Quantitation is performed using an internal standard. For this purpose, prepare a sample solu- tion from an untreated control sample. Fortify this solution, in the same order of magnitude as the anticipated residue, with equal amounts of 3-hydroxy-carbofuran and internal standard carbofuran. The volume of this measuring solution should be equal to that of the analytical sample solution. Inject an aliquot of the measuring solution into the gas chromatograph and determine the ratio, f, of the peak areas (or heights) obtained for the metabolite and the inter- nal standard, f must be determined separately for each sample material.
Add internal standard to the analytical sample solution in the same order of magnitude as the anticipated residue. Inject an aliquot of this solution into the gas chromatograph and per- form the evaluation using the ratio of the peak areas (or heights) obtained for the metabolite and the internal standard.
7.2 Recoveries, limit of detection and limit of determination
The recoveries from untreated control samples of plant material, fortified with carbosulfan and carbofuran at levels of 0.05 to 5 mg/kg, ranged from 65 to 110% and averaged 88%. The limit of detection was 0.02 to 0.03 mg/kg, depending on the sample material, and the limit of determination was 0.05 mg/kg. Carbosulfan and carbofuran in rape seeds were not ex- amined.
The recoveries from soil samples, fortified with carbosulfan and carbofuran at levels of 0.05 to 1 mg/kg, ranged from 86 to 113% and averaged 99%. The limit of detection was 0.02 mg/kg, and the limit of determination was 0.05 mg/kg.
The recoveries from untreated control samples of rape (green matter and seeds) and tomatoes, fortified with 3-hydroxy-carbofuran at levels of 0.1 to 1 mg/kg, ranged from 68 to 115% and averaged 87%. The limit of detection was 0.01 to 0.02 mg/kg, depending on the sample material, and the limit of determination was 0.05 mg/kg.
The average recoveries are given in the Table.
Carbosulfan, Carbofuran 123 Table. Percent recoveries from plant material and soil, fortified with carbosulfan, carbofuran and 3-hydroxy-carbofuran; means from 2 to 5 experiments.
Analytical material Added
mg/kg Carbosulfan Carbofuran 3-Hydroxy- carbofuran Grapes
Head cabbage Lettuce Maize Rape
Green matter Seeds Tomatoes Sugar beet Soil
0.05-5 0.05-0.5 0.05-0.5 0.05-5 0.05-1 0.1-1 0.05-1 0.05-5 0.05-1
65 95 80 103 91
— 103 86 92
88 65 86 83 103
— 88 80 106
— - -
— 79 77 111
— -
7.3 Calculation of residues
The residue R, expressed in mg/kg carbosulfan, carbofuran or 3-hydroxy-carbofuran, is calculated from the following equations:
for carbosulfan and carbofuran: R = WA • VF vEnd
VR1• Y - G
where
G = sample weight (in g)
VEx = total volume of organic phase after addition of solvent or solvent mixture, respec- tively, to filtered extract (in ml)
VR1 = portion of volume VEx used for further cleanup (in ml) VEnd = terminal volume of sample solution from 6.3.2 (in ml)
Y = portion of volume VEnd injected into gas chromatograph (in ul)
WA = amount of carbosulfan or carbofuran, respectively, for Y reac^ from calibration curve (in ng)
for 3-hydroxy-carbofuran: R = S t - E v - YEx * VEnd
Fst.f-vRrvrG
where f =Ik
124 Carbosulfan, Carbofuran and
G = sample weight (in g)
VEx = total volume of filtered extract from 6.2 after addition of hydrochloric acid or water, respectively (in ml)
VR 1 = p o r t i o n of v o l u m e VE x used for further c l e a n u p (in m l ) VE n d = terminal volume of sample solution from 6.4 (in ml)
Vj = portion of volume VEnd injected into gas chromatograph (in ul) St = amount of internal standard injected with sample solution (in ng)
FA = peak area or height for 3-hydroxy-carbofuran obtained from Vj (in integrator counts)
FSt = peak area or height for internal standard carbofuran obtained from Vj (in integrator counts)
¥'A = peak area or height for 3-hydroxy-carbofuran (in integrator counts), and
Fst = peak area or height for internal standard carbofuran (in integrator counts), both from injection of the measuring solution (see 7.1.2)
8 Important points
Carbosulfan, carbofuran and 3-hydroxy-carbofuran have different stabilities in solution. The stability of the standard solutions must therefore be checked at least once a week.
