analyses of rimsulfuronat trace levels. For this reason the present study wasaimed at investigating the stability and persistenceof N-(3-ethylsulfonyl-2-pyridinyl)-4,6-dimethoxy-2-pyridineamine, which seems to be rimsulfuronÏs moststable degradation product,4 so as to develop an ana-lytical procedure to detect it in soil at trace levels. Thedetermination of the major metabolite is necessary

1021997 SCI. Pestic. Sci. 0031-613X/97/$17.50. Printed in Great Britain(

Determination of rimsulfuron metabolites 103

whenever a parent compound degrades in a very shorttime : for instance, the fungicide benomyl degradesrapidly to carbendazim. For this reason only carbenda-zim is determined as the residue in soil and crops. Asimilar situation might be demonstrated for rimsulfu-ron.

2 EXPERIMENTAL METHODS

2.1 Apparatus

Gas chromatography (GLC) analyses were performedusing a Carlo Erba model 8560 chromatograph, Ðttedwith a nitrogen-selective detector (NPD) and an on-column injector. GC/MS analyses were performed usinga Varian Star model 3400 chromatograph, Ðtted with asplit-splitless injector. The mass spectrometer was aVarian Saturn II operating in the electron impact modeand in chemical ionization. HPLC analyses were per-formed using a LDC analytical, Constametric model4100 solvent delivery system Ðtted with a Thermoseparation product, Spectromonitor model 3200 vari-able UV wavelength detector and a Rheodyne injectionsystem. NMR spectra were determined in hexadeuterodimethyl sulfoxide using a Varian NMR(d6-DMSO)spectrometer 300 MHz. IR spectra were determinedusing a Nicolet 205 FT spectrometer. Solid-phaseextractions were performed using Supelco SPE C18 car-tridges (3-ml tubes).

2.2 Reagents and materials

The analytical standard of rimsulfuron was provided byDu Pont Agricultural Products, Wilmington DE, USA.The methylene chloride and acetonitrile were Analar(BDH) grade ; the water, acetonitrile and acetic acidused for HPLC analyses were Hipersolv (BDH) grade.The methanol was a special reagent for pesticideanalysis (BDH).

A loam soil (Fluventic Xerochrept) was used ; themajor properties of this soil are shown in Table 1.

2.3 Synthesis and identiÐcation of N-(3-ethylsulfonyl-2-pyridinyl)-4,6-dimethoxy-2-pyridineamine

Rimsulfuron (1 g) was added to a Tris-HCl bu†er solu-tion (0É1 M, 300 ml) at pH 9 and stirred for three days.The solution was extracted with dichloromethane

TABLE 1Properties of the Soil

Mechanical analyses (%)Organic CEC

matter (%) pH Clay Sand Silt (meq 100 g~1)

1É81 8É05 29É0 35É0 36É0 18

(3] 100 ml) and the organic extract evaporated just todryness in a rotary evaporator (40¡C). The residue wasdissolved in dichloromethane and puriÐed by a chro-matographic column Ðlled with silica gel. The columnwas eluted with hexane] ethyl acetate] acetic acid(20 ] 80 ] 2 by volume). The eluate was evaporatedand the weight of the solid residues was nearly 400 mg.Aliquots of this product were dissolved in methanol andits chemical structure was established by GC/MS, NMRand IR analyses.

2.4 Soil treatment with rimsulfuron

Air-dried soil (100 g), passed through a 1É0-mm sieve,was treated with a methanolic solution (5 ml) contain-ing 2 kg ml~1 of rimsulfuron. The methanol was evapo-rated and a further 900 g of soil was added. In this waythe concentration of rimsulfuron in soil was roughly thesame as that used in Ðeld treatment conditions(0É01 mg kg~1). The soil was then stirred in a mechani-cal shaker for three hours in order to attain an evendistribution of the herbicide, and then placed into tenaerated containers (100 g each). Samples were kept in aclimatic chamber at 25¡C. Distilled water was addeddaily to each sample to maintain soil moisture atapproximately 75% of the Ðeld moisture holding capac-ity. A sample for analysis was removed from thechamber at days 2, 4, 8, 16, 32 and 64. Each sample waskept at [20¡C until analysis.

To determine the stability of rimsulfuron in soil, air-dried soil (100 g), passed through a 1É0-mm sieve, wastreated with a methanolic solution (5 ml) containing20 kg ml~1 of rimsulfuron, then the same procedurewas used.

