TLC DETECTION AND RECOVERY OF ORGANOPHOSPHORUS INSECTICIDE PHOSALONE FROM VISCERA USING COPPER (II) ACETYL ACETONATE.

TLC DETECTION AND RECOVERY OF ORGANOPHOSPHORUS INSECTICIDE PHOSALONE FROM VISCERA USING COPPER (II) ACETYL ACETONATE.

Arun G. Bhoi

Arun’s Institute of  Forensic Sciences, Research and Education, Pune -411 028 (India).

Email : arun.bhoi@gmail.com

(e-J. Foren. Crime Inv. 2013, 9, 2, Art. 1)

Received on : 10^th Jan. 2013.

Accepted on : 16^th Jan. 2013.

 

ABSTRACT

Phosalone (Zolone) has replaced many of the chlorine containing insecticides used for controlling pests on agricultural crops. A number of reagents are reported for the detection and determination of organophosphorus insecticides but these are not specific for the phosalone.  The use of copper(II)   acetylacetonate as a specific spray reagent is reported here for the detection of phosalone on TLC. Phosalone after elution on TLC plates is hydrolysed using sodium hydroxide solution and then sprayed with copper acetylacetonate reagent. Phosalone appears as a brown spot. Other insecticides do not interfere in the detection. The detection limit for phosalone is ca. 2μg. The recovery of phosalone from viscera was found to be 85%.

KEY WORDS : Phosalone, Zolone, insecticide, thin-layer chromatography, TLC, hydrolysis, copper acetylacetonate, recovery, viscera, forensic.

INTRODUCTION

Phosalone (Zolone; phosphorodithioic acid, S-ester with 6-choloro-3-(mercaptomethyl)-2-benzoxazolinone) is a chlorine containing dithiophosphoric acid derivative insecticide. LD₅₀ for phosalone is 120-175 mg/kg orally in rats [1].  It has higher chemical stability and less toxicity for animals as compared to many of the chlorine and phosphorous containing insecticides. Therefore, phosalone has been preferentially used for the protection of seeds, seedlings and agriculture crops. Unfortunately, this has also been misused in many of the homicidal and suicidal cases.

Number of gas chromatographic [2,3], spectrophotometric [4-6] and thin-layer chromatographic [7,8] methods are described in the literature for the detection of phosalone. However, these methods are tedious and need a well equipped instrumental laboratory. The use of copper (II) acetylacetonate as a spray reagent is described here for specific detection of phosalone on silica gel G coated TLC plates.

EXPERIMENTAL

Reagents:

All reagents used were of analytical reagent grade. Glass distilled water  and 95% ethyl alcohol were used throughout.

Sodium hydroxide solution (10%):    10g of NaOH was dissolved in small quantity of distilled water and made  up to 100 ml.

Copper (II) acetylacetone solution (0.25%): 250 mg of copper (II) acetylacetonate was dissolved in chloroform and made up to 100 ml.

Phosalone Standard Solution (1mg/ml) : 10 mg of commercial grade phosalone (99%) was dissolved in 9.9 ml of 95% ethyl alcohol.

PROCEDURE

A standard glass TLC plate of the size of 10cm x 20cm was coated with a slurry of silica gel G in distilled water (1:2 ) to a thickness of 0.25mm. Coated plates were dried in air  and activated at 110 degree centigrade for minimum 1 h. Activated plates were cooled. A 2 μl quantity of a standard solution of phosalone (1mg/1ml) was spotted on TLC plate. The plate was then developed in a pre-saturated flat bottom TLC chamber using n-hexane and acetone (8:2, v/v) as a developing solvent mixture. After the solvent had travelled 10 cm distance on the TLC plate it was taken out, air dried and sprayed with 10% of sodium hydroxide solution and subsequently with 0.25% copper (II) acetylacetonate reagent solution.  A prominent brown spot was visualized at R_f – value 0.60 on the TLC plate for phosalone.

RECOVERY EXPERIMENT OF PHOSALONE FROM VISCERAL MATERIAL

An standard alcoholic solution (1 ml) equivalent to 1 mg of phosalone was added to 50 g  of minced visceral material (stomach – intestine with contents and liver, spleen, kidneys, lungs), homogenized and was kept in situ for about 72 hours. For the extraction of phosalone, the contents were soaked in 100 ml of diethyl ether at least for 15 minutes with intermittent stirring. The ether layer was separated out using separating funnel. The visceral material was re-extracted using 50 ml of diethyl ether, twice. All the ether extracts were combined in a steel capsule and the solvent was allowed to get evaporated at room temperature. The residue (phosalone) left was re-dissolved in 1 ml of an ethanol. A 10 micro liter quantity of it was spotted on activated TLC plate along with 10 micro liter quantity of each of the standard phosalone solutions containing known concentrations of 7.5, 8.0, 8.5, 9.0, 9.5 and 10 mg per 10 ml in ethanol. Te plate was then developed as described in the procedure section and sprayed with 10% sodium hydroxide solution and copper (II) acetylacetonate reagent. The intensity of the brown spot of the residue obtained from visceral material visually agreed with the spot resulting from a standard solution containing 8.5 mg of phosalone per 10 ml. Thus the recovery for phosalone was ca. 85%.

