chapter iii development of method for …shodhganga.inflibnet.ac.in/bitstream/10603/77515/9/09_...

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CHAPTER III

DEVELOPMENT OF METHOD FOR ISOLATION AND

IDENTIFICATION OF NEW PESTICIDES

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CHAPTER - III

SECTION A

NEW CHROMOGENIC SPRAY REAGENT FOR

DETECTION AND IDENTIFICATION OF CARBOSULFAN

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3A.1 Introduction

Carbamates are esters of carbamic acid, having broad spectrum of biological

activity. Carbosulfan (2,3-dihydro-2,2-dimehylbenzofuran-7-yl (dibutylaminothio)

methyl carbamate are carbamate insecticides, used largely against a broad spectrum of

insects in field crops, fruits, vegetables and household flies and mosquitoes. Now a

day it is popular due to its mode of action as systemic insecticide with contact of

stomach action. Owing to its easy availability, is misused for homicidal or suicidal

purposes. This laboratory received considerable number of poisoning cases involving

carbosulfan insecticide in routine forensic work. The detection of carbamates is done

by High performance thin layer chromatography (HPTLC), it is the most simple, rapid

and reliable technique usually used in forensic laboratory for detection and

identification of poison.

A new chromogenic spray reagent for chromatographic detection and

identification of carbosulfan and carbamate insecticide is described by high

performance thin layer chromatographic method. Carbosulfan on alkaline hydrolysis

yields sodium salt of 2,3-dihydro-2,2-dimethylbenzofuran-7-ol, which form purple

color complex with potassium ferricyanide, by thiochrome reaction. Other carbamate,

organophosphorous, organ chlorine, and pyrethroid insecticides and constituents of

viscera (amino acids, proteins, peptides, etc.) do not react with this reagent. The

detection limit of carbosulfan is ca 0.5 µg.

3A.2 Literature Survey

Several different chromogenic reagents are reported in the literature for

detection and identification of carbamate insecticides by TLC and HPTLC. These

spray reagents are diazophenol (after alkaline hydrolysis)1, alkaline fast blue-B

2,

Tollen’s reagent3, 0.5% diazotized p-nitroaniline in 1:4 HCL and then NaOH

solution4 and p-nitro benzene diazonium tetra fluoroborate reagent.

5,6 These reagents

also give coloration with biological impurities, such as amino acids, proteins and

peptides, i.e. they are not specific. The use of phenyl hydrazine hydrochloride in

alkaline media7, ammonium cerium(IV) nitrate,

8 copper(II) chloride followed by

ammonium metavanidate reagents9, diazotized-p-amino-1-naphthol-3-sulfonic acid (J.

acid) and then spraying with NaOH10

followed by observation under 366 nm UV

light11

are reported to be specific for carbaryl only. Sodium hydroxide solution

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followed by 4-amino antipyrine reagent for carbaryl, propoxur and carbofuran are

reported in the literature.12,13

The Bureau of Indian Standard (BIS) has prescribed two

methods namely infrared spectrophotometer method and liquid chromatography

carbosulfan determination.14

In Present communication

we report the use of sodium

hydroxide followed by potassium ferricyanide as selective reagent for detection and

identification of carbosulfan by HPTLC yielding an intense purple color spot. This

reaction is totally based on thiochrome reaction (whose exact structure has not been

elucidated). Alkaline ferricyanide solution is used for detection of thallium16

mixture

of ferric salt, with potassium ferricyanide and arsenous acid is used for detection of

organo-iodine compounds.17

Alkaline ferricyanide solution is also used for detection

of adrenaline and derivative of vitamin B1 (thiochrome reaction).17

We have used this

reagent for HPTLC detection and determination of carbosulfan in biological material,

carbosulfan on oxidation with potassium ferricyanide gives purple violet complex at

Rf 0.72, The hRf value equivalent to 100 Rf and it is hRf 72 , the detection limit is

approximately 1µg.20

There is no specific method for detection and identification of carbosulfan

from biological material. We have developed specific and reliable method for

identification of this insecticide.

3A.3 Present Work

We have developed a new chromogenic spray reagent for chromatographic

detection and identification of carbosulfan20

and carbamate insecticide is described by

high performance thin layer chromatographic method. Carbosulfan on alkaline

hydrolysis yields sodium salt of 2, 3-dihydro-2,2-dimethylbenzofuran-7-ol, which

form purple color complex with potassium ferricyanide, is thiochrome reaction.

3A.4 Experimental Section

Chemicals and Reagents

All reagents used were of analytical reagent grade, distilled water was used

throughout:

I) Stock solution of carbosulfan (1 mg %) was prepared by dissolving

technical grade carbosulfan 25% E.C. (supplied by Dhanuka Group of

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Pesticide division, Gurgaon, Haryana, Marketed this compound by the

name Aatank) in ethanol.

II) Sodium hydroxide solution (10% w/v) was prepared by dissolving 10

gm of sodium hydroxide in 100 ml distilled water.

