ABSTRACT
Introduction
Materials and methods
Treatments with 2,4-D ester
Analysis of coca leaves for 2,4-D
Preparation of calibration curve for 2,4-D ester and 2,4,5-T
Preparation of coca paste from coca leaves spiked with 2,4-D or 2,4-D ester
Analysis of coca paste for 2,4-D and its ester
Observations and experimental results
Conclusions
Author: M. A. ELSOHLY, , E. S. ARAFAT and , A. B. JONES , P.G. VINCENT , B.F. ENGELKE, , J.L. HILTON and , W.A. GENTNER
Pages: 65 to 77
Creation Date: 1984/01/01
The herbicide (2,4-dichlorophenoxy)-acetic acid (2,4-D) is considered to be the most cost-effective for eradicating illicit coca plants. Since coca leaves are chewed by the natives of coca producing areas, and, since coca paste and cocaine are widely abused, a study was untertaken to determine whether 2,4-D is translocated into the leaves and transferred to coca paste when coca plants are treated with this herbicide. The study concluded that basal treatment of coca plants with 2,4-D ester resulted in translocation of 2,4-D into the leaves, and this herbicide was detected in high concentrations one week after treatment. Coca paste prepared from leaves obtained from basally treated plants did not contain 2,4-D. Foliage spraying resulted in extremely high concentrations of 2,4-D in the leaves, with significantly high concentrations of the herbicide found one week after spraying. Trace amounts of 2,4-D were detected in coca paste prepared from plants sprayed by the foliage method.
The use of herbicides for eradication of illicit crops has become one of the effective means of controlling drugs derived from plants. The study group convened by the Narcotics Laboratory of the United Nations Division of Narcotic Drugs identified the use of herbicides as the most efficient and cost-effective means of preventing the harvest of raw materials from illicit fields. 1
Because of the convenience of herbicide use in eradication programmes as opposed to manual eradication, for example by cutting and burning, different herbicides were evaluated for use on coca plants (Erythroxylon coca) . The herbicide (2,4-dichlorophenoxy)-acetic acid (2,4-D) was found to be the most cost-effective for control of this woody plant (W. A. Gentner, unpublished results). This article presents the results of a study that was initiated to determine the following :
1"Methods for the eradication of illicit crops" , report of a study group, Geneva, 25 -27 July 1979 (MNAR/8/1979).
Whether basal spraying of coca plants with butoxyethyl ester of 2,4-D (2,4-D ester) results in translocation of the herbicide to the leaves;
Whether 2-4-D would be transferred to coca paste or cocaine upon processing of coca leaves after the herbicide has been applied by foliage or basal spraying.
Coca plants (Erythroxylon coca) were grown in greenhouse facilities at Beltsville, Maryland, United States.
2,4-D ester and (2,4,5-trichlorophenoxy)-acetic acid (2,4,5-T) were obtained from the laboratories of the United States Environmental Protection Agency at Research Triangle Park, North Carolina. 2,4,5-T was used as an internal standard in the analyses.
2,4-D formulation - a commercial preparation of the 2,4-D ester containing 3.8 lb of acid equivalent per gallon (454 g/l) was used.
Solutions for basal treatments were prepared by diluting 2.09 ml of the 2,4-D commercial formulation with diesel fuel to a total volume of 100 ml. This concentration of 2,4-D was equivalent to 8 lb of active ingredient of the herbicide in a total volume of 100 gallons of diesel fuel (9.6 g/l). The equivalent of 2 lb of the herbicide in a total volume of 100 gallons of diesel fuel (2.4 g/1 ) was achieved by further dilution. Test solutions (0.25 ml) were applied to the lower 1 5 cm of the above-ground portions of the plants. Four plants were used for each treatment. Replicates (leaves) of treatments were individually harvested within one hour after treatment and 24, 48, 72, 96 and
168 hours after treatment. Diesel fuel and absolute checks were included as controls. Leaves were hand-picked and freeze-dried for two days, after which they were ground to a powder in a ball mill and stored in sealed plastic bags at 50 C until analysed. The experimental design was a randomized block which was replicated three times. The experiment was repeated twice.