Extracts from sugar beet obtained in 6.1.1 should be suction-filtered through a layer of filter aid on the fast flow-rate filter paper in order to prevent the filter from being clogged.
The elution range in 6.3.2 should be checked from time to time, as the activity of the Florisil can change on storage.
Blank value problems can occur with 3-hydroxy-carbofuran in rape using a thermionic detector; it is, therefore, preferably determined with a mass-selective detector in the SIM mode using carbofuran as internal standard. For carbosulfan and carbofuran, mass-selective detec- tion is used only to confirm results obtained. The respective mass spectra are given in Fig. 3.
9 References
E. Mollhoff, Uber die Riickstandsanalyse von N-Methylcarbamat-Insektiziden, Pflanzen- schutz-Nachr. 28, 388-395 (1975).
E. Mollhoff, Methode zur gaschromatographischen Bestimmung der Riickstande von Cura- terr in Pflanzen- und Bodenproben unter Beriicksichtigung von Metaboliten, Pflanzenschutz- Nachr. 28, 370-381 (1975).
R. F. Cook, R. P. Stanovick and C. C. Cassil, Determination of carbofuran and its carbamate metabolite residues in corn using a nitrogen-specific gas chromatographic detector, J. Agric.
Food Chem. 17, 277- 282 (1969).
Carbosulfan, Carbofuran 125 FMC Europe SA Agriculture, Chemical Group, Determination of 3-hydroxy-carbofuran crop residues by nitrogen selective gas chromatography, Internal Report INT 35001-2, Brussels 6/79.
FMC Europe SA Agriculture, Chemical Group, Determination of FMC 35001 and carbofuran crop residues by nitrogen selective gas chromatography, Internal Report INT 35001-4, Brussels 6/79.
10 Authors
Federal Biological Research Centre for Agriculture and Forestry, Braunschweig, J. Siebers, H. Kohle and H.-G. Nolting
Chlorflurenol, Flurenol 275-215 Barley (grains and straw), cucumbers, wheat (grains
and straw) Soil, water
Gas-chromatographic determination
(German version published 1987)
1 Introduction
Chemical name
Structural formula
Empirical formula Molar mass Data for Melting point Vapour pressure Solubility
(in 100 ml at 20 °C)
Other properties
Chlorflurenol
2-Chloro-9-hydroxyfluorene- 9-carboxylic acid (IUPAC)
C14H9C1O3
260.68
Chlorflurenol methyl ester 152 °C
6.7-10"5 mbar at 25 °C Virtually insoluble in water;
readily soluble to soluble in most organic solvents, e.g.
readily soluble in acetone (26 g) and methanol (15 g);
soluble in benzene (7 g) and ethanol (8 g);
sparingly soluble in petroleum ether (0.16 g)
Flurenol
9-Hydroxyfluorene-9-carboxylic acid (IUPAC)
COOH C1 4H1 0O3
226.23
Flurenol n-butyl ester 71 °C
10"6 mbar at 25 °C
Virtually insoluble in water;
very readily soluble to readily soluble in most organic solvents, e.g.
very readily soluble in acetone (145 g) and methanol (150 g);
readily soluble in benzene (95 g) and ethanol (70 g);
sparingly soluble in petroleum ether (0.7 g)
Beige, crystalline, practically odourless
Cream coloured, crystalline, odourless
Commercial products contain chlorflurenol as methyl ester and flurenol as n-butyl ester, sometimes as amine salt. As the acids are relatively unstable and the esters are of greater im- portance, the physical data for the esters have been given.
