6.2 High-performance liquid chromatographic determination
6.1.4 Fruit juice, must, wine
Transfer 100 g of the analytical sample (G) to a 1-1 separatory funnel. Add 200 ml acetone and 40 g sodium chloride, and shake the mixture with 100 ml dichloromethane. After phase separation, discard the lower aqueous phase and continue to process the organic phase as described in 6.1.1.
Bitertanol, Triadimefon, Triadimenol 91
6.1.5 Soil
Weigh 50 g soil (G) into a 1-1 round-bottomed flask, add 300 ml methanol-water mixture, and heat under reflux for 4 h. Allow to cool, and filter the suspension with gentle suction through a fast flow-rate filter paper covered with approx. 15 g filter aid in a Buchner porcelain funnel.
Rinse the flask and filter cake twice with 50-ml portions of methanol-water mixture. Allow the filter cake to pull dry, and discard it. Rotary-evaporate the filtrate to its aqueous residue (approx. 100 ml), and transfer to a 250-ml separatory funnel. Rinse the flask with dichloro- methane and shake the aqueous residue three times with dichloromethane (100, 100, 50 ml).
Filter the organic phase successively through a cottonwool plug overlaid with an approx. 3-cm layer of sodium sulphate in a glass funnel. Collect the filtrate in a 500-ml round-bottomed flask. Wash the sodium sulphate three times with 25-ml portions of dichloromethane, and rotary-evaporate the combined filtrates to dryness. For bitertanol, proceed as described in 6.2;
for triadimefon and triadimenol proceed to 6.3.
6.1.6 Water
Extract 400 ml water (G) three times with 200-ml portions of dichloromethane. In the case of only small amounts of water being available (e. g. 100 ml), extract the sample with cor- respondingly smaller portions of dichloromethane. Filter the organic phases successively through a cottonwool plug overlaid with an approx. 3-cm layer of sodium sulphate in a glass funnel. Collect the filtrate in a 1-1 round-bottomed flask. Wash the sodium sulphate three times with 25-ml portions of dichloromethane, and rotary-evaporate the combined filtrates to dryness. Then proceed as described in 6.3.
6.2 Column chromatography
Fill the chromatographic tube, in this order, with 10 ml toluene, a cottonwool plug, 15 g silica gel (mixed to a slurry with toluene, filling height approx. 13 cm), approx. 1 cm sodium sulphate, and a loose glass wool plug. Then drain the toluene down to the top of the sodium sulphate layer.
Dissolve the residue derived from 6.1.1, 6.1.2, 6.1.3, 6.1.4, and (for bitertanol residues) 6.1.5 in 10 ml toluene, transfer the solution onto the column, and allow to percolate to the top of the sodium sulphate. Rinse the flask twice with 10-ml portions of eluting mixture 1. Pre-wash the column with the rinsings followed by a further 80 ml of eluting mixture 1. Next elute the compounds from the column with 100 ml eluting mixture 2 into a 250-ml round-bottomed flask. Rotary-evaporate the eluate to dryness, then proceed as described in 6.3.
6.3 Gel permeation chromatography
Transfer the residue derived from 6.1.5 (for triadimefon and triadimenol), 6.1.6 or 6.2 into a test tube, using a total of 10 ml eluting mixture 3 (VR1) to complete the transfer. Using a 10-ml syringe, load the 5-ml sample loop (VR2) of the gel permeation chromatograph with 7 to 8 ml of the solution. Set the gel permeation chromatograph at the eluting conditions deter- mined beforehand with standard solutions containing approx. 40 [ig/ml of each compound;
cf. Cleanup Method 6, pp. 75 ff, Vol. 1. — Elution volumes ranging from 100 to 130 ml were
92 Bitertanol, Triadimefon, Triadimenol
determined for triadimefon and triadimenol, and from 120 to 140 ml for bitertanol, on Bio- Beads S-X3 polystyrene gel, using eluting mixture 3 as eluant, pumped at a flow rate of 5.0 ml/min.
Collect the appropriate fraction, according to the elution volumes of the compounds, in a 100-ml round-bottomed flask, and rotary-evaporate to dryness. Then proceed to 6.4.
Check the elution ranges from time to time, and determine anew whenever a new gel column is used.
6.4 Gas-chromatographic determination
Dissolve the residue derived from 6.3 in 5 ml ethyl acetate (VEnd) and transfer the solution to a glass stoppered test tube. Inject 5 ul of this solution (Vj) (if necessary, after dilution with ethyl acetate to an appropriate volume) into the gas chromatograph.
