Operating conditions Chromatograph Columns Mobile phase Column inlet pressure Flow rate
Detector Attenuation Recorder Injection volume
Retention time for 5-amino-4- chloropyridazin-3(2H)-one (metabolite B)
HPLC instrument fitted with pump LC-410 and autosampler MSI 660 (Kontron)
Two stainless steel columns, each 3.2 mm i. d. and 10 cm long; packed with Silica Spheri-5 (Brownlee Labs); connected in series
Dichloromethane + methanol + triethylamine 940:60:1 v/v/v
Approx. 80 bar 1.8 ml/min
UV detector Uvikon LC-720 (Kontron) Wavelength 286 nm
0.01 E
10 mV; chart speed 5 mm/min 100 nl
3 min 13 s
7 Evaluation
7.1 Method
Quantitation is performed by the calibration technique. Prepare two calibration curves prior to each series of measurements as follows. Dilute aliquots of the chloridazon standard solu- tions with appropriate amounts of the injection mixture to yield a set of chloridazon measur- ing standards. Inject 1 ul each of these solutions (equivalent to 0.025 to 0.2 ng chloridazon) into the gas chromatograph. Likewise inject 100 ul of each metabolite B standard solution (equivalent to 50 to 200 ng metabolite B) into the high-performance liquid chromatograph.
Plot the heights of the peaks obtained vs. ng chloridazon or ng metabolite B, respectively. Also inject 1-ul or 100-ul aliquots, respectively, of the sample solutions. For the peaks obtained for
Chloridazon 143 these solutions, read the appropriate amounts of chloridazon or metabolite B from the respec- tive calibration curve.
7.2 Recoveries and lowest determined concentration
Recovery experiments were run on different untreated control samples of plant material, soil and water, fortified with chloridazon and metabolite B at levels of 0.05 to 0.5 mg/kg (water 0.5 to 5 |ig/l). The recoveries are given in the Table. The routine limit of determination was 0.05 mg/kg for plant material and soil, and 0.5 |ig/l for water.
Table. Percent recoveries from plant material, soil and water, fortified with chloridazon and metabolite B.
. . . , . . Chloridazon Metabolite B Analytical material
Range Mean Range Mean
71- 76 81-109 86-100 86-104 96-103 83- 87
74 96 92 95 98 85
62- 85 70- 78 77- 83 67-104 73- 91 80- 92
74 73 79 86 81 84 Mangold 63-106 85 8 1 - 9 1 86 Red beet
Foliage Edible root Sugar beet
Foliage Edible root Soil
Water
7.3 Calculation of residues
The residue R, expressed in mg/kg chloridazon or metabolite B, is calculated from the follow- ing equations:
for chloridazon R = W* / \ ' VR,2/ > "
for metabolite B R = ^ % ' V R 4 "V E n d
VR 5 • Vi • G w h e r e
G = s a m p l e weight (in g)
VE x = t o t a l v o l u m e o f extract o b t a i n e d in 6.1.1 o r 6.1.2 (in m l ) VR 1 = p o r t i o n of v o l u m e VE x u s e d for c l e a n u p (in m l )
VR 2 = v o l u m e o f a q u e o u s residue o b t a i n e d from e v a p o r a t i o n in 6.2.1 (in m l )
VR 3 = p o r t i o n o f v o l u m e VR 2 u s e d for c l e a n u p o n t h e E x t r e l u t c o l u m n i n step 6.2.2 (in m l ) VR 4 = v o l u m e o f m e t h a n o l u s e d t o dissolve t h e d r y residue i n 6.3.2 (in m l )
144 Chloridazon
VR5 = portion of volume VR4 injected for gel permeation chromatography (volume of sam- ple loop) (in ml)
VEnd = terminal volume of sample solution from 6.4 or 6.5 (in ml)
Vj = portion of volume VEnd injected into gas chromatograph or high-performance liquid chromatograph (in \i\)
WA = amount of chloridazon or metabolite B, respectively, for V{ read from calibration curve (in ng)
8 Important points
Broad peaks with strong tailing and decreased sensitivity can occur during the gas chromatography of metabolite A (as chloridazon). Shortening the inlet end of the column by approx. 2 cm can greatly restore the sensitivity (see 9. Reference).