2 Outline of method
Residues, which can be present as esters (I), or in the case of flurenol as salts, are extracted under acid conditions with acetone or acetonitrile. If present, the conversion products II and
128 Chlorflurenol, Flurenol
IV, as well as the relatively unstable product III, are also extracted. Next, the extract is washed with toluene or petroleum ether. I is hydrolyzed with potassium hydroxide in the concentrated extract. II is decarboxylated with sulphuric acid to yield compound III which is subsequently oxidized to 2-chlorofluorenone or fluorenone (IV) with chromium trioxide. After column chromatographic cleanup, the oxidation products IV are determined by electron capture gas chromatography.
Chlorflurenol: R2 = Cl; R2 = CH3
Flurenol: R2 = H; R2 = C4H9
3 Apparatus
High-speed blendor fitted with leak-proof glass jar and explosion-proof motor Homogenizer, e.g. Ultra-Turrax (Janke & Kunkel)
Sintered glass filter funnels, porosity 2, 13 cm and 6 cm dia.
Filtration flask, 500-ml
Volumetric flasks, 500-ml, 250-ml, 10-ml and 5-ml Separatory funnels, 2.5-1, 500-ml and 250-ml
Laboratory mechanical shaker, suitable for holding separatory funnels Round-bottomed flasks, 1-1, 250-ml and 100-ml, with ground joints Rotary vacuum evaporator, 40 °C bath temperature
Centrifuge, with 250-ml glass tubes (may be required) Chromatographic tube, 10 mm i.d., 40 cm long
Gas chromatograph equipped with electron capture detector Microsyringe, 10-ul
4 Reagents
Acetone, p.a., fractionally distilled Acetronitrile, p.a., fractionally distilled Petroleum ether, saturated with acetonitrile
Petroleum ether, technical grade, fractionally distilled, boiling range 40-70 °C Toluene, p.a., fractionally distilled
Acetone + water mixture 7:3 v/v
Eluting mixture: petroleum ether + toluene 1:1 v/v
2-Chlorofluorenone standard solutions: 0.01, 0.02, 0.04, 0.08 and 0.1 [ig/ml petroleum ether Fluorenone standard solutions: 0.005, 0.01, 0.02, 0.03, 0.04 and 0.05 ng/ml petroleum ether Sulphuric acid, 1 mol/1 H2SO4 p. a.
Chlorflurenol, Flurenol 129 Potassium hydroxide solution, 10 g/100 ml KOH p. a.
Chromium trioxide solution, 60 g CrO3 p. a. in 100 ml sulphuric acid (1 mol/1) Sodium chloride solution, saturated, washed twice with toluene
Sodium sulphate, p.a., anhydrous, exhaustively extracted with petroleum ether
Aluminium oxide: To 100 g Alumina Woelm A, activity grade I (ICN Biomedicals) in a 300-ml Erlenmeyer flask (with ground joint), add 2 ml water dropwise from a burette, with con- tinuous 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 Florisil, 60-100 mesh, 7% water content: Heat a weighed sample of Florisil to constant weight at 200 °C and allow to cool in a desiccator. To 93 g dried Florisil in a 300-ml Erlenmeyer flask (with ground joint), add 7 ml water dropwise from a burette, with continuous swirling. Im- mediately stopper flask with ground stopper, shake vigorously for 5 min until all lumps have disappeared, next shake for 2 h on a mechanical shaker, and then store in a tightly stoppered container for at least 24 h with occasional swirling
Quartz wool 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 Cereal grains, straw
Homogenize 50 g of the comminuted grains or 20 g straw (G) with 5 ml sulphuric acid and 150 ml acetonitrile in the blendor for 3 min. Suction-filter the homogenate through the 13-cm glass filter funnel and wash the filter cake with approx. 50 ml acetonitrile. Repeat the extrac- tion with a further 150 ml acetonitrile. Wash the filter with 50 ml acetonitrile. Combine the filtrates in a 500-ml volumetric flask and make up to the mark with acetonitrile (VEx). Shake a 100-ml aliquot (VR1) twice in a 250-ml separatory funnel, each time for 5 min, with 50-ml portions of petroleum ether saturated with acetonitrile. Collect the lower acetonitrile phase in a 250-ml round-bottomed flask and rotary-evaporate to 1 -2 ml. Proceed as described in 6.2.