Recovery experiments were run on different untreated control samples of plant material, soil and water, fortified with known amounts of anilazine dissolved in 1-2 ml ethyl acetate. The results are given in the Table.
Table. Percent recoveries from plant material, soil and water, fortified with anilazine; duplicate ex- periments.
Analytical material Added (mg/kg) Range (%) Artichokes 0.02 86-94
0.2 81-85 Aubergines 0.02 89
0.2 84-97 2.0 107-108 20 94-99 Barley
Green matter 0.04 84-93 1.0 87-95 Grains 0.02 96-97 0.2 83-91 Straw 0.04 92-95 0.4 86-87 Beans 0.02 109-111
0.2 87-95
Anilazine 65 Table, (contd.)
Analytical material Added (mg/kg) Range (%) Clover 0.04 79-81 Coffee 0.04 92-96 1.04 72-80 Garlic 0.04 84-85 Hop cones 0.4 82-91 4.0 82-87 Onions 0.04 90-91 Potatoes 0.02 85-98 0.1 72-82 0.2 73-81 1.0 75-79 Radicchio 0.02 91-103
0.2 93-96 2.0 114 Rape green matter 0.04 79-86 Spinach 0.02 97
0.2 94-99 2.0 98-103 20 94-99 Sweet peppers 0.02 86
0.2 85-91 2.0 100-104 20 98-100 Tomatoes 0.02 75-76
0.2 81 2.0 76-79 Turnips
Foliage 0.04 64-70 Edible root 0.04 90-91 Wheat
Green matter 0.04 86-95 0.4 84-90
1.0 76-88c>
10 74-88 a>
0.02 85-91 0.2 78-90
0.04 77-86a>
0.4 81-87a>
0.02 92-101 1.0 91-92 0.02 95-105 1.0 88-91 0.02 87-90 1.0 71-72 Grains
Straw Soil
Standard soil Standard soil
"Laacherhof 2.1 2.2 West'
66 Anilazine Table, (contd.) Analytical material Water
Added (mg/kg) 0.005 0.01 0.05 0.5 Different number of recovery experiments: a) 4, b) 5, c) 6.
The soils used for the recovery
Soil type
Standard soil 2.1 *} Standard soil 2.2*>
"Laacherhof West"
experiments had the
Organic carbon
% 0.31 2.64 1.78
Range (%) 82-93 b>
88-94a>
87-94 90-94
following characteristics:
Particles <0.02 mm
% 8.5 14.5 20.5
PH 6.0 6.0 6.1
*) Standard soils as specified by Biologische Bundesanstalt filr Land- und Forstwirtschaft (BRA), cf. BBA-Richtlinie IV/4-2 (1987), Braunschweig.
The data for water relate to tap water, spring water, drainage water, leaching water, water from lysimeter trials, and water from trials for the determination of fish toxicity.
The limit of determination was 0.02 mg/kg for artichokes, aubergines, beans, cereal grains, potatoes, radicchio, spinach, sweet peppers, tomatoes and soil, 0.4 mg/kg for hop cones, 0.04 mg/kg for other plant material, and 0.005 mg/1 for water.
7.3 Calculation of residues
The residue R, expressed in mg/kg anilazine, is calculated from the following equation:
R
R Fs t. VR 2.V i. G L 0 3 3
where
G = sample weight (in g) or volume (in ml)
VR1 = volume of solution prepared for gel permeation chromatography in 6.3 (in ml) VR2 = p o r t i o n of volume VR1 injected for gel p e r m e a t i o n c h r o m a t o g r a p h y (volume of
sample loop) (in ml)
VE n d = terminal volume of sample solution from 6.4 (in ml)
Vj = p o r t i o n of volume VE n d injected into gas c h r o m a t o g r a p h (in ul) WS t = a m o u n t of dimethoxy anilazine injected with standard solution (in ng) FA = peak area obtained from Vj (in m m2)
FS t = peak area obtained from WSt (in m m2)
1.033 = factor for conversion of dimethoxy anilazine to anilazine
Anilazine 67
8 Important points
To avoid any loss of residues, deep frozen plant material and soil samples should not be thawed prior to starting the analysis.
