R.K.Jain, G.Jayashanker, Md.Afzal, S.Sudhakar, A.K.Srivastava & R.K.Sarin Development of qualitative method for detection of Lorazepam in chocolate R.K.Jain, G.Jayashanker, Md.Afzal, S.Sudhakar, A.K.Srivastava & R.K.Sarin Central Forensic Science Laboratory Directorate of Forensic Science Ministry of Home affairs Government of India Ramanthapur, Hyderabad

Introduction: Benzodiazepine group of drugs like diazepam, lorazepam, nitrazepam are prescribed for insomnia patients as sedative and tranquilizers. Drugs are frequently misused in cases of theft, robbery and even to commit rape in common public places like railways, bus and bus stand. The drugs are camouflaged in edible items to deceive the person to cheat. Forensic scientists often confronted with the problem of detection of the specific drug in the items like biscuit, chocolate, cool drink etc to confirm the drug used to commit the crime.

Materials and methods: All reagents used were of analytical grade. HPLC water, chloroform, acetone, methanol, n- hexane, Ammonia solution from E-Merck India. Lorazepam pure, de ionized double distilled water was used. TLC plate silica gel F 254 were obtained from E-Merck (Darmstadt, Germany) Suspected chocolates and blood were received from the I/O.

Standard solutions Stock solution: Accurately weighed 10 mg of pure Lorazepam dissolved in the methanol, solution made in 10 ml standard flask to a concentration of 1 mg/ml. Working solution: Dilute standard solutions to 1.0, 2.0, 5.0, 10.0  g/ml concentration with methanol from the above stock solution. These solutions have been stored in refrigerator.

Medical findings: The person was unconscious in railway station and later regained in hospital and narrated the scene to the police. Samples like blood were collected by the Medico legal Officer and sent to laboratory, along with the seized chocolates from the scene.

Methods 1) Extraction The chocolates (Coconut crunch 10 nos) were extracted with chloroform in basic condition. A small portion of blood sample was subjected for acidic ether and basic chloroform extraction after deproteinsation with 10% sodium tungstate solution and 1ml of 0.1 N. sulphuric acid. Shaken for ten minutes and then filtered. 10 ml of ether added into the extract in separating funnel, shake vigorously 5 min and separate and the aqueous phase made alkaline with 1 N ammonia solution and extracted with chloroform. The extraction was repeated thrice. Combine the portions of ether extracts and the chloroform extracts; evaporate at room temp and analyzed.

2) TLC: The TLC plate (silica gel F 254 ) of 0.25 mm thickness were used after activation at 105ºC for min and cooled in room temperature. The extract was spotted on TLC plate along with control samples of drug. Solvent system: Chloroform: Acetone (8:2), Rf Ethyl acetate (100), Rf Chloroform: Methanol: ammonia solution:: 90:10:1 Rf-0.60 Spray reagent: Dragon droff:- Orange color spot.

3) GC-MS. GC Column: Capillary column 5% phenyl methyl silicone, 30 mts, 0.25 mm id, 0.25 mm film thickness. GC-MS:- Auto system XL GC, Turbo mass, Auto sampler, 2  l injection, injector port 200ºC, interface 200ºC degree, EI mode, 70 eV, Oven programming: 70ºC hold 1 5ºC to 250ºC hold 1 min Carrier gas helium flow- 1ml/min

Mass spectra of Standard Lorazepam

Library match of Lorazapam

Mass Spectra of Sample compared with Library

4) FT-IR: Nicolet system with KBR pellet method was used for the determination of the drug in tablet and extract form. The IR spectra matched with the library spectra available in the system and conclusively identified lorazepam.

FT-IR Spectra of sample compared with Library

Results and Discussion: Lorazepam a benzodiazepine group drug is easily available in the medical stores. The extract was screened using modified TLC solvent system and confirmation with FTIR and GC-MS. The screening test indicated the presence of lorazepam. The Retention time was found to be 11.5 ±0.2, for 3 injections with mass fragmentation pattern at base peak 239, followed by 274, 302 of the drug lorazepam and compared with library spectra. The IR peaks at wave numbers 1685, 1149, 1317 matched with the library spectra.

In this work we developed a modified TLC method and GC-MS/FT-IR confirmation to detect lorazepam-using simple, rapid, effective and easily adaptable method for screening and qualitative purpose by forensic laboratories. The method was used for detection of lorazepam in body fluids encountered.

References: 1. ECG Clarke-isolation and identification of drugs, 1986, Vol-I, Modi’s medical jurisprudence and toxicology. 3. Pascal Kintz et al, Forensic Science International, 145(2004) British Pharmacopoeia, Pg no

We would like to thank  Director, CFSL, Hyderabad for providing facility and encouragement  Officers and staff of the Division for their co-operation during the study.