Tamp a plug of cottonwool into the bottom of a chromatographic tube, and half-fill the tube with the eluting mixture. Slurry 10 g silica gel with the eluting mixture into the chromato- graphic tube, dislodge any trapped air by stirring with a glass rod, and drain the eluting mix- ture down to the top of the silica gel. Dissolve the residue derived from 6.1.4 or 6.2 in 5 ml of the eluting mixture, and add to the column. Rinse the flask twice with 5-ml portions of the eluting mixture, add the washings separately to the column, allowing each to percolate down to the top of the silica gel layer at a flow rate of approx. 0.7 ml/min. Then elute the column with a further 100 ml of the eluting mixture at the same flow rate until the column has run dry. Rotary-evaporate the eluate to dryness.
6.4 Gas-chromatographic determination
Transfer the residue derived from 6.1.5 or 6.3 into a 50-ml round-bottomed flask using methanol as wash. Rotary-evaporate the solution to dryness, and dissolve the residue in 2.0 ml methanol (VEnd). Inject an aliquot of this solution (Vj) into the gas chromatograph.
Operating conditions
Gas chromatograph Hewlett-Packard 5880 A
Column Glass, 2 mm i.d., 1.8 m long; packed with
6% Apiezon L + 3% OV-17 on Chromosorb W-AW- DMCS, 80-100 mesh
Column temperature 220 °C Injection port temperature 350°C
Metribuzin 249 Detector Thermionic nitrogen-specific detector
Temperature 300 °C
Gas flow rates Helium carrier, 20-30 ml/min Hydrogen, 1-5 ml/min Air, 40-100 ml/min
Attenuation 25
Recorder Chart speed 0.5 inch/min (12.7 mm/min) Linearity range 5-100 ng
Injection volume 1-3 \i\
Retention time for metribuzin approx. 10 min
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 dilutions of the metribuzin standard solution. 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 metribuzin at levels of 0.05 to 0.5 mg/kg. The recoveries are given in the Table, representing the means from 2 to 4 single experiments. The routine limit of deter- mination was 0.05 mg/kg.
Table. Percent recoveries Analytical material
from plant material, soil and
0.
water,
05
fortified with metribuzin.
Metribuzin added (mg/kg)
0.1 0.5
Barley (grains) 85 83 86 Broad beans
Beans Pods Carrots Potatoes (tubers) Tomatoes
Fruits Leaves Wheat
Grains Straw Soil
Standard soil 2.1 Standard soil 2.2 Standard soil 2.3 Water
94 75 89 91 98 98 80 80 80 80 86 85
96 90 88 89 95 81 82 79 80 80 80 89
95 80 82 90 86 99 85 85 83 80 84 90
250 Metribuzin
T h e soils used for t h e recovery experiments h a d t h e following characteristics:
Organic carbon Particles <0.02 mm „
Soil type % % PH
Standard soil 2.1 *> 0.31 8.5 6.0 Standard soil 2.2•> 2.64 14.5 6.0 Standard soil 2.3*> 1.06 24.7 7.0
*> Standard soils as specified by Biologische Bundesanstalt fur Land- und Forstwirtschaft (BBA), cf. BBA-Richtlinie IV/4-2 (1987), Braunschweig.
7.3 Calculation of residues
The residue R, expressed in mg/kg metribuzin, is calculated from the following equation:
p FA-VEnd-Wst
FS t-VrG where
G = sample weight (in g) or volume (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 |iil) WS t = a m o u n t of metribuzin injected with standard solution (in ng) FA = peak area obtained from Vj (in m m2)
Fst = p e a k area obtained from WS t (in m m2)
8 Important points
The entire analytical procedure including the final determinative step should be completed within 2 days to avoid conversion of metribuzin to its metabolites in the extracts.
Acetonitrile must be tested for gas-chromatographic purity prior to use.
During rotary-evaporation of the extracts, the temperature of the water bath must not ex- ceed 50 °C.