Due to the presence of hydrochloric acid in the aqueous solutions from 6.3.1, ferric chloride can be released from the Extrelut column during the cleanup in step 6.3.2. In order to prevent any precipitation in the HPLC column, which would adversely affect the separation efficiency, the ferric chloride is removed by the preceding gel permeation chromatography as described in 6.3.3.
If a gas chromatograph fitted with capillary columns is not available, chloridazon deter- mination can also be performed on packed columns, under the following conditions: Glass column, i.d. 2.5 mm, 90 cm long; packed with 5% Silar-5 CP on Gas Chrom Q, 100-120 mesh; column temperature 240°C; injection port temperature 260°C; 63Ni electron capture detector, temperature 260°C; carrier gas argon + methane 9:1 v/v, 120 ml/min; retention time for chloridazon about 11 min.
In most cases, chloridazon residues can also be determined by HPLC. Operating conditions then differ from those given in 6.5 in the following points: Mobile phase isooctane + isopropanol + methanol 88:10:2 v/v/v; flow rate 2 ml/min, column inlet pressure 20 bar;
attenuation 0.03 E; retention time for chloridazon 6 min 36 s.
9 Reference
F. Kuhlmann, Ruckstandsbestimmung von Pyrazon und seinen Metaboliten in Zuckerriiben, Z. Lebensm. Unters. Forsch. 775, 35-39 (1981).
10 Authors
BASF, Agricultural Research Station, Limburgerhof, W. Keller and S. Otto
Chlorsulfuron, Metsulfuron 664-672 Cereals (green matter, grains and straw)
Soil, water
High-performance liquid chromato- graphic determination
(German version published 1989)
1 Introduction
Chemical name
Structural formula
Chlorsulfuron
l-(2-Chlorophenylsulphonyl)- 3-(4-methoxy-6-methyl-l,3,5- triazin-2-yl)urea (IUPAC)
C1 ff N -
S O2- N H - C - N H — ( / CH,
OCH3
Metsulfuron
(as metsulfuron-methyl) Methyl 2-[3-(4-methoxy-6- methyl-l,3,5-triazin-2- yl)ureidosulphonyl]benzoate (IUPAC)
CH, CO2CH3 O N ^
S O2- N H - C - N H — ( / N OCH,
Empirical formula Molar mass Melting point Vapour pressure Solubility
Other properties
C12H12C1N5O4S 357.78
174-178°C
6.1-10 "6 mbar at 25 °C Very sparingly soluble in water at 25 °C;
slightly soluble in dichloromethane (1-2 g), sparingly soluble in acetone (0.5-1 g) and methanol (0.1-0.2 g),
very sparingly soluble in toluene (0.01-0.1 g), virtually insoluble in n-hexane (<0.01 g), in 100 ml each at 22 °C Hydrolyzed slowly in acid media, heat sensitive, little sensitivity to sunlight and UV radiation
C14H15N5O6S 381.37 163-166°C
7.7-10"5 mbar at 25 °C Virtually insoluble in water at 25 °C;
readily soluble in
dichloromethane (10-20 g), soluble in acetone (2-5 g), sparingly soluble in ethanol and methanol (0.2-0.5 g),
very sparingly soluble in xylene (0.01-0.1 g),
virtually insoluble in n-hexane (<0.01 g),
in 100 ml each at 20 °C Stable in acid and alkaline media, little sensitivity to sunlight and UV radiation.
Metsulfuron is present as the methyl ester in commercial preparations
146 Chlorsulfuron, Metsulfuron
2 Outline of method
Chlorsulfuron and metsulfuron residues are extracted from cereal and soil samples with a slightly alkaline buffer solution. The extract is washed with dichloromethane; the aqueous phase is acidified, and the compounds are partitioned into toluene. Water samples are acidi- fied and extracted with dichloromethane. The extracts are cleaned up using a disposable silica gel cartridge. Chlorsulfuron and metsulfuron are determined by high-performance liquid chromatography using a photoconductivity detector.
3 Apparatus
High-speed blendor fitted with leak-proof glass jar and explosion-proof motor Buchner porcelain funnel, 9 cm dia.