2.5 Stability trials

To determine the stability of rimsulfuron in solventswith di†erent polarities, rimsulfuron (2É5 mg) was dis-solved in methanol (100 ml), acetonitrile (100 ml) anddichloromethane (100 ml), then stored in the dark at25¡C. To determine the stability of rimsulfuron atvarious pH values, rimsulfuron (25 mg) was dissolved inacetonitrile (10 ml) and the solution obtained (1 ml) wasadded to di†erent bu†er solutions (99 ml) and thenstored in the dark at 25¡C. Both types of solution(10 kl) were analysed after 1, 2, 4, 8, 16 and 32 days.Three 0É1 M bu†er solutions were used : acetate bu†erpH 4, monobasic-dibasic phosphate bu†er pH 7 andTris-HCl bu†er pH 9. The same procedure was used tocheck the stability of N-(3-ethylsulfonyl)-2-pyridinyl-4,6-dimethoxy-2-pyridineamine.

2.6 Recovery trials

Air-dried soil (100 g) passed through a 1É0-mm sievewas treated with a methanolic solution (1 ml) contain-

104 Cesare Marucchini, Riccardo L uigetti

ing the amount of rimsulfuron or N-(3-ethylsulfonyl-2-pyridinyl)-4,6-dimethoxy-2-pyridineamine needed toobtain the experimental fortiÐcation level (see Table 2),then extracted, cleaned up and analysed as described inSection 2.7.

2.7 Analytical procedures

Samples from soil treated with rimsulfuron wereanalysed as follows :

2.7.1 Extraction and clean-up procedure of N-(3-ethylsulfonyl-2-pyridinyl)-4,6-dimethoxy-2-pyridineamineDistilled water (20 ml) and acetonitrile (60 ml) wereadded to soil (100 g). The mixture was stirred for 1 h,centrifuged at 6000 rev min~1 for 15 min and the solu-tion Ðltered. The samples were then washed with theextracting solution (100 ml), and centrifuged again asbefore.

The liquid phase was evaporated using a rotaryevaporator at 40¡C. Methanol (1 ml) and distilled water(20 ml) were then added. The mixture was passedthrough a SPE C18 cartridge previously activated withwater (3 ml) and methanol (3 ml) and the eluate dis-carded. After rinsing with water (20 ml), the column wasthen eluted with methanol (6 ml) and the eluate col-lected and evaporated until just dry. The solid residuewas dissolved in methanol (0É2 ml) and this solution(1 kl) was injected into the gas chromatograph equippedwith NPD detector and into the combined gaschromatograph/mass spectrometer.

2.7.2 Extraction and clean-up procedure of rimsulfuronDistilled water adjusted to pH 2É5 with ortho-phosphoric acid (20 ml) and acetonitrile (60 ml) wereadded to soil (100 g). The mixture was stirred for 1 h,centrifuged at 6000 rev min~1 for 15 min and the solu-tion Ðltered.

The samples were then washed with the extractingsolution (100 ml), and centrifuged again as before. Theacetonitrile was evaporated using a rotary evaporatorat 40¡C and the aqueous solution extracted withdichloromethane (2] 30 ml). The organic phase wasevaporated using a rotary evaporator at 40¡C. The solidresidue was dissolved in methanol (1 ml) and this solu-tion (10 kl) was injected into the HPLC system.

2.7.3 Gas chromatography and GC/MSThe column used for gas chromatographic analyses wasa 25 m ] 0É32 mm fused silica capillary column coatedwith 0É25 km OV17. The temperature was preset to aninitial temperature of 50¡C (1 min) ramped at30¡C min~1 to a Ðnal temperature of 270¡C (20 min).The carrier gas used was helium (2É5 ml min~1). Inthese conditions the retention time of N-(3-ethylsulfonyl -2 -pyridinyl ) -4 ,6 -dimethoxy-2 -pyridine -amine was 20É2 min.

The column used for GC/MS analysis was a30 m ] 0É25 mm fused silica capillary column coatedwith 0É25 km DB5. The temperature was preset to aninitial temperature of 60¡C (1 min) ramped at15¡C min~1 to a Ðnal temperature of 260¡C (5 min).The carrier gas was helium (1 ml min~1), the injectortemperature was 270¡C, the ionization voltage was70 eV and the emission current 10 kA. Methane wasused as the ionizing agent. In these conditions theretention time of N-(3-ethylsulfonyl-2-pyridinyl)-4,6-dimethoxy-2-pyridineamine was 19É4 min.

2.7.4 HPL CThe column used was an octadecyl (C 18)25 cm ] 4É6 mm. The elution solvents used were (A)acetic acid 0É1 M and (B) acetonitrile ; the following gra-dient was used (A : B, linear shifts between all points) :0 minÈ70 : 30, 5 minÈ40 : 60, 20 minÈ40 : 60,25 minÈ0 : 100. The Ñow rate was 1 ml min~1, thedetection wavelength was 254 nm. In these conditionsthe retention time of rimsulfuron was 14É1 min, whilstthe retention time of N-(3-ethylsulfonyl-2-pyridinyl)-4,6-dimethoxy-2-pyridineamine was 20É3 min.