Operating conditions Gas chromatograph Column 1
Column 2
Injection port temperature Detector
Gas flow rates Attenuation Recorder Linearity range Injection volume
Conditions for bitertanol:
Column temperature Carrier gas flow rates Retention times for bitertanol
Conditions for triadimefon and triadimenol:
Column temperature Carrier gas flow rates Retention times for triadimefon triadimenol
Column 1 245 °C
Nitrogen, 55 ml/min 3 min 54 s
215 °C
Nitrogen, 40 ml/min 3 min
3 min 54 s
Varian 3700
Glass, 3 mm i.d., 1.8 m long; packed with 1.5% SP- 2250 + 1.95% SP-2401 on Supelcoport, 100-120 mesh Glass, 3 mm i.d., 1.8 m long; packed with 3.8% SE- 30 on Chromosorb W-HP, 80-100 mesh
280 °C
Thermionic nitrogen-specific detector Temperature 35O°C
Hydrogen, 4.5 ml/min Air, 175 ml/min 10-11
1 mV; chart speed 5 mm/min Bitertanol 1-50 ng
Triadimefon 0.5-50 ng Triadimenol 1-100 ng 5ul
Column 2
255 °C
Nitrogen, 30 ml/min 2 min 54 s
195 °C
Nitrogen, 35 ml/min 3 min 18 s
4 min 6 s
Bitertanol, Triadimefon, Triadimenol 93
7 Evaluation
7.1 Method
Quantitation is performed by measuring the peak areas of the sample solutions and compar- ing them with the peak areas obtained for the compound standard solutions. Equal volumes of the sample solutions and the standard solutions should be injected; additionally, the peaks of the solutions should exhibit comparable areas.
7.2 Recoveries and lowest determined concentration
Recovery experiments were run on different untreated control samples of plant material, soil, and water, fortified with known amounts of the compounds 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 bitertanol, triadimefon and triadimenol; duplicate experiments.
Analytical material ,, Bitertanol Triadimefon Triadimenol Apples
Fruit 0.02-0.05 89-110e> 84-91 94-99
92-99
85-86
Juice 0.02-0.05 87-105c> 84-87 99-100
Pulp 0.02-0.05 96-108b> 80-81 92-93
Apricots Artichokes Bananas
Fruit 0.01-0.05 83-89b) 87-91 91-93
85-103
89-108
Peel 0.01-0.05 88-95b> 95-99 92
82-85
92-96 Barley
Green matter 0.04-0.08 82-89 90-105b> 90-104b>
87-97 87-89 86-87 87-91
Grains 0.04-0.08 87-91 84-91b> 90-93 b>
92-93 95 77-83 85-89
Straw 0.04-0.08 95-104 89-99b> 92-104b>
90-102 87-95
85_89b) 93-95b>
Beans
0.02-0.05 0.4-0.5 1.0-5.0 0.02-0.05 0.02-0.05 0.05 0.5 0.05 0.5 0.01-0.05 0.4-0.5 1.0 0.01-0.05 0.4-0.5 1.0 0.04-0.08 0.2-0.4 1.0-4.0 0.04-0.08 0.4-0.8 1.0-2.0 0.04-0.08 0.4-0.8 1.0-4.0 0.05 0.5
89-110e>
80-100c>
102-107 87-105c>
96-108b>
89-92 87-92 95 80-86 83-89b)
82-90 88-95b>
85-92
82-89 92-93 87-91 86-91 95-104 86-97 85-92 83-86
94 Bitertanol, Triadimefon, Triadimenol Table, (contd.)
Analytical material Cherries
Fruit Juice Cucumbers
Grapes
Hop cones Melons
Must Peaches
Peanuts Kernels Shells Pears
Fruit
Juice Plums Rye
Green matter
Grains Straw Sugar beet
Foliage
Edible root
Added mg/kg 0.05 0.5 0.05 0.5 0.02-0.05 0.1-0.5 1.0 0.02-0.05 0.4 1.0 0.3-0.5 3.0-5.0 0.02-0.05 0.4 1.0 0.02-0.05 0.02-0.05 0.25-0.5 1.0 0.02-0.05 0.5 0.05 0.5 0.02-0.05 0.1-0.5 1.0 0.02-0.05 0.05 0.5 0.04-0.08 0.4-0.8 1.0-2.0 0.04-0.08 1.0-2.0 0.04-0.08 1.0-2.0 0.02-0.05 0.4-0.5 1.0 0.02-0.05 0.4-0.5 1.0
Bitertanol
82-90b>
89-102b>
87-91 93-97 89-105b>
85-98b>
93-99 86-96 93-100 85-94b>
83-92b>
88-89 91-92 86-90 90-104^
87-110ô) 86-100b>
93-105 82-83
98-104b>
84-94 86-88 91-104b>
86-88 83-93
Triadimefon
90-94 88-92 95 86-88 84-88 81-86 88-97 90-92 86-93 86 83
81-91 100-101
80-81
75_98b) 83-89 86-90 82-103 92-96 90-103b>
78-79 80 85-86 88-91 89-100
Triadimenol
84-87 93-95 107-110
92-100 101-104 85-93 93-102 97-98 85-86 100-103
96-97
87-95 85 89-90
81-97b>
91-96 94-102 92-106 94-98 91-101b>
78-86 90-93 96-98 82-87 94-105
Bitertanol, Triadimefon, Triadimenol 95 Table, (contd.)