When processing extracts from plant material as described in 6.1.1, the aqueous residue resulting from concentrating the filtrate, on prolonged standing, tends to solidify to a jelly-like mass, which dissolves in the sodium chloride solution only after warming to approx. 50 °C.
Therefore, sodium chloride solution should be added to the aqueous residue without delay.
9 References
R. Brennecke, Methode zur gaschromatographischen Bestimmung von ®Dyrene-Ruckstanden in Pflanzenmaterial, Boden und Wasser, Pflanzenschutz-Nachr. 38, 11-32 (1985).
C. E. Mendoza, P. J. Wales and G. V. Hatina, Alkoxy derivatives of Dyrene: Identification and carboxylesterase inhibition, J. Agric. Food Chem. 19, 41-45 (1971).
P. J. Wales and C. E. Mendoza, Investigation on determination and confirmation of Dyrene added to plant extracts: GLC and TLC of Dyrene and products of its reaction in methanolic sodium hydroxide, J. Assoc. Off. Anal. Chem. 53, 509-513 (1970).
10 Author
Bayer AG, Agrochemicals Sector, Research and Development, Institute for Product Infor- mation and Residue Analysis, Monheim Agrochemicals Centre, Leverkusen, Bayerwerk, R. Brennecke
Benomyl, Carbendazim,
Thiophanate-methyl 261-378-370 Lettuce, wheat (grains and straw) High-performance
liquid chromato- graphic determination
(German version published 1987)
1 Introduction
Benomyl Chemical name
Structural formula Empirical formula Molar mass Melting point Boiling point Solubility Other properties Carbendazim Chemical name
Methyl l-(butylcarbamoyl)benzimidazol-2-ylcarbamate (IUPAC)
a>CONH(CH2)3CH3
C14H18N4O3
290.32
Undergoes decomposition on heating Not distillable
Virtually insoluble in water
Decomposed in acid and alkaline media
Methyl benzimidazol-2-ylcarbamate (IUPAC)
Structural formula N/V-NHCOOCH3
Empirical formula Molar mass Melting point Boiling point Solubility
(in 100 ml at 20 °C)
C9H9N3O2
191.19
307-312°C (with decomposition) Not distillable
Virtually insoluble in water at pH 7;
readily soluble in acetic acid and dimethylformamide;
sparingly soluble in ethanol (30 mg);
70 Benomyl, Carbendazim, Thiophanate-methyl
Other properties
virtually insoluble in benzene (3.6 mg) and dichloromethane (6.8 mg)
Stable to light and acids, decomposed in alkaline media
Thiophanate-methyl Chemical name
Structural formula
Empirical formula Molar mass Melting point Boiling point Solubility
(in 100 ml at 20 °C)
Other properties
Dimethyl 4,4'-(o-phenylene)bis(3-thioallophanate) (IUPAC)
NHCSNHCOOCH, NHCSNHCOOCH3 C12H14N4O4S2
342.40
178 °C (with decomposition) Not distillable
Virtually insoluble in water;
soluble in acetone (5.8 mg);
slightly soluble in acetonitrile (2.4 g), chloroform (2.6 g), ethyl acetate (1.2 g) and methanol (2.9 g)
Stable to light, decomposed in acid and alkaline media
2 Outline of method
In plant tissues, benomyl and thiophanate-methyl are partially converted to carbendazim.
Therefore, in this method the sum of the three compounds is determined and expressed as carbendazim.
The residues are extracted from the plant material with methanol or aqueous methanol, whereby any existing benomyl is converted to carbendazim. An aliquot of the extract is con- centrated in the presence of dimethylformamide, and any residual thiophanate-methyl is con- verted to carbendazim with ammonia. The reaction mixture is diluted with buffer solution (pH 7) and extracted with dichloromethane. The dichloromethane phase is cleaned up by col- umn chromatography on acidic aluminium oxide, and carbendazim is determined by high- performance liquid chromatography using a UV detector.