The commercial formulation of 2,4-D ester was applied with an experimental endless belt sprayer at rates of 0, 2, 4, 8 and 1 6 pounds per acre (respectively 0, 2.25 , 4.5, 9 and 18 kilograms per hectare) in a total volume of 20 gallons per acre (l .9 hl/ha). Harvest times and methods for drying, milling, and storage were the same as those described for basal treatments. The experimental design was a randomized block which was replicated three times.
Coca leaves were analysed according to the following protocol:
To 0. 1 g of coca leaves (powdered) in a 50 ml-centrifuge tube, add 0. 1 ml of methanol containing 10 µg of 2,4, 5-T per ml ;
Add 1 0 ml of 0. 1 N Ba (OH) 2 in aqueous methanol ( 1 : 2);
Place sample in a water bath for 10 minutes at 60 o C,;
Filter and wash with 5 ml of methanol, collecting the filtrate in a 50 ml centrifuge to be;
Place tube in a 60o C water bath and reduce volume to approximately 5 ml with N 2;
Extract with three 5-ml portions of ether, vortexing for one minute after each addition of ether. Discard the ether washes;
Acidify the aqueous phase by the addition of 1 N H2S04to a pH of approximately 2;
Extract with two 5-ml portions of ether, vortexing for one minute after each addition of ether (centrifuge to achieve phase separation), transferring the ether phase to a l 5 ml centrifuge tube and discarding the aqueous phase ;
Evaporate to dryness under N 2 at 60 o C and dissolve residue in l.0 ml of methanol;
Remove 0.5 ml of this solution and save;
To the residue of the remaining 0.5 ml, add 0.5 ml of saturated NaHCO 3 solution and sonicate or vortex;
Extract the bicarbonate solution with three 5-ml portions of ether, vortexing for one minute each addition of ether and discarding the ether washes;
Acidify the aqueous phase by the addition of 1 N H2S04to a pH of approximately 2;
Extract with two 15-ml portions of ether, combinating the ether extracts in a 15-ml centrifuge tube and discarding the aqueous phase;
Evaporate to dryness under N 2 at 60 o C.
The methyl esters of both herbicides were prepared before gas chromatographic (GC) analyses. Different methods were investigated for derivatization. The following method was found to be most successful:
Add 0.05 ml of a 1 : 40 mixture of a tetramethylammonium hydroxide solution (25 per cent in water) and dimethyl sulfoxide to the residue obtained under o above ;
Add 0.0 1 ml of iodomethane, vortex, and allow sample to stand at room temperature for five minutes. Sufficient iodomethane must be added to make the solution cloudy ;
Terminate the reaction by the addition of 0.5 ml of 0. 1 N HCl ;
Extract the methylated derivatives of 2,4-D and 2,4,5-T by the addition of l ml hexane;
Remove the hexane layer, adjust the volume of the extract as required and inject an aliquot into the gas chromatograph.
Column : 2.44 m x 2 mm glass column packed with 3 per cent OV-17 on Gas Chrom Q 100 - 200 mesh was used under the following conditions : column temperature, 185 0 C ; injector temperature, 250 o C ; detector temperature, gas, 95 per cent argon and 5 per cent methane ; flow rate, 30 ml/min ; detector, electron capture (EC) using a Hewlett Packard model 5710 A gas chromatograph equipped with 63Ni EC detector.
Aliquots of 2,4-D ester corresponding to 1 , 2, 3, 4, 5, 6, 7, 10, 15, and 20 µg of 2,4-D and 1 µg of 2,4,5-T were added to 0.1 g of powdered coca leaves. The spiked material was then analysed following the previously outlined procedure.
The ratios of the peak heights of 2,4-D and the internal standard peaks (2,4,5-T) were then plotted in relation to concentration. The results are shown in figure I.
Dried powdered coca leaves prepared from coca plants treated with 2,4-D ester by both methods of application of the herbicide were spiked with coca paste (about 70 per cent cocaine) in order to have a sufficient concentration of cocaine for isolation.