III) Potassium ferricyanide reagent was prepared by dissolve 5 gm of

potassium ferricyanide K3[Fe(CN)]6 in 100 ml distilled water.

Extraction of carbosulfan from biological materials

In a portion of about 100 gm each of various biological tissues (stomach,

intestine, liver, spleen and kidney) containing carbosulfan, 10 gm ammonium sulphate

was added and were minced individually in an aqueous solution. Each biological

sample was extracted with a separating funnel with 150 ml of chloroform : alcohol

(7:3) mixture. The extract was transferred into an evaporating dish. The aqueous

phase was re-extracted two to three times with 50 ml of chloroform : alcohol (7:3)

mixture. The extracts were combined and the solvent was evaporated at room

temperature. The residue was dissolved in 2 ml of ethanol. A known volume (10 �l)

of the solution was spotted on HPTLC plates together with standard solution of

carbosulfan insecticides. The plates were developed and sprayed with 10 % sodium

hydroxide solution followed by potassium ferricyanide spray reagent successively to

give purple violet color spot. This reaction is thiochrome reaction.

Chromatography

Chromatography was performed on 10 cm × 10 cm silica gel F254 HPTLC

plate (Merck, Darmstadt, Germany #1.05729 OB397077) a Desaga (Heidelberg,

Germany) 30 TLC applicator, spotting volume 5 ml, spotting rate 10 s/ml was used to

apply standard carbosulfan stock solution, standard solution of other

organophosphorous (dimethoate, phosphomidon, dichlorvos, malathion, parathion,

methylparathion, phorate, chlorpyriphos, triazophos), organochlorine insecticide

(endosulfan, DDT, BHC), carbamate (baygon, carbaryl, carbofuran), synthetic

pyrethroid (Fenvalrate, cypermethrin, deltamethrin), proinsecticide (indoxacarb) and

extract from visceral tissue spiked with carbosulfan solution (5 ml stock solution).

The HPTLC plate was then developed in previously saturated HPTLC chamber with

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n-hexane: acetone (4:1) as mobile phase to distance of 10 cm. After development the

plate was removed from the chamber, dried in air, and sprayed with 10% sodium

hydroxide solution followed by potassium ferricyanide reagent successively. A typical

chromatogram is shown schematically in figure 1 and color photograph in figure 2.

The purple colored spot of the oxidized product of standard carbosulfan and of

carbosulfan extracted from viscera were extracted from the HPTLC plate with

ethanol. The visible spectrum of the extracted colored compound formed between

carbosulfan and potassium ferricyanide was recorded in ethanol by means of Specord

S-100 UV-Vis Spectrometer (Carl Zeiss Jena).

Recovery Experiment

A 1 mg amount of carbosulfan in ethanol was added to 50 gm of minced

visceral tissue, mixed them well and kept for a day. The insecticides were then

extracted separately with chloroform: alcohol (7:3) mixture as described under

extraction section. The solvent was evaporated at room temperature and the residues

were dissolved separately in 1 ml of ethanol. A 10 �l volume of solution was spotted

on activated thin layer plates together with 10 �l standard carbosulfan solutions

containing known concentrations of 7.0, 8.0 and 9.0, 9.5 and 10.0 mg per 10 ml in

ethanol. The plates were then developed as described in the procedure section and

sprayed with 10% sodium hydroxide solution followed by potassium ferricyanide

solution. The intensity of purple color spots developed for the visceral extracts were

compared with known standards and were found to agree with the spot resulting from

the carbosulfan of 9 mg/ 10 ml (average of 5 experiments). Hence the recovery for

each insecticide is 90%.

Semi-quantitative determination of Carbosulfan

Carbosulfan was semi quantitatively determined in biological and non-

biological materials by HPTLC with visual assessment. Carbosulfan was extracted by

using chloroform: alcohol (7:3) from known amount of (50 gm) of biological sample

such as viscera, blood, stomach-wash etc. and non-biological materials such as grains,

food materials, water sample, soil etc. as described under ‘extraction of carbosulfan’.

The extract was then evaporated at room temperature and the residue was dissolved in

1-2 ml ethanol. A 10 µl volume of this extract was spotted on HPTLC plate together

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with 10 µl each of standard solution of technical carbosulfan containing known

concentration of 1, 5, 10, 15, 20, 25…. mg per 10 ml in ethanol. The plate was then

developed as described under “chromatography procedure” and sprayed with 10%

sodium hydroxide solution followed by potassium ferricyanide solution. The intensity

of purple colored spot was developed for the extract of unknown concentration, was

visually compared with those of known standards. From this the amount of

carbosulfan present in the total extract and that in the 100 gm of viscera was

determined. Since visual assessment was done, it is a semi-quantitative determination.

3A.5 Results and Discussion

On alkaline hydrolysis of carbosulfan yields sodium salt of 3-

hydroxycarbofuran13

which forms purple color complex with potassium ferricyanide.

