A new chromogenic spray reagent for detection of

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46. Global Journal of Analytical Chemistry | Volume 2 | Issue 1 | January 2011 ... advance bud-break by 2-4 weeks and harvest by. 2-3 weeks. Hydrogen .... and M. Bochmann, Advance Inorganic. Chemistry, John Wiley & Sons, Inc, 6th ed.,.
GLOBAL JOURNAL OF ANALYTICAL CHEMISTRY

A new chromogenic spray reagent for detection of hydrogen cyanamide Krishna V. Kulkarnia,*, Davanand B. Shindeb, Dhananjay V. Manec, Raghunath B. Toched,*, Manchk V.Garada a

b

Directorate of Forensic Science Laboratory, Vidyanagari, Kalina, Santacruz (E), Mumbai, India Department of Chemical Technology, Dr Babasaheb Ambedkar Marathwada University, Aurangabad, India c Department of Chemistry, Chatrapati Shivaji College, Omerga, India d Department of Chemistry, K.T.H.M. College, Nashik, India *

Authors for correspondence: Krishna V. Kulkarni, email: [email protected] Raghunath B. Toche, email: [email protected] Received 23 Sep 2010; Accepted 8 Nov 2010; Available Online 24 Jan 2011

Abstract A new inexpensive chromogenic spray reagent for HPTLC was developed for the detection of hydrogen cyanamide in biological materials. Hydrogen cyanamide in alkaline medium followed by the spray of sodium nitroprusside and potassium ferricyanide gives pink color complex. Such pink color complex is not observed with other insecticides such as carbamate, organophosphorus, organochlorine, and pyrethroid insecticides and even the constituents of viscera that consist of amino acids, proteins. The detection limit of hydrogen cyanamide is ca. 0.5 g. Keywords: Chromogenic reagent; Forensic science; HPTLC; Spray reagent; Hydrogen cyanamide

1. Introduction Hydrogen cyanamide (H2NCN) is a plant growth regulator to promote bud-break of grape and other plants specially grown in tropical and subtropical regions where cold weather is unavailable. It has the same effect for fruits and potatoes in green house. Normally, it can advance bud-break by 2-4 weeks and harvest by 2-3 weeks. Hydrogen cyanamide can also be used as germicide, defoliator and herbicide. It is highly toxic having adverse health effects on contact including severe irritation and ulceration of the eyes, skin, and respiratory tract. This also inhibits aldehyde dehydrogenase and can produce acetaldehyde syndrome such as vomiting, parasympathetic hyperactivity, dyspnea, hypotension and confusion, when exposure coincides with alcohol [1-3]. Nashik region (Maharashtra State, India) is famous for grape production. Farmers in this region regularly use hydrogen cyanamide to get high crop yield. The regional forensic

laboratory, Nashik receives a considerable number of poisoning cases due to hydrogen cyanamide. The HPTLC (High Performance Thin Layer Chromatography) is a simple, rapid and reliable technique usually used in forensic laboratory for the detection of poison. In literature, few chromogenic reagents such as Dragandroff`s reagent, Iodoplatinate reagent are used for the detection of hydrogen cyanamide using TLC and HPTLC techniques [4-8]. In the present communication, we report an inexpensive, non-toxic chromogenic spray reagent for the rapid detection of hydrogen cyanamide in the viscera of the suicide victims. 2. Experimental 2.1. Chemicals and Reagents All reagents used were of analytical reagent grade. Distilled water was used throughout the experiment. The stock solution of hydrogen cyanamide (1% w/v) was prepared by

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GLOBAL JOURNAL OF ANALYTICAL CHEMISTRY dissolving technical grade hydrogen cyanamide (Degussa Corp.) in ethanol. Sodium hydroxide solution (10% w/v) was prepared by dissolving 10 g of sodium hydroxide in 100 ml distilled water. Sodium nitroprusside solution (10% w/v) was prepared by dissolving 10 g of sodium nitroprusside in 100 ml distilled water. Potassium ferricyanide solution (10% w/v) was prepared by dissolving 10 g of potassium ferricyanide K3[Fe(CN)6] in 100 ml distilled water. 2.2. Extraction of hydrogen cyanamide from biological materials In a portion of about 100 g of each of various biological tissues (stomach, intestine, liver, spleen and kidney) contaminated with hydrogen cyanamide, 10 g of ammonium sulphate was added and 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 evaporating dish. The aqueous phase was re-extracted for 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 and a known volume (10 μl) of the solution was spotted on HPTLC plates together with standard solution of hydrogen cyanamide insecticide. The plates were developed and sprayed with 10% sodium hydroxide solution followed by reagent containing sodium nitroprusside and potassium ferricyanide, which gives pink complex. 2.3. Chromatography Chromatography was performed on 10 cm × 10 cm Silica gel F254 HPTLC plate (Merck, Darmstadt, Germany #1.05729 OB397077). Desaga (Heidelberg, Germany) AS30 TLC applicator, spotting volume 5 ml and spotting rate 10 s ml-1 were used to apply standard hydrogen cyanamide stock solution. Standard solution of other organophosphorus insecticides (dimethoate, phosphomidon, dichlorvos, malathion, parathion, methyl parathion, phorate, chlorpyriphos, triazophos), organochloro insecticides (endosulfan, DDT, BHC), carbamate insecticides (baygon, carbaryl, carbofuran), pyrethroid insecticides (fenvalerate, cypermethrin, deltamethrin), oxidizing proinsecticide (Indoxicarb) and extract from visceral tissue spiked with hydrogen cyanamide solution were individually used. The HPTLC plate was then developed in previously saturated

Figure 1. HPTLC chromatogram.