3 Apparatus
Beater-cross mill
Homogenizer, e.g. Ultra-Turrax (Janke & Kunkel) Knife mill
Wide neck bottle, 500-ml, with screw cap Laboratory mechanical shaker
Benomyl, Carbendazim, Thiophanate-methyl 71 Buchner porcelain funnel, 6 cm dia.
Filter paper, 6 cm dia., e.g. MN 713 (Macherey-Nagel) Filtration vessel after Witt
Volumetric flask, 250-ml
Round-bottomed flasks, 500-ml and 250-ml, with ground joints
Rotary vacuum evaporator, 40-45°C, 58-65 °C and 80-85°C bath temperature Water bath, 80 °C temperature
Separatory funnel, 500-ml
Chromatographic tube with sintered glass disk, 16 mm i.d., 30 cm long, with PTFE stopcock Test tubes, 10-ml, graduated
High-performance liquid chromatograph equipped with UV detector Microsyringe, 10-|il
4 Reagents
Dichloromethane, distilled in glass
N,N-Dimethylformamide, p. a. (Fluka No. 40250) Ethanol, absolute, p. a. (Merck No. 983)
Ethyl acetate, p. a. (Merck No. 9623)
n-Hexane, for residue analysis (Merck No. 4371) Methanol, distilled in glass
Eluting mixture 1: n-hexane + ethyl acetate 8:2 v/v Eluting mixture 2: n-hexane + ethyl acetate 7:3 v/v
Mobile phase: ethanol + n-hexane + phosphoric acid 700:300:0.2 v/v/v
Carbendazim standard solutions: dilute a solution of 10 mg/ml carbendazim in ethanol to 0.03, 0.09, 0.24 and 0.6 ng/ml with mobile phase
ortho-Phosphoric acid 85%, p. a. (Merck No. 573)
Ammonium hydroxide solution 25%, p. a. (Merck No. 5432) Sodium chloride solution, saturated
Phosphate buffer solution (pH 7): 0.041 mol/1 Na2HPO4 + 0.028 mol/1 KH2PO4 Sodium sulphate, p.a., anhydrous
Aluminium oxide, activity grade V: To 100 g Alumina Woelm A Super I (ICN Biomedicals) in a 300-ml Erlenmeyer flask (with ground joint), add 19 ml water dropwise from a burette, with continuous swirling. Immediately stopper flask with ground stopper, shake vigorously until all lumps have disappeared, and then store in a tightly stoppered container for at least 2 h Dry ice
Cottonwool
5 Sampling and sample preparation
The analytical sample is taken and prepared as described on pp. 17 ff and pp. 21 f, Vol. 1. Let- tuce is pre-homogenized, wheat grains are finely ground in the beater-cross mill in the presence of dry ice. Wheat straw is coarsely cut in the knife mill, and also finely ground in the beater- cross mill in the presence of dry ice.
72 Benomyl, Carbendazim, Thiophanate-methyl
6 Procedure
6.1 Extraction 6.1.1 Wheat grains
Weigh 25 g of the analytical sample (G) into the wide neck bottle and add 35 ml water. Mix for about 1-2 min with a glass rod, add 150 ml methanol, and shake the mixture for 30 min on the mechanical shaker. Filter the mixture with gentle suction through a filter paper in the Buchner porcelain funnel and collect the filtrate in the 250-ml volumetric flask which stands in the filtration vessel. Rinse the bottle and the filter cake with 60 ml methanol, suction-filter the rinsings also into the volumetric flask, and make up to the mark with methanol (VEx).
6.1.2 Wheat straw
Weigh 5 g of the analytical sample (G) into the wide neck bottle and add 25 ml water. Mix for about 1-2 min with a glass rod, rinse the glas rod with a little methanol which is given into the bottle, and leave to stand for about 10 min. Add 150 ml methanol and shake the mix- ture for 30 min on the mechanical shaker. Then proceed as described in 6.1.1.
6.1.3 Lettuce
Transfer 25 g of the analytical sample (G) into the wide neck bottle with 150 ml methanol and homogenize at high speed. Rinse the mixer blade with 25 ml methanol which is given into the bottle, and shake the mixture for 30 min on the mechanical shaker. Then proceed as described in 6.1.1.