3 RESULTS AND DISCUSSION

The chemical structure of the product obtained fromthe hydrolysis of rimsulfuron (compound 3, Fig. 1) wasestablished by GC/MS, NMR and IR analyses. Themass spectrum obtained by chemical ionization (Fig.2(a)) shows that the most intense signals were those atm/e 325 and at m/e 231. The former is the protonatedmolecular ion of compound 3, whilst the latter is consis-tent with the loss of the ethylsulfonyl group from thision. The signal at m/e 216 in the mass spectrum

Fig. 1 Transformation of rimsulfuron in soil.

Determination of rimsulfuron metabolites 105

Fig. 2. (a) Chemical ionization and (b) electron impact mass spectra of compound 3.

obtained by electron ionization (Fig. (2b)) is consistentwith the loss of from the parent ion 231 ; theÈCH3signal at m/e 188 was formed by the loss of a methylgroup from the parent ion followed by the loss of aCxO group.

In the [1H]NMR spectrum (in the ethyld6-DMSO),protons absorbed at d 1É11 and 3É4(ÈCH3) (ÈCH2È)ppm and the coupling constant was 7É3 Hz. The

groups appear as a singlet at d 3É8 ppm, whileÈOCH3

the pyrimidinic proton resonated at d 5É8 ppm. Thearomatic pyridinic protons appear as double doubletsat d 7É38 (J \ 4É8, 7É8 Hz), 8É23 (J \ 1É8, 7É8 Hz) and8É68 (J \ 1É8, 4É8 Hz) and NH as a singlet at d9É18 ppm. In the [13C]NMR spectrum, it is noteworthythat the signals at d 6É9, 53É8, 82É5, 119É6, 139É7 and153É7 in the Attached Proton Test (APT) correspondedto and xCHÈ, whilst those at d 48É9, 123É4,ÈCH3150, 158É2 and 171É7 were associated with the ÈCH2È

106 Cesare Marucchini, Riccardo L uigetti

Fig. 3. Degradation of rimsulfuron at (2) pH 4, 7 and(+)(E) 9.

and quaternary carbon atoms. The assignments weremade on the basis of : (a) the reported shifts of substit-uents, i.e. sulfonyl and amino groups, on aromatic andheteroaromatic compounds ; (b) the comparison ofchemical shifts of pyridine derivatives and benzensul-fonyl compounds. Moreover the IR spectrum givesfurther proof of the presence of compound 3, ratherthan compound 2, since no C\ O bond is present.

Preliminary trials showed that compound 3 was amore stable compound than rimsulfuron, both in soiland in solvents with di†erent polarities (data notshown). In water the compound was stable at pH values

Fig. 4. Compound 3 detected in soil after treatment with rim-sulfuron at 0É01 mg kg~1.

TABLE 2Recoveries of Rimsulfuron and Compound 3 Added to the

Soil before Extraction

Rimsulfuron Compound 3

Added Recovery Added Recovery(mg kg~1) (%) (^SD)a (mg kg~1) (%) (^SD)a

10 82 (^4É9) 1 90 (^3É1)1 84 (^4É5) 0É1 85 (^4É0)0É1 78 (^5É1) 0É01 85 (^3É8)0É01 76 (^5É0) 0É001 84 (^4É2)

a n \ 3.

between 4 and 9. Analytical standards in the varioussolvents and at the given pH values showed no changesover the 32-day stability trial period ; however, rimsulfu-ron degraded rapidly at all the pH values tested (Fig. 3).Compound 3 was detected within two days in the soiltreated with rimsulfuron at 0É01 mg kg~1 under theconditions described in Section 2.4. This compoundproved to be stable under gas-chromatographic condi-tions enabling detection by gas chromatographic andGC/MS analytical methods, as proposed in this paper.In this experiment it was possible to detect compound 3up to 64 days after treatment (Fig. 4), whilst the rimsul-furon was no longer detectable by HPLC with UVdetection, in soil treated at 0É1 mg kg~1, two days aftertreatment because of its rapid degradation in soil. Thelimit of sensitivity of the analytical method used todetermine the degradation of rimsulfuron was0É01 mg kg~1 and recoveries varied from 76% for0É01 mg kg~1 to 84% for 1 mg kg~1 (Table 2).

The efficiency of the analytical procedure describedfor the analysis of compound 3 residues in soil is indi-cated by the recoveries of the compound added tountreated samples (Table 2). Recoveries varied from84% for 0É001 mg kg~1 to 90% for 1 mg kg~1. On thebasis of the chromatograms of the numerous blankstested, a value of 0É001 mg kg~1 can be Ðxed as thelower limit of determination. Compared to othermethods described in literature4,6 this analytical pro-cedure would appear to be highly cost-e†ective and sen-sitive.

ACKNOWLEDGEMENT

The authors would like to thank Professor GianniPorzi, Department of Chemistry, Bologna Universityfor his assistance in IR and NMR spectroscopy.

REFERENCES

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Determination of rimsulfuron metabolites 107

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