Filter paper, 9 cm dia., fast flow rate (Schleicher & Schull) Filtration flask, 500-ml
Laboratory mechanical shaker Laboratory centrifuge, 3800 r.p.m.
Centrifuge tubes, 500-ml and 250-ml Volumetric flask, 100-ml
Graduated cylinders, 500-ml, 250-ml, 100-ml, 50-ml and 10-ml Separatory funnels, 2-1 and 500-ml
Magnetic stirrer, with stirring rod Beakers, 400-ml and 250-ml pH Meter
Round-bottomed flasks, 500-ml, 250-ml and 50-ml, with ground joints Volumetric pipets, 50-ml, 25-ml, 2-ml and 1-ml
Rotary vacuum evaporator, 45 °C bath temperature Glass funnel, 9 cm dia.
Glass syringe, 10-ml, with Luer-lock fitting Test tubes, 10-ml, with ground stoppers
Vacuum filtration unit, with 0.45 fim membrane filter Ultrasonic bath
High-performance liquid chromatograph equipped with photoconductivity detector Microsyringe, 100-ul
4 Reagents
Acetone, p. a.
Cyclohexane, HPLC quality Dichloromethane, p. a.
Diethyl ether, p. a.
Ethyl acetate, p. a.
Glacial acetic acid, HPLC quality
Chlorsulfuron, Metsulfuron 147
Methanol, HPLC quality
2-Propanol (isopropanol), HPLC quality Toluene, p. a.
Water, bi-distilled
Extraction solution: acetone + buffer solution B 4:1 v/v
Mobile phase: Prepare a mixture consisting of 690 ml cyclohexane + 195 ml isopropanol + 115 ml methanol + 2 ml glacial acetic acid + 1 ml of a glacial acetic acid-water mixture (9:1 v/v). Mix well, and de-gas before use in a vacuum filtration unit using water jet pump suction
Conditioning solution: Prepare a mixture consisting of 200 ml isopropanol + 200 ml methanol + 200 ml glacial acetic acid + 20 ml water. Mix well, and de-gas before use in a vacuum filtration unit using water jet pump suction
Stock solutions: 10 mg/100 ml each of chlorsulfuron and metsulfuron-methyl in ethyl acetate.
Pipet 1 ml of each stock solution into a 100-ml volumetric flask. Remove the solvent with a gentle stream of nitrogen, dissolve the residue and make up to the mark with mobile phase Chlorsulfuron and metsulfuron-methyl standard solutions: 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5 M-g/ml of each in mobile phase, de-gassed in an ultrasonic bath. The solutions are stable for approx. two weeks when stored in a refrigerator
Hydrochloric acid, cone., 10 g/100 g and 1 mol/1 HC1 p. a.
Sodium sulphate, p.a., anhydrous
Buffer solution A: 10.6 g/1 sodium carbonate anhydrous p. a. and 8.4 g/1 sodium hydrogen carbonate anhydrous p. a.
Buffer solution B: 0.82 g/1 sodium acetate anhydrous p. a.
Silica gel disposable cartridge: Sep-Pak Cartridge Silica (Millipore No. 51900) Nitrogen, re-purified
5 Sampling and sample preparation
The analytical sample is taken and prepared as described on pp. 17 ff and pp. 21 f, Vol. 1. For water samples, observe the guidelines given on pp. 23 ff, Vol. 1.
6 Procedure
6.1 Extraction 6.1.1 Cereals
Homogenize 10 g of the analytical sample (G) (25 g of grains) with 150 ml extraction solution in the mixer for 2 min. Suction-filter the supernatant liquid through a fast-flow rate filter paper in a Buchner porcelain funnel. Homogenize the residue in the glass jar and suction-filter twice more as above, each time using 120 ml extraction solution (for cereal grains, use 100 ml each time). Finally, rinse the glass jar and the filter with a further 80 ml of extraction solution.
Transfer the filtrate to a volumetric flask and make up to a definite volume, e.g. 500 ml (VEx). Next transfer a tenth of this solution (VR1) to a 500-ml separatory funnel, add 100 ml buffer solution A, and proceed to step 6.2.1.