Coca paste was prepared by a method similar to that used by clandestine coca paste producers. The isolation procedure was as follows :
Dry powdered coca leaves (about 7 - 10 g samples) were moistened with water ;
To each moistened sample was added 0.5 g of coca paste (70 per cent cocaine) ;
To each sample was added 1 .7 g of sodium carbonate and the mixture was frequently stirred with a spatula for two hours ;
To each sample was then added 60 ml of kerosene and stirring was continued for 6 - 8 hours ;
The mixtures were then filtered and the leaves discarded ;
To the kerosene extract was added 20 ml of diluted sulfuric acid (l .7 ml H2 SO4/250 ml water) and the mixture was stirred for 3() minutes ;
The aqueous sulfuric acid layer was separated and divided into two equal portions ;
To one portion was added 5 drops of 25 per cent aqueous potassium permanganate (or the amount required for the permanganate colour to persist for a second or so before disappearing) and the mixture was then filtered ;
To the filtrate obtained from step h and to the second portion obtained from step gwas added ammonium hydroxide to render the solution basic ;
After the basic solution had stood for a time, a buff-coloured precipitate of coca paste (cocaine) formed, which was then separated by filtration ;
The mixtures of dried coca paste obtained from control coca leaves and coca leaves sprayed by the basal or foliage method of herbicide application were then analysed for 2,4-D and its ester as described below. Coca paste obtained with and without KmnO 4 purification was analysed.
Coca paste was analysed as follows :
Weigh accurately about 25 mg of coca paste and transfer to a separatory funnel ;
Dissolve the paste in 5 ml of dilute ; H 2S0 4
Extract the acid solution with three 10-ml portions of ether ;
Dry the ether extract over anhydrous Na 2 SO 4 and then evaporate the solution;
Dissolve the residue in 1.0 ml of methanol.
Inject an aliquot of the methanol solution (1 µl) directly into the gas chromatograph under the conditions outlined above. Under these conditions, 2,4-D ester has a retention time of 20 minutes.
Analysis for 2,4-D
Transfer the methanol solution obtained in step e to a conical test tube;
Evaporate the methanol, add 0.5 ml of saturated sodiumbicarbonate and extract with three 1 -ml portions of ether ;
Discard the ether layer and acidify the bicarbonate layer with dilute H 2 SO 4 ;
Extract the acidic solution with two 2-ml portions of ether;
Evaporate the solvent and derivatize the residue as previously outlined ;
Inject the derivatized solution into the gas chromatograph under the conditions previously outlined.
In an effort to reduce the traffic in illicit drugs, the use of herbicides has been evaluated for the control of cannabis plants, opium poppies (Papaver somniferum ) 1 and most recently for coca plants Erythroxylon coca. The herbicide 2,4-D has been selected for coca (W. A. Gentner, unpublished results). Since coca leaves are chewed by the natives of coca-producing countries of South America, it is important to know whether 2,4-D translocates into the leaves upon treatment of the coca plants with the herbicide. In addition, since coca paste or cocaine, the main product of coca leaves, is illicitly used by millions of people around the world, it is important to determine whether 2,4-D residues in leaves will be transferred to coca paste. The 2,4-D ester was applied to coca plants by basal treatment (treating the lower part of the trunk of the plant) and by foliage spraying (application directly onto the leaves). In each case, the herbicide was applied at several rates, as indicated in the experimental section, with three replicates at each application rate. Samples of the leaves were collected immediately after treatment and after 24, 48, 72, 96 and l 68 hours. The leaves were dried, milled and stored at 5 o C until analysed. Analyses were carried out within two weeks from the date of collection of the leaves in all cases.
A GC procedure was developed for the analysis of 2,4-D and its ester (combined) in coca leaves and in coca paste. EC detection was used with 2,4,5-T as internal standard for 2,4-D determinations. A calibration curve for esters of 2,4-D and 2,4,5-T was prepared using coca leaves spiked with the standards of these herbicides. Figure I shows a linear relationship (r - 0.999) between the peak height ratio and the concentration of the methyl ester derivatives of 2,4-D and 2,4,5 -T. The hydrolysis step in the extraction procedure was included to convert the ester to the free acid, which was extracted with the internal standard. The mixture was methylated prior to GC analysis. Sufficient resolution of the two compounds was achieved using 3 per cent OV-17 column with minimum interference of other constituents in the plant material (figure II). Under the conditions used for this analysis, concentrations down to 1 ppm could readily be detected. Results of the analysis are presented in tables 1 and 2.