Distinguished purple color spot from standard carbosulfan and carbosulfan extracted

from visceral were observed at hRF 72 whereas no spots were observed for other

organophosphorous, organochlorine, carbamates and pyrethroid insecticide. From the

recovery experiment it was observed that the intensity determined by densitometry of

the purple spot developed for the visceral extract was comparable with that of the spot

corresponding to 9 mg carbosulfan per 10 ml ethanol (average from 3 experiments).

Hence, the recovery was 90%. This reaction is totally based on thiochrome reaction19

.

Merits of the Reagent

The reagent reported is selective for carbosulfan among other insecticide. This

reagent do not give positive reaction with organochlorine insecticide such as BHC,

DDT and endosulfan; organophosphorous insecticides such as dimethoate, phorate,

metasystox, methyl parathion, ethyl parathion, thiometon, quinalphos, chlorpyriphos,

phosphamidon, dimecron and monocrotophos. Pyrethroid insecticides such as

cypermethrin, fenvalerate and deltamethrin and narcotic substances such as morphine,

heroin does not give positive reaction. Constituents of viscera i.e. amino acids, protein

peptide, etc. which are co-extracted with the insecticide do not interfere. The reported

regent for HPTLC detection and identification of carbosulfan is simple, sensitive and

can be routinely used for the detection and semi quantitative determination of residual

carbosulfan in biological material investigated in forensic work.

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0.72 No Spots Were Located

I II III IV V VI VII VIII IX X

Reaction Mechanism:

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Scheme 1

Fig 1

HPTLC Chromatogram obtained from:

I) Standard carbosulfan II) Carbosulfan from Visceral extract III) Blank Viscera IV)

Dimethoate V) Malathion VI) Phosphomidon (organophosphorous insecticide) VII)

Encosulfan (organochloro insecticide) VIII) Propoxur (carbamate insecticide) IX)

Cypermethrin (pyrethroid insecticide) X) Diazepam (drug).

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Fig 2: Color photograph of HPTLC plate


I) Standard carbosulfan II) Carbosulfan from visceral extract III) Blank Viscera

I II III

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3A.6 References

1. Randerath, K., Thin-layer chromatography, Academic press, New York, 1965,

176.

2. Tiwari, S. N.; Singh, R., Brochure of the autumn school of Forensic Science,

Chandigarh, India, 1979, 4.

3. Kawale, G. B.; Jogalekar, V. D., Current Science, 1976, 45.

4. Bose, D.; Shivhare, P.; Gupta, V. K., Journal of Planar Chromatography,

1994, 7, 415.

5. McGinnis, S. C.; Sharma, J., Journal of Liquid Chromatography, 1994, 17,

151.

6. Rathore, H. S.; Khan, H. A.; Sharma, R., Journal of Planar Chromatography,

1991, 4, 494.

7. Patil, V. B.; Shingare, M. S., Journal of Chromatography, 1993, 653, 181.

8. Patil, V. B.; Shingare, M. S., Analyst, 1994, 119, 415.

9. Padlikar, S. V.; Shinde, S. S.; Shinde, B. M., Analyst, 1988, 113, 1747.

10. Bose, D.; Shivhare, P.; Gupta, V. K., Journal of Planar Chromatography,

1999, 7, 415.

11. Jork, H.; Winner, H. (Editor), TLC Report, GIT verlag GmbH, Darmstadt,

Germany, 1986,7.

12. Sevalkar, M. T.; Patil, V. B.; Garad, M.V., Journal of Planar

Chromatography, 2000, 13, 235.

13. Clive, Tomlin, The Pesticide Manual, Ed. 10, Crop. Protection Publication,

2004, 154.

14. Indian Standard Carbosulfan Technical-Specification, BIS 2001, Bureau of

Indian Standard, New Delhi, 2001, 1.

15. Indian Standard Carbosulfan ECl-Specification, BIS 2001, Bureau of Indian

Standard, New Delhi, 2001, 1.

16. Fritz, Feigl, Spot Test in Inorganic analysis, Ed. 7, Elsevier Publication Pvt.

Ltd., New Delhi, 1983, 476.

17. Fritz, Feigl, Spot Test in Organic analysis, Ed. 7, Elsevier Publication Pvt.


18. Stahl, E., Thin layer chromatography, Springer Berlin, N. Y., 1969, Reagent

No. 109.

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19. Williams, D. V., Scientific Foundations of Clinical Biochemistry, Vol. I,

William Heinemann Medical Books Ltd. London, 1990, 89.

20. Kulkarni, K. V.; Shinde D. B.; Garad, M.V.; Mane, D.V., Journal of Planar

Chromatography, 2010, 5, 373.

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CHAPTER - III

SECTION B

A NEW CHROMOGENIC SPRAY REAGENT FOR

THE DETECTION AND IDENTIFICATION OF INDOXACARB

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3B.1 Introduction

Oxidizines are the newly discovered group of insecticides, highly toxic to

animal and insects, wildly used in agriculture for the protection of fruits and

vegetables from insects.1 Indoxacarb is an oxidizine class organic compounds act as

sodium channel blocker. Indoxacarb effectively block the neuronal sodium channels

in insect body2. Hence it shows outstanding field insecticide activity.