HPTLC chamber with n-hexane : acetone (4:1) as mobile phase to a 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 containing sodium nitroprusside and potassium ferricyanide. A typical chromatogram is shown in Figure 1. The pink color spots of the standard hydrogen cyanamide and of hydrogen cyanamide extracted from viscera were observed on the HPTLC plates. The visible spectrum of the extracted colored compound formed between hydrogen cyanamide and reagent containing sodium nitroprusside and potassium ferricyanide, was recorded in ethanol using specord S-100 UV- Vis Spectrometer (Carl Zeiss Jena). 3. Results and Discussion On alkaline hydrolysis, hydrogen cyanamide reacts with sodium nitroprusside potassium ferricyanide to yield pink color complex. Scheme 1 shows the possible reaction suggested for the formation of this compound. Pink color spot of standard hydrogen cyanamide and hydrogen cyanamide from visceral extract were observed at hRf 72 on HPTLC, whereas no spots were observed for other organophosphorus, organochloro, carbamate and pyrethroid insecticides. The interaction of linear NO complexes with nucleophiles such as OH-, OR, SR-, NH2R, and weak nucleophiles e.g. H2NCN in basic medium usually occurs at the N atom but attacks

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GLOBAL JOURNAL OF ANALYTICAL CHEMISTRY

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Scheme 1.

on O may occur [9]. A classic example is that of nitroprusside with hydroxyl ion to give a nitro complex or with H2NCN in basic medium to give hydrogen cyanamide complex. These types of complexes with thiols give red coloration, while with NHR (R=CN) give pink color complexes [10] at hRF with the detection limit is approximately 1µg pink coloration. Potassium ferricyanide acts as reducing agent in the reaction. 3.1. Recovery Experiment 1.0 mg amount of hydrogen cyanamide in ethanol was added to 50 g of minced visceral tissue, mixed well and kept for a day. The insecticides were then extracted separately with chloroform : alcohol (7:3) mixture as described under section 2.2. The solvent was evaporated at room temperature and the residues were dissolved separately in 1.0 ml of ethanol. 10 μl of the solution was spotted on activated thin layer plates together with 10 μl standard hydrogen cyanamide 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 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%.

3.2. Semi-quantitative determination of hydrogen cyanamide Hydrogen cyanamide was extracted by chloroform : alcohol (7:3) mixure from known amount of (ca 50.0g) biological sample such as viscera, blood, stomach-wash and non-biological materials such as grains, food materials, water sample, soil as described under section 2.2. The extract was then evaporated at room temperature and the residue was dissolved in 2 ml ethanol. 10 l of this extract was spotted on HPTLC plate together with 10 l each of standard solutions of hydrogen cyanamide containing known concentrations of 1, 5, 10, 15, 20, 25…..mg per 10 ml in ethanol. The plate was then developed and sprayed with 10% sodium hydroxide solution followed by reagent containing sodium nitroprusside and potassium ferricyanide. The intensity of pink colored spot developed for the extract of unknown concentration, was visually compared with those of known standards to approximately determine hydrogen cyanamide present in the extract. 4. Conclusions The reagent reported is selective for hydrogen cyanamide among other insecticides. This reagent does not give positive reaction with organochlorine insecticides (such as BHC, DDT and endosulfan), organophosphorus insecticides (such as dimethoate, phorate, metasystox, methyl parathion, ethyl parathion, thiometon, quinalphos, dalf, chlorpyriphos, phosphamidon, dimecron and monocrotophos), Pyrethroid insecticides (such as cypermethrin, fenvalerate

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GLOBAL JOURNAL OF ANALYTICAL CHEMISTRY and deltamethrin) and narcotic substances (such as morphine, heroin). The constituents of viscera such as amino acids and proteins, which are co-extracted with the insecticide, do not interfere. The reported regent for HPTLC detection of hydrogen cyanamide can be routinely used for the detection and semiquantitative determination of residual hydrogen cyanamide in biological materials investigated in forensic work. Acknowledgement The authors are grateful to Dr. Mrs. Krishnamurty, Ex-Director of Forensic Science Laboratories, State of Maharashtra, Mumbai and HOD, Department of Chemistry, Dr. B. A. Marathwada University, Aurangabad, India for her keen interest and valuable guidance. References 1.

Fritz Feigl, Spot Test in Organic analysis, 7th Ed., Elsevier (1983) pp. 592-593. 2. Hathaway GJ, Proctor NH, Hughes JP, Proctor and Hughes' chemical hazards of the workplace, 4th ed., John Wile & Sons, New York (1996). 3. The Merck Index, 13th ed., Merck, Rathway, NJ (1988). 4. Fritz Feigl, Spot Test in Organic analysis, 7th Ed., Elsevier (1983) pp. 476-477. 5. Egon Stahl, Thin layer Chromatography, 2nd ed., (Indian Reprint) (2005). 6. Randerath, K., Thin-layer chromatography, Academic press, New York (1965). 7. Deitrich R.A, Troxell P.A., Biochem. Parmacol., 25 (1976) 2733. 8. P. Kaur, S. Upadhya and V.K. Gupta, Analyst, 112 (1987) 1681. 9. H. Tanaka et al., Inorg. Chem., 31 (1992) 1971. 10. F. A. Cotton, G. Wilkinson, C. A. Murillo and M. Bochmann, Advance Inorganic Chemistry, John Wiley & Sons, Inc, 6th ed., (1999) pp. 326-346.

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