Development of qualitative method for detection of Lorazepam in chocolate R'K'Jain, G'Jayashanker, M

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Development of qualitative method for detection
of Lorazepam in chocolateR.K.Jain,
G.Jayashanker, Md.Afzal, S.Sudhakar,
A.K.Srivastava R.K.SarinCentral Forensic
Science LaboratoryDirectorate of Forensic
ScienceMinistry of Home affairsGovernment of
IndiaRamanthapur, Hyderabad-500013
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IntroductionBenzodiazepine 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.
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Materials and methodsAll 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 F254 were obtained from E-Merck
(Darmstadt, Germany)Suspected chocolates and
blood were received from the I/O.
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Standard solutionsStock 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
solutionDilute 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.
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Medical findingsThe 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.
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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.
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2) TLCThe 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 (82),
Rf- 0.37Ethyl acetate (100),
Rf- 0.55Chloroform Methanol
ammonia solution 90101

Rf-0.60Spray reagent Dragon droff-
Orange color spot.
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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 _at_ 5ºC to
250ºC hold 1 min Carrier gas helium flow-
1ml/min
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Mass spectra of Standard Lorazepam
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Library match of Lorazapam
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Mass Spectra of Sample compared with Library
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4) FT-IRNicolet 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.
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FT-IR Spectra of sample compared with Library
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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.
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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.
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References1. ECG Clarke-isolation and
identification of drugs, 1986, Vol-I,
465.2. Modis medical jurisprudence and
toxicology.3. Pascal Kintz et al, Forensic
Science International, 145(2004)
131-135.4. British Pharmacopoeia, Pg no
1014-1015.
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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.

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Thank U All