A New Chromogenic Spray Reagent for Detection and Identification of Monocrotophos Krishna V. Kulkarni*, Davanand B. Shinde, Dhananjay V. Mane, and Manchak V. Garad
Key Words Forensic science HPTLC Monocrotophos N-Methylacetoacetamide Monomethylamide
Summary
– diazotized sulfanilamide/sulfanilic acid [3, 4]; and
A new chromogenic spray reagent is described for chromatographic detection and identification of monocrotophos, an organophosphorus insecticide by HPTLC. Monocrotophos on alkaline hydrolysis yields one molecule each of O,O-dimethylphosphoric acid and Nmethylacetoacetamide. After acidification, N-methylacetoacetamide gives the enol form of monomethylamide which reacts with ferric ions to yield a purple complex. Other organophosphorus, organochlorine, pyrethroid, and carbamate insecticides and constituents of viscera (amino acids, proteins, peptides, etc.) do not react with this reagent. The detection limit for monocrotophos is ca 0.5 μg.
– chloranil reagent [4]. Other reagents not specific for monocrotophos have been reported for detection of organophosphorus compounds including monocrotophos [5, 6] and a spectrophotometric method for analysis of monocrotophos is also reported in the literature [7–10]. These reagents and methods are not selective, however, so highly selective and sensitive reagents are needed for analysis of monocrotophos. In this communication we discuss alkaline hydrolysis followed by use of acidic methanolic ferric chloride as spray reagent specifically for detection and identification of monocrotophos by HPTLC in forensic toxicology.
1 Introduction Homicidal, suicidal, and accidental use of insecticides in India is very common because of to their ready availability. Organophosphorus insecticides are widely applied in agriculture and are highly toxic to animals and humans. Monocrotophos (Azodrine, Nuvacron) is widely used for crop protection. During 2006 the Forensic Science Laboratory, Maharashtra, India, detected 663 cases of organophosphorus poisoning of which 275 were human poisoning by monocrotophos. The increasing number of biological samples for poison detection need versatile, sensitive, and selective reagents. Thin-layer chromatography (TLC) and high-performance thin-layer chromatography (HPTLC) are methods of choice because of their speed, low cost, and versatility. The literature contains several reports of chromogenic reagents for detection of monocrotophos by TLC, for example: – 50% potassium iodate in 1:1 ethanol–HCl [1]; – cupric acetate in dilute HCl followed by potassium iodide [2, 3];
K.V. Kulkarni and M.V. Garad, Regional Forensic Science Laboratory, Dindori Road, Panchvati, Nasik-431004, India; D.B. Shinde, Department of Chemical Technology, Dr Babasaheb Ambedkar Marathwada University, Aurangabad431001, India; and D.V. Mane, Department of Chemistry, Chatrapati Shivaji College, Omerga-413606, India. E-mail:
[email protected]
Journal of Planar Chromatography 22 (2009) 2, 133–135 0933-4173/$ 20.00 © Akadémiai Kiadó, Budapest
2 Experimental 2.1 Reagents and Chemicals
All reagents and chemicals used were of analytical reagent grade. Glass-distilled water was used throughout. A stock solution of monocrotophos (1 mg mL–1) was prepared by dissolving 13.5 mg 74% Technical grade monocrotophos (Hindustan Ciba–Geigy, Mumbai, India) in 10 mL ethanol. Sodium carbonate solution (20%) was prepared by dissolving 20 g sodium carbonate in 100 mL distilled water. Methanolic ferric chloride solution was prepared by dissolving 4 g ferric chloride in 100 mL AR-grade methanol then adding 5 mL glacial acetic acid. 2.2 Extraction of Biological Material
Ammonium sulfate (10 g) was added to visceral tissue (stomach, intestine, liver, spleen and kidney; 50 g) containing monocrotophos and the sample was individually minced in water. Each sample was then extracted with 150 mL ethyl acetate in a separating funnel, by shaking for 2–3 min. The ethyl acetate extract was transferred to an evaporating dish and the aqueous phase was re-extracted with ethyl acetate (2–3 × 50 mL). The combined ethyl acetate extract was evaporated to dryness by a steam DOI: 10.1556/JPC.22.2009.2.10
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Detection Reagent for Monocrotophos
The purple spots of the ferric chloride derivatives of monocrotophos standard and monocrotophos extracted from viscera were extracted from the HPTLC plate with ethanol. The visible spectrum of the extracted colored compound formed between monocrotophos and ferric chloride reagent was recorded in ethanol by means of a Specord S-100 UV–visible spectrometer (Carl Zeiss Jena). 2.4 Determination of Recovery
I
II
III
IV
V
VI
Figure 1 Chromatogram obtained from: (I) monocrotophos from extract of viscera; (II) monocrotophos standard; (III) malathion; (IV) dimethoate; (V) phosphomidon (all organophosphorus insecticides); (VI) blank viscera.