Supplementary studies have shown that the following procedure may also be employed for the extraction of soil samples (see step 6.1.4): After extraction of the sample with the metha- nol-water mixture, acetonitrile and dichloromethane, combine the supernatants in a 1-1 round- bottomed flask, and rotary-evaporate to an aqueous residue. Add 50 ml water, and transfer the solution into a 500-ml separatory funnel. Rinse the round-bottomed flask with a further 50 ml of water, and add the washing to the separatory funnel. Extract the contents three times with dichloromethane (150, 150, 100 ml), shaking the funnel each time. Filter the combined dichloromethane phases through approx. 10 g sodium sulphate into a 1-1 round-bottomed flask, and rotary-evaporate to dryness. Discard the phase left in the separatory funnel. Then proceed to step 6.3.
Metribuzln 251
9 References
C. W. Stanley and S. A. Schumann, A gas chromatographic method for the determination of Bay 94337 residues in potatoes, soybeans, and corn, Chemagro Report No. 25838 (1969).
E G. von Stryk, Determination of residues of Bay 94337 (4-amino-3-methylthio-6-tert.butyl- l,2,4-triazin-5-one), J. Chromatogr. 56, 345-348 (1971).
J. S. Thornton and C. W. Stanley, Gas chromatographic determination of Sencor and metabolites in crops and soil, J. Agric. Food Chem. 25, 380-386 (1977).
H. J. Jarczyk, Methode zur gaschromatographischen Bestimmung von ®Sencor-Ruckstanden in Pflanzenmaterial, Boden und Wasser mit N-spezifischem Detektor, Pflanzenschutz-Nachr.
36, 63-72 (1983).
10 Author
Bayer AG, Agrochemicals Sector, Research and Development, Institute for Product Informa- tion and Residue Analysis, Monheim Agrochemicals Centre, Leverkusen, Bayerwerk, H. J. Jarczyk
Nitrothal-isopropyl 416
Apples Soil, water
Gas-chromatographic determination
(German version published 1989)
1 Introduction
Nitrothal-isopropyl
Chemical name Di-isopropyl 5-nitroisophthalate (IUPAC)
Structural formula
CO-O-CH
CO-O-CH
Empirical formula Molar mass Melting point Boiling point Vapour pressure Solubility
Other properties
Metabolite I (Monoester) Chemical name
C14H17NO6
295.30 65 °C No data
<0.1 -10"6 mbar at 20 °C Very sparingly soluble in water;
readily soluble in acetone, benzene, chloroform and ethyl acetate
Yellow crystals, steam volatile, hydrolyzed in alkaline media
Isopropyl 5-nitroisophthalate
/CH3
Structural formula
Empirical formula Molar mass Melting point Boiling point Vapour pressure Solubility
O2N - ^ J >
CUHUNO6
253.22 178 °C No data No data No data
C O - O - C H /
COOH
254 Nitrothal-isopropyl
Metabolite II (Acid)
Chemical name 5-Nitroisophthalic acid
/COOH
Structural formula 02N H Q
COOH
Empirical formula C8H5NO6 Molar mass 211.13 Melting point 255 °C Boiling point No data Vapour pressure No data
Solubility Slightly soluble in water (approx. 2g/100ml at 25 °C);
soluble in ethanol and in dilute alkaline solutions
2 Outline of method
Nitrothal-isopropyl residues are steam-distilled from the analytical sample in a Bleidner appa- ratus, simultaneously being extracted from the condensate into isooctane. The metabolites re- maining in the aqueous phase are extracted after acidification into tert.butyl methyl ether and methylated with diazomethane. The methylated metabolites are also steam-distilled in the Bleidner apparatus and extracted into isooctane. Nitrothal-isopropyl and its methylated me- tabolites are determined by electron-capture gas chromatography.