Concentration of the herbicide (2.4-D equivalent) (ppm)
The method was used to analyse the level of herbicide residues in leaves obtained from basally treated plants. It is clear from the data that 2,4-D translocates into the leaves following basal treatment of the plants with its ester. The 2,4-D starts to appear in the leaves shortly after treatment. The concentration increased with time and it reached its maximum after 72 hours. However, significant amounts of the herbicide persisted seven days after treatment, as reflected in figure III.
|
Time elapsed between treatment and collection |
||||||
|---|---|---|---|---|---|---|
|
Replicate number |
1h |
24h |
48h |
72h |
96h |
168h |
| 103 | 6.5 | 17.0 | 14.0 | 26.0 | 7.5 | 7.0 |
| 203 | 2.5 | 23.0 | 36.5 | 36.0 | 23.5 | 25.0 |
| 303 | 4.5 | 10.5 | 9.0 | 22.0 | 11.5 | 6.5 |
|
Average
|
4.5 | 16.8 | 19.8 | 28.0 | 14.2 | 12.8 |
|
Standard deviation
|
2.0 | 6.2 | 14.6 | 7.2 | 8.3 | 10.5 |
|
Time elapsed between treatment and collection |
||||||
|---|---|---|---|---|---|---|
|
Replicate number |
1h |
24h |
48h |
72h |
96h |
168h |
| 104 | 16.5 | 45 | 35.5 | 41.5 | 42.0 | 26.5 |
| 204 | 5.5 | 148 | 133.0 | 183.0 | 85.0 | 76.0 |
| 304 | 3.5 | 43 | 105.0 | 118.0 | - | 64.5 |
|
Average
|
8.5 | 78.6 | 91.2 | 114.2 | 63.5 | 55.7 |
|
Standard deviation
|
7.0 | 60 | 50 | 70 | 30 | 26 |
The concentration of 2,4-D in coca leaves collected at different times after foliage spraying of coca plants with 2,4-D ester is shown in tables 3 - 6. Highest concentrations of 2,4-D on the leaves were found immediately after treatment. The concentrations fell steadily with time and levelled at 48 - 72 hours. The change in 2,4-D concentration as a function of time is reflected in figure IV. The data also indicated that very high levels of 2,4-D were found in the leaves seven days after spraying, with the lowest concentration of about 1 ,000 ppm at the 2 lb per acre rate (2.3 kg/ha) and almost 4,000 ppm at the 16 lb per acre rate (18.1 kg/ha).
In addition to the analysis of 2,4-D in the leaves, coca paste was separated and analysed for the herbicide. Leaves obtained from coca plants treated basally with solutions containing 8 lb of active ingredient per 1 00 gallons of diesel fuel (9.6 g/l) were collected 72 hours after spraying, while leaves obtained from plants sprayed at 16 lb per acre ( 18.1 kg/ha) by foliage application were collected immediately after spraying. The samples were used for the preparation of coca paste since they showed the highest 2,4-D content (see tables 2 and 6). The samples (about 10 g each) were spiked with coca paste to facilitate the extraction and recovery of cocaine. Isolation was done by a procedure designed to simulate the method used in the preparation of illicit coca paste. Isolated coca paste was then analysed for 2,4-D. Analysis showed that coca paste prepared from basally treated plants contained no detectable amount (less than 1 ppm) of the herbicide. However, trace amounts of 2,4-D were detected in coca paste prepared from plants treated by foliage spray. Amounts of 2,4-D were calculated to be less than 1 µg in coca paste recovered from l g of coca leaves, whether KmnO 4 was used during the preparation of the paste or not.
Note: The points shown are the average of three replicates at each time. For standard deviation. see tables 1 and 2.