1

Indoxacarb (Avant) is {7-chloro-3-(methoxycarbonyl-(4-trifloro-methoxy-

phenyl)-carbomoyl)-2,5-dihydro-indeno carboxylic acid methyl ester}3,4

is largely

used for protection of crop. The ending carb in the name doesn’t mean that it belongs

to the carbamate class of insecticide. Owing to its easy availability and extensive use

in agriculture by the farmers, are often misused for homicidal or suicidal purposes.

Homicidal, suicidal and accidental uses of these insecticides are very common in

India. There is extensive burden on forensic laboratories due increasing number of

biological samples for poison detection. Hence, the forensic toxicology needs to

develop new rapid analytical technique of Indoxacarb analysis in biological & non-

biological material. High performance thin layer chromatography (HPTLC) is the

method of choice because of its speed, and versatility.

3B.2 Literature Survey

Unavailability of specific reagent for detection and identification of

indoxacarb from biological samples and limitations of Dragandroff’s reagent inspired

us to search new reagent. Drgandroff’s reagent gives positive test to all compounds

containing basic nitrogen. By several trials it was observed that diacetylmonoxime in

strong aqueous acidic medium is the selective and specific reagent for HPTLC

detection and determination of Indoxacarb. Indoxacarb 1 is hydrolyzed in 20%

H2SO4 yield 3, which undergo further hydrolyses to its oxadiazine derivative 4. The

oxadiazine under acidic condition react with diacetylmonoxime 5 and furnish azide

derivative of oxadiazine 6, which is being sensitive to heat and light turns black on

heating (scheme-1).

Only indoxacarb 1 on hydrolysis can form oxadiazine, which further can form

its azide derivative, indicates the specificity of the reagent. We have used this reagent

for HPTLC detection and determination of indoxacarb in biological material, because

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indoxacarb reacts with diacetylmonoxime on heating produce black color, hRf 42, 90.

The detection limit is approximately 0.5 µg.

3B.3 Present Work

A new chromogenic spray reagent for chromatographic detection and

identification of indoxacarb, an oxidizine insecticide is described by high

performance thin layer chromatographic method. Indoxacarb on acid hydrolysis yield

its oxadiazine derivative. The oxadiazine under acidic condition react with

diacetylmonoxime and furnish azide derivative of oxadiazine which is being sensitive

to heat and light turns black on heating. Other organophosphorous, organochlorine,

pyrethroid and carbamate insecticide and constituents of viscera (amino acid, proteins,

peptides, etc.) do not react with this reagent. The detection limit of indoxacarb is ca

0.5 �g.

3B.4 Experimental Section

Chemicals and Reagent

All analytical reagent grade reagents were used for the experiment. Distilled

water was used throughout the experiment:

I) Stock solution of Indoxacarb (1.0 mg %) was prepared by dissolving

technical grade indoxacarb in ethanol.

II) Sulphuric acid solution (20% w/v) was prepared by dissolving 20 ml of

concentrated sulphuric acid in 100 ml distilled water.

III) Diacetylmonoxime reagent was prepared by dissolving 2 gm of

diacetylmonoxime in 100 ml distilled water.

Extraction of Indoxacarb from Biological Materials


intestine, liver, spleen and kidney) containing the above mentioned insecticides, 10

gm ammonium sulphate was added and were minced individually in an aqueous

solution. Each biological sample was extracted with 150 ml of ethyl acetate. The

extract after concentration by evaporation was stirred in water and the aqueous phase

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was re-extracted two to three times with 50 ml ethyl acetate. The extracts were

combined and the solvent was evaporated at room temperature. The residue was

dissolved in 2 ml of ethanol. A known volume (10 �l) of the solution was spotted on

HPTLC plates together with standard solution of indoxacarb insecticides.

Chromatography

Chromatography was performed on 10 cm × 10 cm silica gel F254 HPTLC


Germany). As 30 TLC applicator, spotting volume 5 ml, spotting rate 10 s/ml was

used to apply standard indoxacarb stock solution, standard solutions of other


methylparathion, phorate, chlorpyriphos, triazophos), organochloro insecticide

(endosulfan, DDT, BHC) carbamate insecticide (baygon, carbaryl, Carbofuran),

pyrethroid insecticide (fenvalrate, cypermethrin, deltamethrin), and extract from

visceral tissue spiked with indoxacarb solution (5 ml stock solution). The HPTLC

plate was then developed in previously saturated HPTLC chamber with n-hexane:

acetone (4:1) as mobile phase to distance of 10 cm. After development, the plate was

dried in air, sprayed with 20% sulphuric acid solution followed by diacetylmonoxime

reagent, and was kept in oven at 60 0C for 10 minutes. The Black colored spots for

authentic and sample under study (extracted from viscera) were observed on HPTLC

plate (Figure-1) at hRf 42, 90.