Analytical material Sweet peppers
Tomatoes
Wheat Green matter
Grains Straw Wine
Soil Water
*) Equivalent to 5, Different number of
The soils used for
Soil type
Standard soil 2.1*) Standard soil 2.2* >
Standard soil 2.3*)
Alluvial soil
Added mg/kg 0.02-0.05 0.4 1.0 0.02-0.05 0.4 1.0 0.04-0.08 0.4-0.8 1.0-2.0 0.04-0.08 0.4-0.8 0.04-0.08 0.4-0.8 0.02-0.05 0.4 1.0 0.04-0.08 0.4-0.8 0.005*)
o.oi*)
0.5*>
Bitertanol
83-107d) 79-101d>
91-114e>
96-105 104 10, and 500 ng/1, respectively.
* recovery experiments: a) 3, b) 4, c) 6,
the recovery experiments had the
Organic carbon
°/o 0.31 2.64 1.06 1.47
Triadimefon 88-91 82-87 90-97 89-94
90-110b) 85-91 80-87 90-94 89 84-104a) 96-105 102-109a)
87-94a) 96-103°) 94-108°) 88-108e) 97-112
d) 7, e) 8, ° 10, ô> 12.
Triadimenol 97-105 95-100 90-96 100-106
83-98b) 90-99
83-101b>
93-99 98-99 93-1093) 103-112
92-1033) 87-1033) 104-108c) 94_1 nc) 93-103°) 94
following characteristics:
Particles < 0.02 mm
% p H
8.5 14.5 24.7 26.6
6.0 6.0 7.0 7.0
*) Standard soils as specified by Biologische Bundesanstalt fur Land- und Forstwirtschaft (BBA), cf.
BBA-Richtlinie IV/4-2 (1987), Braunschweig.
The data for water relate to tap, spring, well, lysimeter, ground, and drainage waters as well as to water used for fish toxicity studies.
The routine limit of determination for bitertanol was 0.01 mg/kg in bananas, 0.02 mg/kg in apples, pears and peanut kernels, 0.05 mg/kg in other plant material and soil, and 0.005 mg/1 in water.
The routine limit of determination for triadimefon was 0.02 to 0.04 mg/kg in plant material, 0.04 mg/kg in soil, and 0.005 mg/1 in water. For triadimenol, it was 0.05 to 0.08 mg/kg in plant material, 0.08 mg/kg in soil, and 0.005 mg/1 in water.
96 Bitertanol, Triadimefon, Triadimenol 7.3 Calculation of residues
The residue R, expressed in mg/kg, of an identified compound is calculated from the follow- ing equation:
R_ FA-VR 1-VE n d-WS t
F s , - VR 2- VrG 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 = portion of volume VR1 injected for gel permeation chromatography (volume of sam-
ple loop) (in ml)
VEnd = terminal volume of sample solution from 6.4 (in ml) (if necessary, take account of a dilution)
Vj = portion of volume VEnd injected into gas chromatograph (in ul)
Wst = amount of bitertanol, triadimefon or triadimenol, respectively, injected with standard solution (in ng)
FA = peak area obtained from Vj (in mm2 or integrator counts) FSt = peak area obtained from WSt (in mm2 or integrator counts)
8 Important points
Triadimefon is reduced to triadimenol in plants, soil and water. Therefore, both compounds can appear as residues after the application of triadimefon.
Use both gas chromatographic columns to analyze sample solutions with a high content of plant co-extractives (e. g. from cereal samples). As an additional gas-chromatographic column the following can be used: Glass column, 3 mm i.d., 1.8 m long; packed with 4% SE-30 + 6% OV-210 on Chromosorb W-HP, 80-100 mesh.