RESULTS AND DISCUSSION

Alkaline sodium hydroxide solution when sprayed on TLC plate it rapidly hydrolyses Phosalone producing sodium salt of 6-chloro-benzenexaazolone, diethyl dithiophosphoric  acid and formaldehyde. It is postulated that the sodium salt formed, in turn, reacts with copper acetylacetonate and produces the brown coloured complex. The reagent used is selective for phosalone. The detection limit is calculated as 2 microgram. Other organophosphorus insecticides such as thimet, phosphamidon, fenitrothion, nuvan, monocrotophos, ekalux, solvirex and organochlorine insecticides such as, BHC, DDT, endosulfan, aldrin, endrin, dieldrin did not give any coloured spots. Dimethoate, malathion, methyl parathion and pyrethroids insecticides did not interfere by way of colour and R_f – values. Carbamate insecticides have been studied, separately, with the same reagents.

Since many of the reagents described for the detection of the insecticides did not respond to phosalone, the reagent studied here can be used for the detection and semi-quantitative determination of phosalone in the extracts of biological material (viscera) submitted in forensic laboratories and commercial samples.

AKNOWLEDGEMENTS

Author’s thanks are due to Prof. (Dr.) S. B. Kulkarni, Head (Ex.), Dept.  Of Chemistry, University of Pune, Maharashtra (India) and the Director, Directorate of Forensic Science Labs. M. S., Mumbai (India).

REFERENCES

[1] Martha W., The Merck Index, An Encyclopedia of Chemicals and Drugs, Merck and Co., Inc., USA, 1976, p-955.

[2] Spenser W. F., Iwata Y., Kilgore W. W. and Knaak J. B.; Bull. Environ. Contam. Toxicol., 1977, 18, 656.

[3] Their H. P., Stijve T. and Discrense H.; Lebenmittelchem. Gerichtl. Chem., 1989, 43, 121 (Ger.).

[4] Syoyama M., Miyachi Y. and Sakakibara J.; Bunseki Kagaku, 1976, 25, 179 (Japanese).

[5] Novikova K. F. and Mel’fser F. R.; Zh. Analit. Khim., 1971, 26, 1651 (Russ.).

[6] Makarova S. V., Khenelnitsakaya V. N. and Eliseeva M. A.; Zar. Lab., 1974, 40, 792 (Russ.).

[7] Rodinova T. V., Belskaya G. G., Ivanov V. M. and Makarova S. V.; Zh. Anal. Khim.; 1987, 42, 1125 (Russ.).

[8] Sevelkar M. T., Patil V. B. and Katkar H. N.; J. Assoc. Off. Ana. Chem., 1991, 74, 545.

 

 

ACCIDENTAL LIGATURE STRANGULATION (ALS) : SURVIVAL OF A MINOR

ACCIDENTAL LIGATURE STRANGULATION : SURVIVAL OF A MINOR

Vilas P. Wadile (M. D.), Sarika V. Wadile (MBBS, DGO)

Jeevak Hospital, Dhule, M. S. (India)

Deepali D. More (MBBS, DCH)

More Hospital, Dapoli -Ratnagiri, M. S. (India)

Arun G. Bhoi

Arun’s Institute of  Forensic Sciences, Research and Education, Pune -411 028 (India).

Email : arun.bhoi@gmail.com

(e-J. Foren. Crime Inv. 2013, 9, 1, Art. 1)

Received on : 12^th Dec. 2012.

Accepted on : 16^th Dec. 2012.

ABSTRACT

Accidental ligature strangulation (ALS) is uncommon, as also, survival of a victim in such cases is rare. The ALS is reported here which occurred to a minor girl while travelling on a M/cycle, however, she survived through it. The prominent ligature mark was observed on the backside of her neck. Observations revealed the presence of the petechiae, too.

KEY WORDS

Accident, ligature, strangulation, petechiae, forensic.

INTRODUCTION

Few cases of accidental ligature strangulation (ALS) are reported in the literature. Kohli et. al. [1] and Aggarwal et. al. [2] have discussed accidental strangulation resulted by chunni in a rickshaw. ALS occurred by massage device has been reported by Deidikar R. D. [3]. Gupta et. al. [4] have reported the case of ALS by sari in M/cycle. ALS by crop thrasher [5], electric grinder [6] and ironing machine [7] are also explored in the literature. Strangulation resulted in domestic violence is reported by Richard [8]. Suicidal ALS is explained by Li D. R. et. al. [9]. Human hair has also, stood the cause of strangulation [10]. The case of incomplete strangulation [11] is explained by Amit et. al. Saurabh and others [12] have reported the survival in an accidental strangulation.

We report here the survival of a minor girl went through a  strangle in incomplete accidental ligature strangulation.