Recovery Experiment

Indoxacarb (1 mg) in ethanol was added to minced visceral tissues (50 gm)

mixed them well and kept for a day. The insecticides were then extracted separately

with ethyl acetate as described. The solvent was evaporated at room temperature, the

residues was dissolved separately in of ethanol (1 ml). The solution (10 �l) was

spotted on activated thin layer plates together with standard indoxacarb solutions (10

�l) containing known concentrations of 7, 8 and 9, 9.5 and 10 mg per 10 ml in

ethanol. The plates were then developed as described and sprayed with sulphuric acid

solution (20%) followed by diacetylmonoxime reagent successively. The plate was

kept in oven at 60 0C for 10 minutes. The intensity of black colored spots developed

for the visceral extracts were compared with known standards and were found to be in

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agreement with the spots resulting from the Indoxacarb of 9 mg/ 10 ml (average of 5

experiments). Hence the recovery for each insecticide was Ca. 90%.

Semi-quantitative determination of Indoxacarb

Indoxacarb was semi quantitatively determined in biological and/or non-

biological materials by HPTLC with visual assessment. Indoxacarb was extracted by

ethyl acetate from known amount of (Ca. 50gm) biological sample such as viscera,

blood, stomach–wash etc. and non-biological materials such as grains, food materials,

water sample, soil etc. as described under ‘extraction of indoxacarb’. The extract was

then evaporated at room temperature and the residue was dissolved in 1-2 ml ethanol.

A 10 µl volume of this extract was spotted on an activated TLC plate together with 10

µl each of standard solution of technical indoxacarb containing known concentration

of 1, 5, 10, 15, 20, 25…. mg per 10 ml in ethanol. The plate was then developed as

described under ‘chromatography procedure’ and sprayed with 20% sulphuric acid

solution followed by diacetylmonoxime reagent, and was kept in oven at 60 0C for 10

minutes. The Black colored spots were observed on HPTLC plate at hRf 42, 90. A

typical chromatogram is shown schematically in figure 1.The intensity of the spot

developed for the extract, of unknown concentration, was visually compared with

those of known standards. From this the amount of indoxacarb present in the total

extract and that in the 100 gm of viscera was determined. Since visual assessment, it

is a semi-quantitative determination.

3B.5 Results and Discussion

Condensation of indoxacarb with diacetylmonooxime gives a black brown

color. Distinguished black brown spot from standard indoxacarb and indoxacarb

extracted from visceral extract were observed at hRf 42, 90, whereas no spot were

observed for other organophosphorous, organochlorine, and carbamate and pyrethroid

insecticide. From recovery experiment it was observed that the intensity determined

by densitometry of the black spot developed for the visceral extract was comparable

with that of the spot corresponding to 9 mg indoxacarb per 10 ml Ethanol (average

from 3 experiments). Hence, the recovery was 90%. Complex method is originally

based on method applied for estimation of urea9. Strong acid solution containing

oxidizing agent and is believed to proceed. An oxidizing agent is use to destroy the

hydroxylamine. Diacrtylmonoxime condenses with hydroxylamine to produce

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diacetyldioxime (dimetylglyxime) the well known reagent for nickel10

reported in

literature.

3B.6 Conclusion

The reagent reported is selective for Indoxacarb among other insecticide. This

reagent does not give positive reaction with organochlorine insecticide such as BHC,

DDT and endosulfan; organ phosphorus insecticides such as dimethoate, phorate,

metasystox, methyl parathion, ethyl parathion, thiometon, quinalphos, dalf,

chlorpyriphos, phosphamidon, dimecron and monocrotophos. Pyrethroid insecticide

such as cypermethrin, fenvalerate and deltamethrin and narcotic substances such as

morphine, heroins do not give positive reaction. Constituents of viscera i.e. amino

acids, proteinpeptide, etc. which are co-extracted with the insecticide do not interfere.

The reported regent for HPTLC detection and identification of Indoxacarb

insecticide is inexpensive, harmless, selective and specific, can be routinely used for

the detection and semi quantitative determination of residual indoxacarb in biological

material under investigation in forensic work.

Reaction:

Scheme-1

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0.90 0.90

0.22 0.22 No Spots Were Located

� � � � � � � � � �


Fig 1


I) Standard indoxacarb II) Indoxacarb from visceral extract III) Blank viscera IV)

Dimethoate V) Malathion VI) Phosphomidon (organophosphorous insecticide) VII)

Encosulfan (organochloro insecticide) VIII) Propoxur (carbamate insecticide) IX)

Cypermethrin (pyrethroid insecticide) X) Diazepam (drug).

P a g e | 86

3B.7 References

1. Timan, P. G., Pest Management Science, 2002, 58, 92.

2. Lapied, B.; Gerelleav, F., British Journal of Pharmacology, 2000, 132(2),

587.

3. Barr, C. L., Product Evaluation., S.W. ento supplement, 2002, 25, 47.

4. Dept. of Primary Industries and Fisheries, Brisbane, Australia, Merits of JMPR

Metting, 2002.

5. Tifton, G. A., USDA, ARS, Crop Production and Management Research

article, Society of Chemical Industry, USA, 2002.