Concentration of 2,4-D in coca leaves after basal treatment of coca plants with 2,4-D ester at the equivalent of 2 ( A ) and 8 ( B ) 1b of active ingredient per 100 gallons of diesel fuel [2.4 (A) and 9.6 ( B ) g/1]
|
Time elapsed between treatment and collection |
||||||
|---|---|---|---|---|---|---|
|
Replicate number |
1h |
24h |
48h |
72h |
96h |
168h |
| 102 | 1730 | 2180 |
l 350
|
740 | 740 | 1610 |
| 202 | 1 130 | 1190 | 760 | 1060 | 570 | 910 |
| 302 | 1540 | 1430 | 1040 | 1520 | 1 550 | 680 |
|
Average
|
1467 | 1600 | 1050 | 1106 | 953 | 1066 |
|
Standard deviation
|
306 | 516 | 295 | 392 | 523 | 484 |
|
a Total volume of 20 gallons per acre (l.9hl/ha).
|
||||||
a Total volume of 20 gallons per acre (l.9hl/ha).
|
Time elapsed between treatment and collection |
||||||
|---|---|---|---|---|---|---|
|
Replicate number |
1h |
24h |
48h |
72h |
96h |
168h |
| 103 | 3360 | 1560 | 2240 | 1920 | 1740 | 1620 |
| 203 | 5800 | 2260 | 2240 |
l780
|
2120 | 2700 |
| 303 | 3960 | 1640 | 1000 | 1920 | 1160 | 1640 |
|
Average
|
4373 | 1820 | 1826 | 1873 | 1673 | 1986 |
|
Standard deviation
|
1271 | 383 | 716 | 80 | 483 | 618 |
a Total volume of 20 gallons per acre (l.9hl/ha).
|
Time elapsed between treatment and collection |
||||||
|---|---|---|---|---|---|---|
|
Replicate number |
1h |
24h |
48h |
72h |
96h |
168h |
| 104 | 5760 | 3056 | 2760 | 2200 | 2880 | 2040 |
| 204 | 4984 | 2720 | 2080 | 2520 | 2000 | 2280 |
| 304 | 7760 | 3760 | 1504 | 2560 | 2160 | 4000 |
|
Average
|
6168 | 3178 | 2114 | 2426 | 2346 | 2773 |
|
Standard deviation
|
1432 | 530 | 628 | 197 | 468 | 1069 |
a Total volume of 20 gallons per acre (l.9hl/ha).
|
Time elapsed between treatment and collection |
||||||
|---|---|---|---|---|---|---|
|
Replicate number |
1h |
24h |
48h |
72h |
96h |
168h |
| 105 | 4480 | 3920 | 2720 | 2080 | 4560 | 3680 |
| 205 | 10960 | 7520 | 5920 | 2640 | 3280 | 2480 |
| 305 | 7520 | 6800 | 2880 | 3280 | 5760 | 5440 |
|
Average
|
7653 | 6080 | 3840 | 2666 | 4533 | 3866 |
|
Standard deviation
|
3242 | 1904 | 1803 | 600 | 1240 | 1488 |
a Total volume of 20 gallons per acre (l.9hl/ha).
Note: The points shown are the average of three replicates at each time. For standard deviation. see tables 3 - 6
Basal treatment of coca plants with 2,4-D ester resulted in translocation of 2,4-D into the leaves.
The concentration of 2,4-D in the leaves increased with time, reaching its maximum level 72 hours after treatment and then declining at 96 hours. Such was the case whether the plants were treated with solutions containing 2 lb or 8 lb of active ingredient per 100 gallons of diesel fuel (2.4 g or 9.6 g per litre).
2,4-D was still detectable in high concentrations one week after treatment.
2,4-D was not detected in coca paste made from leaves obtained from basally treated plants.
The authors are grateful to the Environmental Protection Agency (EPA), Research Triangle Park, North Carolina, United States, for providing the analytical samples of 2,4-D ester and 2,4,5 -T. This research was supported in part by the United States Department of State, under Contract No. 2071-210109, and the Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, University, Mississippi, United States.
Foliage spray of coca plants with 2,4-D ester resulted in extremely high concentrations of 2,4-D in the leaves even at rates as low as 2 lb per acre (2.3 kg/ha).
The concentration of 2,4-D was highest immediately after spraying, dropping by about 50 per cent within 48 - 72 hours and thereafter remaining almost constant for the duration of the experiment (one week).
Significantly high concentrations of 2,4-D persisted in the leaves one week after spraying.
Trace amounts of 2,4-D were detected in coca paste prepared from plants sprayed by the foliage application method.
