Download Development of qualitative method for detection of

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-500013
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, nhexane, Ammonia solution from E-Merck India.
Lorazepam pure, de ionized double distilled water
was used.
TLC plate silica gel F254 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 F254 ) of 0.25 mm thickness were
used after activation at 105ºC for 30-40 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- 0.37
Ethyl acetate (100),
Rf- 0.55
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 min @ 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, 465.
2. Modi’s medical jurisprudence and
toxicology.
3. Pascal Kintz et al, Forensic Science
International, 145(2004) 131-135.
4. British Pharmacopoeia, Pg no 1014-1015.
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.