148 Chlorsulfuron, Metsulfuron
6.1.2 Soil
Weigh 50 g of the analytical sample (G) into a 500-ml centrifuge tube, add 100 ml buffer solu- tion A, and vigorously shake on a mechanical shaker for 1 h. Centrifuge the suspension at 3800 r.p.m. for 15 min, and transfer the supernatant liquid into a 500-ml separatory funnel.
Repeat the extraction once more, combine the extracts, and proceed to step 6.2.1.
6.1.3 Water
Transfer 2 1 water (G) into a separatory funnel, adjust the pH to 3-4 with hydrochloric acid (1 mol/1), and extract the water three times with 100-ml portions of dichloromethane. Filter the combined dichloromethane phases through a layer of sodium sulphate, contained in a fun- nel, into a 500-ml round-bottomed flask. Rinse the funnel with 50 ml dichloromethane. Add 1 ml glacial acetic acid and rotary-evaporate to approx. 1 ml. Transfer the residue into a 50-ml round-bottomed flask, using four 5-ml portions of mobile phase to complete the transfer, and rotary-evaporate to 1-2 ml. Evaporate the solution to dryness, using a gentle stream of nitrogen, and proceed to step 6.2.2.
6.2 Cleanup
6.2.1 Liquid-liquid partition (only cereals and soil)
Shake the extracts derived from 6.1.1 or 6.1.2 twice with 50-ml portions of dichloromethane (for soil extracts, use two 50-ml portions of diethyl ether) for 3 min. Discard the organic phases. Break emulsions, if required, by centrifugation. Transfer the aqueous phase to a beaker and acidify, with vigorous stirring, to pH 5-6 (pH meter) with concentrated hydrochloric acid. Further, adjust the pH to 3.5 using hydrochloric acid (10% w/w) (see 8. Important points). Transfer the solution back to the separatory funnel, rinse the beaker with 5 ml water and 50 ml toluene, also add the rinsings to the separatory funnel, and shake for 3 min. Separate the aqueous layer, drain the organic phase into a 250-ml centrifuge tube, rinse the separatory funnel with 5 ml water, and add the rinsings to the centrifuge tube. Repeat the extraction twice, each time using 50 ml toluene and adding the organic phases and water rinsings to the centrifuge tube. Add a further 10 ml of water to the centrifuge tube if the phase boundary is difficult to see after the third extraction.
Centrifuge for 15 min at 3800 r.p.m., then transfer the upper toluene phase into a 250-ml round-bottomed flask, using a 50-ml volumetric pipet. Add 30 ml toluene to the aqueous phase remaining in the centrifuge tube, mix, centrifuge for 5 min, and likewise pipet off the top layer into the 250-ml round-bottomed flask. Add 1 ml glacial acetic acid to the combined toluene phases, and rotary-evaporate to approx. 1 ml. Transfer the residue into a 50-ml round- bottomed flask, using four 5-ml portions of mobile phase to complete the transfer, and rotary- evaporate to 1 -2 ml. Evaporate the solution to dryness, using a gentle stream of nitrogen, and proceed to step 6.2.2.
6.2.2 Silica gel cartridge
Draw 10 ml of the mobile phase into the glass syringe, attach a silica gel cartridge to the syr- inge, and force the mobile phase through to condition the cartridge packing. Repeat the condi-
Chlorsulfuron, Metsulfuron 149
tioning with a further 10-ml portion of mobile phase. Next detach the cartridge, pull the plunger out of the syringe, and re-attach the cartridge. Dissolve the residue derived from 6.1.3 or 6.2.1 in 1 ml mobile phase and transfer the solution quantitatively into the syringe with the aid of a Pasteur pipet. Rinse the 50-ml flask with 1 ml mobile phase and also add the rinsings to the syringe. Re-insert the plunger into the syringe and force the liquid through the cartridge, collecting the eluate in a 10-ml test tube. Detach the cartridge, remove the plunger from the syringe, and re-attach the cartridge. Force a further 5 ml mobile phase through the cartridge, proceeding in a similar manner as described above, and collect the eluate in the same test tube.
Evaporate the solution to dryness, using a gentle stream of nitrogen.