On the gas-chromatographic columns described here, neither the two diastereoisomers of bitertanol nor those of triadimenol will be separated; one peak will be obtained with a shoulder appearing to a greater or lesser degree.
9 References
R. Brennecke, Methode zur gaschromatographischen Bestimmung von Riickstanden der Fungizide ®Bayleton und ®Bayfidan in Pflanzenmaterial, Boden und Wasser, Pflanzen- schutz-Nachr. 37, 66-91 (1984).
R. Brennecke, Methode zur gaschromatographischen Bestimmung des Fungizids ®Baycor in Pflanzenmaterial, Boden und Wasser, Pflanzenschutz-Nachr. 38, 33-54 (1985).
R. Brennecke and K. Vogeler, Methode zur gaschromatographischen Bestimmung von Riickstanden verschiedener Fungizide in Wasser, Pflanzenschutz-Nachr. 37, 44-65 (1984).
Bitertanol, Triadimefon, Triadimenol 97
W. Specht and M. Tillkes, Gaschromatographische Bestimmung von Riickstanden an Pflanzenbehandlungsmitteln nach Clean-up iiber Gelchromatographie und Mini-Kieselgel- Saulenchromatographie. 2. Mitt.: Bestimmung der Fungizide Bitertanol, Fluotrimazol, Fuberidazol, Imazalil, Rabenzazole, Triadimefon und Triadimenol in Pflanzen und Boden, Pflanzenschutz-Nachr. 55, 61-85 (1980).
10 Author
Bayer AG, Agrochemicals Sector, Research and Development, Institute for Product Informa- tion and Residue Analysis, Monheim Agrochemicals Centre, Leverkusen, Bayerwerk, R. Brennecke
Bromoxynil, Ioxynil 264-212
Barley (grains and straw), wheat (grains and green matter) Gas-chromatographic Soil, water determination (German version published 1989)
1 Introduction
Chemical name
Structural formula Empirical formula Molar mass Melting point Boiling point Vapour pressure Solubility
(in 100 ml at 20 °C)
Other properties
Bromoxynil 3,5-Dibromo-4-
hydroxybenzonitrile (IUPAC)
Ioxynil
3,5-Diiodo-4-hydroxybenzonitrile (IUPAC)
Br
C7H3Br2NO 276.93
194-195°C, octanoate 45-46°C Not distillable
<10"5 mbar at 20°C Very sparingly soluble in water;
readily soluble in acetone (17 g),
soluble in methanol (9 g)
Commercial products contain bromoxynil and ioxynil mostly as octanoates or alkali metal salts
C7H3I2NO 370.92 212-213 °C, octanoate 59-60 °C Not distillable
<10"5 mbar at 20 °C Virtually insoluble in water;
soluble in acetone (7 g) and methanol (2 g)
2 Outline of method
After refluxing the analytical sample with methanolic potassium hydroxide solution, brom- oxynil and ioxynil residues are partitioned into dichloromethane. The extract is cleaned up by acid-base partitioning, whereupon the compounds are methylated with diazomethane. The methyl ethers are chromatographed on a Florisil column and are determined by electron cap- ture gas chromatography.
3 Apparatus
High-speed blendor, e.g. Waring Blendor
Round-bottomed flasks, 1-1, 500-ml, 250-ml and 100-ml, with ground joints
100 Bromoxynil, loxynil
Heating mantles, 1-1 and 500-ml Reflux condenser (Dimroth type) Buchner porcelain funnel, 9 cm dia.
Filter paper, 9 cm dia., fast flow rate
Rotary vacuum evaporator, 40 °C bath temperature Glass funnel
Fluted filter paper, 15 cm dia. (Schleicher & Schull) Separatory funnels, 1-1 and 500-ml
Membrane filter, e.g. SM 11605-025 N, 0.65-u.m (Sartorius), and Millex-HV4 filter unit (Millipore)
Glass syringe, 10-ml, with Luer-lock fitting
Automated instrument for gel permeation chromatography, e.g. GPC Autoprep 1002 A (Analytical Bio-Chemistry Laboratories) (see Cleanup Method 6, pp. 75 ff, Vol. 1)
Chromatographic tube, 15 mm i.d., 40 cm long Methylation apparatus, see Fig. 1, p. 130, Vol. 1
Graduated cylinders, 1-1, 500-ml, 250-ml, 100-ml and 50-ml Gas chromatograph equipped with electron capture detector Microsyringe, 10-ul
4 Reagents
Acetone, high purity
Cyclohexane, for residue analysis Dichloromethane, techn. pure, dist.