HISTORY

A father was carrying his daughter aged about 10 years on the M/cycle to a nearby place. Being winter days the girl had tied headscarf around her head. She was sitting backside of her father on the M/cycle. On the way of destination the M/cycle got a jerk and bumped heavily by which the girl fell down as she was sitting comfortably behind her father on the M/cycle. While falling down the girl’s headscarf got entangled in a rear wheel of M/cycle and she got strangled. By the motion of the wheel the headscarf got pulled heavily resulting in strangling of  girl’s neck. Father immediately stopped the M/cycle and tried to remove the headscarf from the wheel forcibly to make his daughter free from the trap.

EXPERIMENTAL

PROCEDURE : OBSERVATIONS

Strangulation leads to the constriction of a body organ or duct which leads to ligature and the stoppage of the body fluids like blood. Usually ligature marks (bruising of skin) are caused by materials like ropes, wires, cords, garrote, clothes like sari, chunni and scarf involved in the act of strangulation. In severe cases bleeding, stoppage of respiration and blood flow is noticed. Injuries to head, damage to cartilage may also, be seen.

In the subject matter presented here, the patient’s father briefed that she showed slight signs of suffocation when the incidence took place. Examination of a patient showed presence of a prominent ligature mark to the backside of the neck with peeling of skin (epithelial layer) exposing inner muscular tissues, there from (Fig.1).

Figure 1: Prominent ligature mark to the neck back with peeled skin and exposed tissues.

Redness was also, seen around the neck periphery, on the cheek and face. Petechiae (redness due to blood) was seen in patient’s eyes. It was very much prominent in the left eye as in Fig.2.

Figure 2:  Petechiae seen in patient’s eyes with prominence in the left eye.

The ligature was found slightly extending to the frontal throat portion and curved, too. (Fig.3).

Figure 3:   Ligature mark curved and extending towards throat.

The patient examined, herein, did not show swelling nearby ligature, head injuries, bleeding through ears. ENT and surgical findings were normal. No dislocation/damage was noticed to the cartilage bone.

TREATMENT  

Patient was treated with anti-inflammatory and antibiotic drugs, only and  showed the speedy recovery and found very normal in a week’s time.

RESULTS, DISCUSSION AND CONCLUSION

The slight suffocation noticed by patient’s father must have been resulted by the partial constriction of a wind pipe. The mark produced on neck back is seen prominently as ligature strangulation had got enhanced by abrasion – friction resulted by headscarf while the father tried to remove it forcibly from around the neck of the patient girl. Further, the pressure generated at ligature strangulation site had suppressed the blood flow around neck periphery resulting in redness on the face, cheek and neck. Petechiae (redness due to blood) seen in patient’s eyes was also, the result of the same.

It is ascertained that the falling of a girl in head down – face up position created deep abrasion – ligature mark encircling the back portion of the neck as in Fig.1. The ligature found on the neck back slightly extending to the frontal throat portion and curved (Fig.3) is definitely rare and is suggestive of accidental ligature strangulation.

The patient’s immediate survival is  because of immediate steps taken by her conscious father to stop the speedy M/cycle and to make the girl free from the entangled scarf in the M/cycle by removing it out from the rear wheel which restricted the stopping of respiration and blockage of blood flow.

The proper treatment of anti-inflammatory and antibiotic drugs given to the minor brought her to the normalcy in a short span of a time. Thus, the risk of accidental strangulation which could have lead to fatal and complete strangulation was turned in the saving of the life of a minor girl.

REFERENCES

[1]. Kohli A., Verma S. K. and Agarwal B. B., Forensic Sci. Int. 1996 March 5, 78, 1, 7-11.

[2]. Aggarwal N. K. and Agarwal B. B.,  Med. Sci. Law 1988 July, 38, 3, 263-5.

[3]. Deidikar R. D., Am. J. Forensic Med. Pathol. 1999 Dec., 20, 4, 354-6.

[4]. Gupta B. D., Jain C. B. and Datta R. B., Med. Sci. Law 2004 Oct., 44, 4, 359-64.

[5]. Dixit P. G. and Kukde H. G., Journal of Forensic and Legal Medicine, 2008 May, 15, 4, 263-5.

[6]. Shetty Mahabalesh and Shetty Suresh B., J. Clinical Forensic Medicine 2006 April, 13, 3, 748-750.

[7]. Dogan K. H., Demirci S., Gunaydin G. and bunken B., J. Forensic Sci. 2010 Jan, 55, 1, 251 -3.

[8]. Investigating Domestic Violence Strangultion, Richard, Bluesheepdog.com, 2007 Nov. 9.

[9]. Zhao D., Ishikawa T., Quan L., Li D. R., Michine T and Maeda H., Leg. Med (Tokyo) Nov. 2008, 10, 6, 310-15.

[10]. Milkovich S. M., Owens J, Stool D., Chen X. and Beran M., Int. J. Pediatr. Otorhinolaryngol. 2005Dec., 69, 12, 1621-8.

[11]. Agarwal Amit, Ninave Sudhir, Shrivastav Tripti, Sunkara Anil and Agarwal Sachin, Journal of Forensic and Legal Medicine, 2009 Aug., 16, 6, 350-1.

[12]. Chattopadhyay Saurabh and Pal Indranil, Journal of Forensic and Legal Medicine, 2008 Jan., 15, 1, 53-55.