of air at room temperate. The residue was then dissolved in a minimum volume of ethanol (2 mL). This solution was treated as stock sample solution. 2.3 Chromatography
Chromatography was performed on 10 cm × 10 cm silica gel F254 HPTLC plates (Merck, Darmstadt, Germany; #1.05729 OB397077). A Desaga (Heidelberg, Germany) AS 30 TLC applicator was used to apply samples to the plates. The spotting volume was 5 μL and the spotting rate 10 s μL–1. The samples applied were: – standard stock solution of monocrotophos (1 mg mL–1); – standard solutions of other organophosphorus insecticides, for example dimethoate, phosphomidon, dichlorovas, malathion, ethyl parathion, methyl parathion, and phorate; – standard solutions of organochlorine insecticides, for example endosulfan, DDT, and BHC; – standard solutions of carbamate insecticides for example propoxur (baygon), carbaryl, carbofuran, and carbosulfan;
A sample of monocrotophos (10 mg) in ethanol was added to 50 g minced visceral tissue, mixed well, and left for a day. The biological material was then extracted with ethyl acetate as described above. The extract was evaporated to dryness and the residue was dissolved in 10 mL ethanol. This solution (10 μL) was spotted on an HPTLC plate with 10 μL of standard solutions of monocrotophos in ethanol of known concentration (5, 6, 7, 8, 9, 10 mg per 10 mL). The plate was then developed and sprayed with reagent as described above. The intensity of the purple spot developed for the visceral extract was visually comparable with the spot corresponding to 9 mg monocrotophos per 10 mL ethanol (average from three experiments); hence the recovery was ca 90%. 2.5 Semi-Quantitative Analysis of Monocrotophos
Monocrotophos was analyzed semi-quantitatively in biological and/or non-biological materials by HPTLC with visual assessment. Monocrotophos was extracted with ethyl acetate from a known amount (ca 50 g) of a biological sample, for example viscera, blood, stomach-wash, etc., or non-biological materials such as grain, food materials, water samples, soil etc., as described above. The extract was then evaporated at room temperature and the residue was dissolved in 1–2 mL ethanol. This extract (10 μL) was spotted on an HPTLC plate with 10 μL of standard solutions of monocrotophos in ethanol of known concentration (1, 5, 10, 15, 20, 25 … mg per 10 mL). The plate was then developed and sprayed with reagent as described above. The intensity of the spot developed for the extract of unknown concentration was compared visually with those from the standards. The amount of monocrotophos present in the total extract and that in 100 g viscera could thus be calculated. Because visual assessment was used, this is semi-quantitative determination.
– standard solutions of pyrethroid insecticides, for example fenvalerate, cypermethrin, and deltamethrin; – a standard solution of the neonicotinoid insecticide imidacloprid;
3 Results and Discussion
– a standard solution of the oxidizine insecticide indoxacarb; and
Monocrotophos is classified by the World Health Organization is highly hazardous and it has been responsible for deaths resulting from accidental or intentional exposure. It is highly toxic orally and by inhalation or absorption through the skin. The acute oral toxicity to rats (LD50) is 23 mg kg–1 (males) and 18 mg kg–1 (females).The acute dermal toxicity to rats is 364 mg kg–1. Severe poisoning affects the central nervous system. Like other organophosphorus pesticides, monocrotophos is potent cholinesterase inhibitor [11]. Monocrotophos 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 health [12]. Owing to its high poisoning capacity, characterization of this insecticide toxicologically is needed by forensic laboratories.