6. Khambly; Bhupinder, P. S.; Pesticide Outlook, 2002, 13, 49.

7. Tilman, P., Pest Management Science, 2002, 58(1), 92.

8. Merck; Merck Index, Merek and Co. Inc., N. J., 1997, Ed.12, 893.

9. William, D. L., Scientific Foundation of Clinical Biochemistry, William

Heinemao Medical Book ltd., London, 1978, I, 25.

10. Fritz, Feigl, Spot test in Inorganic analysis, Ed. 6, Elsevier Publisher Ltd.,

New Delhi, 1972, 343.

11. Fritz, Feigl, Spot test in Organic analysis, Ed. 7, Elsevier Publisher Ltd., New

Delhi, 2005, 263.

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CHAPTER - III

SECTION C

NEW CHROMOGENIC SPRAY REAGENT FOR

DETECTION AND IDENTIFICATION OF

HYDROGEN CYNAMIDE

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3C.1 Introduction

Hydrogen cyanamide is used in agriculture as a plant growth regulator and is

applied to many deciduous plants to stimulate uniform bud break after dormancy,

resulting in uniform flowering and maturity. Dormex®

(Degussa A. G., Trostberg,

Germany), a pesticide producing company has started the production of hydrogen

cyanamide in Italy in 2000. The product is sold as (50% by weight) passing

atmospheric nitrogen over heated calcium carbide in the presence of catalyst produces

this cyanamide. It is highly toxic having adverse health effects from contact include

severe irritation and ulceration of the eyes, skin, and respiratory tract. This also

inhibits aldehyde dehydrogenase and can produce acetaldehyde syndrome (e.g.

vomiting, parasympathetic hyperactivity, dyspnea, hypotension, and confusion) when

exposure coincides with alcohol.1

Nashik region (Maharastra State) is famous for grape production and use of

hydrogen cyanamide is very common in this region. This laboratory is receiving

considerable number of poisoning cases involving hydrogen cyanamide insecticide in

routine forensic work. The detection of hydrogen cyanamide is achieved by high

performance thin layer chromatography (HPTLC) is the most simple, rapid and

reliable technique, usually used in forensic laboratory for detection and identification

of poison.2,3

3C.2 Literature Survey

Very few chromogenic reagents are plasticized as reported in the literature for

detection and identification of hydrogen cyanamide by TLC and HPTLC.4,5,6

In Present communication explain the use of sodium hydroxide followed by

combination of reagent containing sodium nitroprusside and potassium ferricyanide,

as selective reagent for detection and identification of hydrogen cyanamide by

HPTLC yielding an intense pink color spot. This reagent can be used for HPTLC

detection and determination of hydrogen cyanamide in biological material. Hydrogen

cyanamide on alkaline hydrolysis with sodium hydroxide followed by reagent

containing sodium nitroprusside and potassium ferrocyanide give pink complex at hRf

the detection limit is approximately 1µg.

P a g e | 89

Here, the hydrogen cyanamide being inorganic compound given formation of

hydroxylamine and sodium cyanide. The sodium nitroprusside and potassium

ferricyanide may form unstable complex K3[Fe(CN)5NO], which with hydroxylamine

form new complex of K3[Fe(H2NOH)(CN)(NO)] pink color.

3C.3 Present Work

The hydrogen cyanamide is a plant growth regulator, widely used in

agriculture. The present communication demonstrates the development of new

selective and specific chromogenic spray reagent for chromatographic detection and

identification of hydrogen cyanamide. Hydrogen cyanamide on alkaline hydrolysis

followed by spray of sodium nitroprusside and potassium ferricyanide furnish pink

color complex. Such pink color complex do not observed for other insecticide such as

carbamate, organophosphorous, organochlorine, and pyrethroid insecticides and even

constituents of viscera (amino acids, proteins, peptides, etc.) do not react with this

reagent. The detection limit of Hydrogen cyanamide is ca 0.5 µg.

3C.4 Experimental Section

Chemicals and Reagents

All reagents used were of analytical reagent grade. Distilled water was used

throughout the experiment:

I) Stock solution of hydrogen cyanamide (1 mg %) was prepared by

dissolving technical grade Hydrogen cyanamide (supplied by Degussa

group of pesticide division) in ethanol.

II) Sodium hydroxide solution (10% w/v) was prepared by dissolving 10

gm of sodium hydroxide in 100 ml distilled water.

III) Sodium nitroprusside solution (10% w/v) was prepared by dissolving

10 gm of sodium nitroprusside in 100 ml distilled water.

IV) Potassium ferricyanide solution (10% w/v) was prepared by dissolving

10 gm of potassium ferricyanide K3[Fe(CN)]6 in 100 ml distilled

water.

P a g e | 90

Extraction of Hydrogen cyanamide from Biological Materials


intestine, liver, spleen and kidney) containing hydrogen cyanamide, 10 gm

ammonium sulphate was added and were minced individually in an aqueous solution.