3 Apparatus
Round-bottomed flasks, 500-ml and 50-ml, with ground joints
Bleidner apparatus, modified by W. Heizler, for distillation and extraction; see Fig. 1, p. 243, Vol. 1
Hotplate with magnetic stirrer, e.g. IKA Combimag RCT (Janke & Kunkel) Aluminium foil
Graduated cylinder, 500-ml, with glass stopper
Laboratory centrifuge, explosion-proof, with 250-ml glass tubes Water bath, 60 °C temperature
Separatory funnel, 500-ml
Rotary vacuum evaporator, 20°C, 30°C and 50-60°C bath temperature Methylation apparatus, see Fig. 1, p. 130, Vol. 1
Volumetric flasks, 100-ml, 50-ml and 10-ml
Gas chromatograph equipped with electron capture detector Microsyringe, 10-ul
N itrothal-isopropy I 255
4 Reagents
tert.Butyl methyl ether, dist.
2,2,4-Trimethyl pentane (isooctane), dist.
Standard solutions: Mixtures containing 0.2, 0.4 and 0.6 ng/ml each of nitrothal-isopropyl, dimethyl 5-nitroisophthalate and isopropyl methyl 5-nitroisophthalate
Compound and metabolite solutions for fortification experiments: Dissolve 10 mg nitro- thal-isopropyl, 10 mg metabolite I and 10 mg metabolite II individually in 10 ml methanol.
From each of these solutions, pipet 1 ml into a 100-ml volumetric flask and make up to vol- ume with water
Diazomethane solution in diethyl ether (for apparatus, see Fig. 1, p. 130, Vol. 1):
Dissolve 2 g N-methyl-N-nitroso-p-toluenesulphonamide (Merck No. 808406) in 60 ml diethyl ether and transfer to the dropping funnel. Slowly add this solution dropwise to 10 ml methanolic potassium hydroxide solution contained in the reaction vessel standing in a 60 °C water bath. Distil the generated diazomethane and the diethyl ether through a descending con- denser into a receiver which is cooled with an ice + sodium chloride freezing mixture ortho-Phosphoric acid, p.a., 85% (Merck No. 573)
Boric acid, p. a. (Merck No. 165)
Methanolic potassium hydroxide solution, 10 g/100 ml KOH p. a. in methanol + water mix- ture 9:1 v/v
Sodium chloride solution, saturated
Isopropyl 5-nitroisophthalate (metabolite I): Add 4 g nitrothal-isopropyl to 10 ml concen- trated sulphuric acid at room temperature and stir (on a magnetic stirrer) until it is dissolved.
Add 20 g ice and continue to stir strongly when a white crystalline precipitate containing oily lumps is formed. Add water, and suction-filter the precipitate. Dissolve the precipitate in chlo- roform and a little acetone and extract the solution with water. With stirring, add sodium hy- drogen carbonate solution portionwise to the chloroform phase until the aqueous phase has a pH of 4.5 (pH indicator paper). Shake the mixture vigorously, separate off the organic phase, and extract the aqueous phase with chloroform once more. Wash the combined chloro- form phases with water, dry on sodium sulphate, and rotary-evaporate to dryness. Dissolve the residue in a little acetone and dilute the solution with n-hexane to form a precipitate. Suc- tion-filter the precipitate and extract repeatedly with boiling n-hexane to remove unreacted starting material. Dissolve the crystalline residue in acetone, treat with activated charcoal, pre- cipitate with water and suction-filter. Dry over phosphorus pentoxide and paraffin in a desic- cator. Yield: approx. 450 mg (m.p. 178°C)
Isopropyl methyl 5-nitroisophthalate: Dissolve 200 mg metabolite I in a little diethyl ether and add diazomethane solution until a slight yellow colour remains. Evaporate the solution to dry- ness. Scratch the remaining oily residue with a glass rod to initiate crystallization, and dry the crystalline mass over phosphorus pentoxide in a desiccator (m.p. 48°C)
Dimethyl 5-nitroisophthalate (Aldrich No. 23,736-1) 5-Nitroisophthalic acid (Fluka No. 73450) (metabolite II) Argon + methane mixture 9:1 v/v
Helium, pure
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.
256 Nitrothal-isopropyl
6 Procedure