– solutions of monocrotophos extracted from viscera. Plates were then developed with chloroform–acetone 7:3 as mobile phase in an HPTLC chamber previously saturated with mobile phase vapor for 30 min. The development distance was 10 cm. After development the plate was removed, left to dry at room temperature for 10 min, and sprayed with 20% sodium carbonate then acidic methanolic ferric chloride reagent. Purple spots were observed at RF 0.45 for standard monocrotophos and monocrotophos extracted from viscera, whereas no spots were observed for organophosphorus, organochlorine, carbamate, and pyrethroid insecticides (Figure 1).
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Detection Reagent for Monocrotophos
A spectrophotometric method for analysis of monocrotophos by alkaline hydrolysis followed by reaction with acidic ferric chloride is reported in the literature [7]. It can, however, be applied for pure samples only. Extracts of biological samples contain proteins which interfere with spectrophotometry. If, with few modifications, if it is applied in HPTLC, however, it will be helpful for identification of monocrotophos in the presence of other pesticides with good sensitivity. Monocrotophos on alkaline hydrolysis yields one molecule each of O,O-dimethylphosphoric acid and N-methylacetoacetamide. On acidification, N-methylacetoacetamide gives the enol form of monomethylamide which reacts with ferric ions to yield a purple complex. N-methylacetoacetamide, which contains, reactive methylene group, is acidic in nature, and hence the enol form is more stable; the enol is also stabilized by intramolecular hydrogen bonding [6]. It is the enol form of the monomethylamide which produces color with the ferric ion. Figure 2 shows the reaction mechanism suggested for formation of this compound. Purple spots from monocrotophos standard and from monocrotophos extracted from viscera were observed at hRF 45. The trans form is less strongly absorbed in HPTLC than the cis form.
Reaction mechanism
Monocrotophos Alkaline hydrolysis
Dimethylphosphoric acid
Keto form
enol form of monomethylamide
trans form cis form Probable color complex with enol form
Figure 2 Suggested reaction mechanism.
No spots were observed for other organophosphorus, organochlorine, carbamate, and pyrethroid insecticides. From recovery experiments 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 monocrotophos per 10 mL ethanol (average from three experiments). Hence recovery was ca 90%. The absorption maximum of the UV–visible spectrum of the purple monocrotophos–ferric chloride compound extracted from the HPTLC plate was in the visible range at 544 nm. The reagent reported is selective for monocrotophos among other insecticides. Other organophosphorus insecticides (dimethoate, phosphomidon, dichlorvos, malathion, ethyl parathion, methyl parathion, phorate), organochlorine insecticides (endosulfan, DDT, and BHC), carbamate insecticides (propoxur (baygon), carbaryl, carbofuran, carbosulfan), pyrethroid insecticides (fenvalerate, cypermethrin and deltamethrin), the neonicotinoid insecticide imidacloprid, and the oxidizine insecticide indoxacarb, did not give colored spots. Other constituents of viscera, e.g. amino acids, peptides, etc., which are coextracted with the insecticides, do not interfere. The various chromogenic reagents described for screening of organophosphorus insecticides in biological materials by TLC or HPTLC failed to give a colored reaction with monocrotophos, hence the need to screen biological samples for the presence of monocrotophos in forensic toxicology. The reagent described here is sensitive and selective for monocrotophos and hence can be routinely used for the detection and semiquantitative analysis of residual monocrotophos in biological materials in forensic toxicology. Acknowledgments
The authors are grateful to Dr Mrs Krishnamurthy, Director of Forensic Science Laboratories, State of Maharashtra, Mumbai,
Journal of Planar Chromatography 22 (2009) 2
India, and Head of Department, Department of Chemistry, Dr B.A. Marathwada University, Aurangabad, India, and to Dr Raghunath Toche, Associate Professor, Department of Chemistry, KTHM College, Nashik for their keen interest and valuable guidance.
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