Each biological sample was extracted with a separating funnel with 150 ml of

chloroform: alcohol (7:3) mixture. The extract was transferred into an evaporating

dish. The aqueous phase was re-extracted two to three times with 50 ml of

chloroform: alcohol (7:3) mixture. The extracts were combined and the solvent was

evaporated at room temperature. The residue was dissolved in 2 ml of ethanol. A

known volume (10 �l) of the solution was spotted on HPTLC plates together with

standard solution of hydrogen cyanamide insecticides. The plates were developed and

sprayed with 10% sodium hydroxide solution followed by reagent containing sodium

nitroprusside and potassium ferricyanide, gives pink complex.

Chromatography

Chromatography was performed on 10 cm × 10 cm Silica gel F254 HPTLC


Germany). As 30 TLC applicator ,spotting volume 5 ml, spotting rate 10 s/ml was

used to apply standard hydrogen cyanamide stock solution, standard solution of other


methylparathion, phorate, chlorpyriphos, triazophos), organochloro insecticide

(endosulfan, DDT, BHC) carbamate insecticide (baygon, carbaryl, carbofuran),

pyrethroid insecticide (fenvalrate, cypermethrin, deltamethrin), oxidizing

proinsecticide (indoxicarb) and extract from visceral tissue spiked with hydrogen

cyanamide solution (5 ml stock solution). The HPTLC plate was then developed in

previously saturated HPTLC chamber with n-hexane: acetone (4:1) as mobile phase to

distance of 10 cm. After development the plate was removed from the chamber, dried

in air, and sprayed with 10% sodium hydroxide solution followed by reagent

cantaining sodium nitropruside and potassium ferricyanide successively. A typical

chromatogram is shown schematically in figure 1 and color photograph in figure 2.

The pink colored spot of the standard Hydrogen cyanamide and of Hydrogen

cyanamide extracted from viscera were extracted from the HPTLC plate with ethanol.

The visible spectrum of the extracted colored compound formed between hydrogen

P a g e | 91

cyanamide and reagent containing sodium nitropruside and potassium ferricyanide,

was recorded in ethanol by means of specord S-100 UV-Vis Spectrometer (Carl Zeiss

Jena).

Recovery Experiment

A 1 mg amount of Hydrogen cyanamide in ethanol was added to 50 gm of

minced visceral tissue, mixed them well and kept for a day. The insecticides were

then extracted separately with chloroform: alcohol (7:3) mixture as described under

extraction section. The solvent was evaporated at room temperature and the residues

were dissolved separately in 1.0 ml of ethanol. A 10 �l volume of solution was

spotted on activated thin layer plates together with 10 �l standard Hydrogen

cyanamide solutions containing known concentrations of 7, 8 and 9, 9.5 and 10 mg

per 10 ml in ethanol. The plates were then developed as described in the procedure

section and sprayed with 10% sodium hydroxide solution followed by reagent

containing sodium nitroprusside and potassium ferricyanide. The intensity of pink

color spots developed for the visceral extracts were compared with known standards

and were found to agree with the spot resulting from the hydrogen cyanamide of 9

mg/ 10 ml (average of 5 experiments). Hence the recovery for each insecticide is Ca.

90%.

Semi-quantitative determination Hydrogen Cyanamide

Hydrogen cyanamide was semi quantitatively determined in biological and or

non-biological materials by HPTLC with visual assessment. Hydrogen cyanamide

was extracted by chloroform: alcohol (7:3) from known amount of (Ca 50 gm)

biological sample such as viscera, blood, stomach-wash etc. and non-biological

materials such as grains, food materials, water sample, soil etc. as described under

‘extraction of hydrogen cyanamide’. The extract was then evaporated at room

temperature and the residue was dissolved in 1-2 ml ethanol. A 10 µl volume of this

extract was spotted on HPTLC plate together with 10 µl each of standard solution of

technical hydrogen cyanamide containing known concentration of 1, 5, 10, 15, 20,

25…. mg per 10 ml in ethanol. The plate was then developed as described under

‘chromatography procedure’ and sprayed with 10% sodium hydroxide solution

followed by reagent containing sodium nitroprusside and potassium ferrocyanide. The

P a g e | 92

intensity of pink colored spot was developed for the extract of unknown

concentration, was visually compared with those of known standards. From this the

amount of hydrogen cyanamide present in the total extract and that in the 100 gm of

viscera was determined. Since visual assessment, it is a semi-quantitative

determination.

3C.5 Results and Discussion

Hydrogen cyanamide is a plant growth regulator to promote bud break of

grape and other plants specially grown in tropical and subtropical where cold weather

in uneven. It has the same effect for fruit and potatoes in green house. Normally it can

advance 2-4 week for bud-break and 2-3 week for harvest. Cyanamide can also be

used as germicide, defoliator and herbicide.5

Hydrogen cyanamide did not concentrate in any tissue in the rat. There was no

indication of hydrogen cyanamide being converted to cyanide in vivo in rats or

human. The principal metabolite excreted in urine of laboratory animal and human

was N-acetylcyanamide. It is highly toxic orally, as well by inhalation or absorption

through skin. The acute oral toxicity for rats (LD 50) is 150 mg/kg (male & female).

