USE OF SIMPLIFIED TLC FOR THE DETECTION OF OXAZEPAM AND DIAZEPAM TRANQUILIZERS IN EATABLES

USE  OF  SIMPLIFIED  TLC  FOR  THE  DETECTION  OF  OXAZEPAM  AND  DIAZEPAM  TRANQUILIZERS  IN EATABLES

Arun G.  Bhoi

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

 Email : arun.bhoi@gmail.com

C. S. KAMAT

 Regional Forensic Science Laboratory, Nasik (India)

 

( e-J. Foren. Crime Inv. 2011, 2, 1,  Art. 1, 9^th June 2011)

 

 ABSTRACT

               Benzodiazepines like oxazepam, diazepam are malafidely used by the opportunist thieves to steal the belongings of the pilgrims and/or travelers by mixing it homogeneously with eatables. The method of extracting these drugs from ammonical mass of eatables using chloroform is described, here in, with the aid of easy solvent system and iodine-potassium iodide (alc.) and copper sulphate (aq.) solutions. Oxazepam, diazepam, lorazepam, nitrazepam give brown spots at respective R_f-values with this reagent. The detection limit is 1μg.

KEY WORDS : Oxazepam, diazepam, lorazepam, nitrazepam, tranquilizer, sweets, extraction, tlc, iodine, copper sulphate.

INTRODUCTION

             In an outskirt areas of the holy places the complaints of thefts are raised by the pilgrims. Also, the police receive complaints of lifting the belongings of the people while on journey. To succeed in stealing the money and valuables, a specific and wise modus operandi is exercised by the thieves. They distribute, among the devotees / travelers, sweets, cream biscuits, eatables such as pedhas, burfi, kachori etc. which are premixed with benzodiazepines the sleep inducing drugs, under the farce of “Mahaprasada of the Lord”. The benzodiazepines produce sedation and unconsciousness in 5-10 min.(1). The tranquilizers such as oxazepam and diazepam are preferred for this purpose. Sometimes these have been mixed with drinks like tea, coffee, soft drinks and served to devotees/ pilgrims. As they chew – swallow the eatables, slowly and slowly, enter in the state of drowsiness. Taking this as the best occasion their valuable belongings are stolen by the opportunist thieves. Many methods and techniques have already been reported for the detection and estimation of the tranquilizers (2,3,4). However, a simple technique is described here for the extraction of oxazepam and diazepam tranquilizers from the eatables and their detection on Silica Gel G coated TLC plates using iodine-potassium iodide (alc.) and copper sulphate (aq.) solutions, as spray reagents.

EXPERIMENTAL

Equipments:

1. Standard thin-layer chromatographic equipment
2. Filtration assembly
3. Separating funnels (500 ml cap.)

Reagents :

 (A) Iodine – Potassium iodide alcoholic reagent:

10 g of Iodine and 10 g potassium iodide dissolved in 100 ml of  95 % ethanol.

(B) 10 % Copper sulphate aq. solution : 10 g of copper suphate was dissolved in distilled water and was made up to 100 ml.

All reagents and solvents used were of analytical reagent grade.

Standard Solutions :

              Standard solutions of  oxazepam, diazepam, lorazepam and nitrazedpam drugs procured from market were prepared by dissolving weight equivalent to 10 mg of respective active ingredients in 10 ml of 95% ethyl alcohol separately.

Preparation of Synthetic samples :

Powdered masses of tablets/capsules constiturting 10 mg of oxazepam diazepam were homogeneously mixed with 10 gm of sweets (pedhas) separately, as also, with 10 g of spicy/chili powder preparations, taken in separate big sized, wide mouth test tubes.

Procedure:

Extraction :

25 ml of diluted ammonia solution (1 part 25 % ammonia + 1 part distilled water) was added to all the synthetic samples and were allowed to stand for at least three hours with intermittent shaking and swirling and then filtered through the glass wool, separately. The residues left on the glass wool were washed at least thrice with 10 ml of dilute ammonia (5%), every time. The filtrates of two different synthetic samples were transferred to separate separating funnels, then mixed and extracted with 50 ml of solvent chloroform. The extraction process was repeated thrice, so as to achieve the maximum recovery of the tranquilizers of interest. The solvent from extracts was allowed to get evaporated and the concentrated aliquots (10 ml) were used for further analytical purposes.

Thin-layer Chromatography :

             The glass plates (20 cm x 20 cm) were coated with slurry of silica Gel G with water (1:2) to a thickness of 0.25 mm. The plates were air dried at room temperature and then activated at 105⁰ C – 110⁰ C at least for half an hour. Aliquots of each of the synthetic samples were spotted on the activated TLC plate ( equivalent to 5μg). The reference tranquilizers oxazepam and diazepam along with additional control samples of lorazepam and nitrazepam (hyphotic), 5 μg  each were also spotted on TLC plate. The plates were developed in a pre-saturated chamber containing solvent system Benzene, Acetonitrile , Methanol (8:1:1 v/v). After the solvent front had travelled 10 cm distance from the original spotting line, the plate was taken out from the chamber and dried in air. The spots were visualized on TLC by spraying with (A) Iodine- Potassium iodide reagent fllowed by (B) 10 % copper sulphate aq. solution.

RESULTS AND DISCUSSION

             The brown spots were observed at specific R_f-values for oxazepam, diazepam, lorazepam and nitrazepam, immediately after spraying the above reagents. The corresponding R_f x 100 values are cited in the Table – 1 given below. The reagents has a sensitivity to detect the oxazepam and diazepam to the level of 1 μg,  where as routinely used Dragendorff’s reagent does not responds to this limit.

 TABLE- 1 :    Showing R_f x 100 values of the tranquilizers / hypnotics studied .

Sr. No.	Drug	Rf x 100 value
1	Oxazepam	40
2	Diazepam	85
3	Lorazepam	45 (*)
4	Nitrazepam	65 (*)

(*) Synthetic samples are not studied being its non usability for the purpose mentioned above .

Furthermore, no interferences were noticed either by additives or constituting substances from the eatables. Though this method has also been proved helpful to detect other tranquilizers/hypnotics such as lorazepam, nitrazepam, no synthetic samples of these were prepared with eatables for the detailed study looking at their remote possibility of use for the purpose mentioned, herein. The spray reagent described here can also be used for the detection of tranquilizers in the biological materials referred to Forensic Laboratory.

ACKNOWLEDGEMENT

The authors are grateful to Director, Directorate of Forensic Science Laboratories, M.S., Bombay-400 098.

REFERENCES 

 (1) Tripathi K. D., Essentials of Medical Pharmacology, Third Edition, Jaypee Brothers, Medical Publishers Pvt. Ltd., New Delhi, India, 1994, p-3,  .

(2) E.G.C. Clarke, Isolation And Identification of Drugs in Pharmaceuticals, body fluids and post mortem material, First Edition 1969, The Pharmaceutical Press, LONDON, Reprint – 1974, p – 294, 443, 458.

(3) Fartushnyi, A.F., Muzhanovskii, Sukhin, Sedov, Kvasov,E.B. Farm. Zh. (Kiev), Sep-Oct, 1986(5),47-50 (Ukrain).

(4)  Yan, E.; Jin, X. (  Yiyao Gongye, 1986,17 (5), 21-23 (ch).