Diethyl ether, high purity, dried over calcium chloride Ethyl acetate, for residue analysis
n-Hexane, high purity Methanol, high purity Toluene, high purity
2,2,4-Trimethyl pentane (isooctane), p. a.
Eluting mixture 1: cyclohexane + ethyl acetate 1:1 v/v Eluting mixture 2: n-hexane + toluene 8:2 v/v Eluting mixture 3: n-hexane + toluene 2:8 v/v
Compound standard solutions: 1.46, 14.6, 146 and 1460 u.g/ml bromoxynil octanoate or 1.34, 13.4, 134 and 1340 |iig/ml ioxynil octanoate (equivalent to 1, 10, 100 and 1000 ng/ml brom- oxynil or ioxynil) in methanol
Derivative standard solutions: bromoxynil methyl ether or ioxynil methyl ether (equivalent to 0.01 to 0.1 ng/ml bromoxynil or ioxynil) in isooctane.
Reflux 728 [ig bromoxynil octanoate or 670 |ug ioxynil octanoate (equivalent to 500 fig bromoxynil or ioxynil) for 1 h in 200 ml methanolic potassium hydroxide solution. Allow to cool and rinse the condenser with 20 ml methanol. Filter through a fluted filter paper and wash with 20 ml methanol. Add 20 ml water and rotary-evaporate to an aqueous residue.
Transfer the residue into a 250-ml separatory funnel, using 100 ml water to complete the transfer, acidify with 3 ml sulphuric acid, and extract the compound with dichloromethane (see 6.2.1). Perform the methylation as described in 6.2.3. Remove the solvent, dissolve the residue in isooctane, and dilute this solution to the concentrations given above
Bromoxynil, loxynil 101
Glacial acetic acid, p. a.
Sulphuric acid, p.a., cone.
Ethanolic potassium hydroxide solution: Dissolve 7 g KOH p.a. in 10 ml water and make up to 100 ml with ethanol
Methanolic potassium hydroxide solution: 3 g/100 ml KOH p.a.
Sodium sulphate, p.a., anhydrous
Bio-Beads S-X3, 200-400 mesh (Bio-Rad Laboratories No. 152-2750)
Florisil, 60-100 mesh, deactivated with 5% water: Heat a weighed sample of Florisil for at least 8 h at 130 °C and allow to cool in a desiccator. To 100 g dried Florisil in a 300-ml Erlenmeyer flask (with ground joint), add 5 ml water dropwise from a burette, with con- tinuous swirling. Immediately stopper flask with ground stopper, shake vigorously for 5 min until all lumps have disappeared; next shake for at least 20 min on a mechanical shaker, and then store in a tightly stoppered container for at least 24 h with occasional swirling Filter aid, e.g. Celite 545
Diazomethane solution in diethyl ether (for apparatus see Fig. 1, p. 130, Vol. 1):
Dissolve 1.2 g N-methyl-N-nitroso-p-toluenesulphonamide in 10 ml diethyl ether and transfer to the dropping funnel. Slowly add this solution dropwise to 5 ml ethanolic potassium hydrox- ide solution contained in the reaction vessel, and sweep the generated diazomethane into 20 ml diethyl ether, using a gentle stream of nitrogen, while the receiver containing the ether is cooled in an ice + sodium chloride freezing mixture
Cottonwool, exhaustively extracted with dichloromethane Helium
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 Plant material (except grains)
Reflux 50 g of finely cut plant green matter or 25 g of chopped straw (G) with 400 ml methanolic potassium hydroxide solution for 1 h. Allow to cool and rinse the condenser with 20 ml methanol. Filter the mixture through a fast flow-rate filter paper covered with filter aid in a Buchner porcelain funnel and wash the filter cake with 80 ml methanol. Add 20 ml water to the filtrate and rotary-evaporate to approx. 100 ml.
6.1.2 Cereal grains, soil
Reflux 50 g of finely ground grains or 50 g soil (G) for 1 h with 200 ml methanolic potassium hydroxide solution. Then proceed as described in 6.1.1.
102 Bromoxynil, loxynil
6.1.3 Water
Reflux 500 ml of the water sample (G) with 200 ml methanolic potassium hydroxide solution for 1 h. Allow to cool and rinse the condenser with 20 ml methanol. Filter the solution through a fluted filter paper, wash the filter with 30 ml methanol, and rotary-evaporate the filtrate to an aqueous residue. Then proceed as described in 6.2.1.