The acute dermal toxicity for a rat is 742 mg/kg, severe poisoning will affect on

central nervous system. Hydrogen cyanamide is a widely used and extremely

dangerous insecticide. Its low cost and many applications will present a challenge to

users looking for safer alternatives, or measures, which will protect health1. Owing to

its high poisoning capacity, characterization of this insecticide from toxicological

point of is need for Forensic laboratory.

Hydrogen cyanamide on alkaline hydrolysis yields molecules each of which

reacts with potassium ferrocyanamide to yield pink color complex. Fig.1 shows the

possible reaction suggested for the formation of this compound. Pink color spot from

standard hydrogen cyanamide and hydrogen cyanamide from visceral extract were

observed at hRf 72 on HPTLC, whereas no spots were observed for other

organophosphorous, organochloro, carbamate and pyrethroid insecticide.

From recovery experiment it was observed that the intensity determined by

determined by densitometry of the pink spot developed for the visceral extract was

P a g e | 93

comparable with that of the spot corresponding to 9 mg hydrogen cyanamide per 10

ml ethanol (average from three experiments). Hence the recovery was Ca. 90%.

The absorption maximum of the UV-Visible spectrum of the pink color

compound extracted from the HPTLC plate was in the visible range at 544 nm.

Merits of the Reagent

The reagent reported is selective for hydrogen cyanamide among other

insecticide. This reagent do not give positive reaction with organochlorine insecticide

such as BHC, DDT and endosulfan; organophosphorous insecticides such as

dimethoate, phorate, metasystox, methyl parathion, ethyl parathion, thiometon,

quinalphos, dalf, chlorpyriphos, phosphamidon, dimecron and monocrotophos.

Pyrethroid insecticides such as cypermethrin, fenvalerate and deltamethrin and

narcotic substances such as morphine, heroin does not give positive reaction.

Constituents of viscera i.e. amino acids, protein, peptide, etc. which are co-extracted

with the insecticide do not interfere.

The reported regent for HPTLC detection and identification of hydrogen

cyanamide is simple, sensitive and can be routinely used for the detection and semi

quantitative determination of residual hydrogen cyanamide in biological material

investigated in forensic work.

Chemical Reaction:

P a g e | 94

0.72 No Spots Were Located


Fig 1


I) Standard Hydrogen cyanamide II) Hydrogen cyanamide from visceral extract III)

Blank viscera IV) Dimetholate V) Malathion VI) Phosphomidon (organophosphorous

insecticide) VII) Encosulfan (organochloro insecticide) VIII) Propoxur (carbamate

insceticide) IX) Cypermethrin (pyrethroid insecticide) X) Diazepam (drug).

P a g e | 95

Fig. 2 Color photograph of HPTLC Chromatogram obtained from:

I) Standard hydrogen cyanamide II) Hydrogen cyanamide from visceral extract III)

Blank viscera IV) Dimethoate v) Malathion VI) Phosphomidon (organophosphorous

insecticide) VII) Encosulfan (organochloro insecticide) VIII) Propoxur (carbamate

insecticide) IX) Cypermethrin (pyrethroid insecticide) X) Diazepam (drug).

P a g e | 96

3C.6 References

1. Hathaway, G. J.; Proctor, N. H.; Hughes, J. P., Proctor and Hughe’s chemical

hazards of the workplace, New York, Ed. 4, John Wile & Sons, Inc., 1996,

170.

2. Fritz, Feigl; Spot Test in Organic analysis, Ed. 7, Elsevier Publications Pvt.


3. Merck; The Merck Index, Merck Rathway, N. J., 1988, Ed. 13, 2713.

4. Egon, Stahl, Thin layer Chromatography, Indian Reprint, Ed. 2, 2005, 551.

5. Wanjie., Wanjie international Co. Ltd., HTML document, 2005.

6. Randerath, K., Thin-layer chromatography, Academic press, New York, 1965,

176.

7. Fritz, feigl, Spot Test in Inorganic analysis, Ed. 6., Elsevier Publication Pvt.


8. Sicilia, D., Analyst, 1999, 124, 615.

9. Deitrich, R. A.; Troxell, P. A., Biochem Parmacol, 1976, 25, 2733.

10. Kaur, P.; Upadhya, S.; Gupta,V. K., Analyst , 1987, 112, 1681.

11. Agrawal, V.; Cherian, L.; Gupta, V. K.; International Journal of

Environmental Analytical Chemistry, 1991, 45, 235.

12. Amlathe, S.; Gupta, V. K., Journal of Analytical Chemistry, 1990, 338, 615.

13. Rubio, S.; Gomer, A.; Valcarcel, M., Talenta, 1984, 31, 783.

14. Dolizine, T. W.; Espositro, G. G.; Rinehart, D. S., Analytical Chemistry, 1982,

54, 470.

15. Fielden, P. R.; Smith, S. J.; Analyst, 1986, 111, 695.

16. Endecrott, B. R., Journal of Analytical Toxicology, 1996, 20, 189.

17. Miralles, E., Analyst, 1997, 122, 553.

18. Sicilia, D.; Talenta, 1991, 38, 1147.

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