Analysis of Narcotic and Psychotropic Substances

Unit : 4
Analysis of Narcotic and Psychotropic Substances
Presented By :
Vishal Donda (101PHSPPC2122004)
Shreyaskumar J. Patel (101PHSPPC2122014)
M.Sc Pharmaceutical Chemistry
Sem - III
Submitted To:
Dr. Mahesh Vasava
Asst. Professor,
School of Pharmacy,
NFSU

A controlled substance is generally a drug or chemical whose manufacture, possession and use
is regulated by a government, such as illicitly used drugs or prescription medications that are
designated by law.

Introduction
• In addition to drugs from illicit sources, the forensic
scientist will sometimes be faced with casework
involving samples diverted from legitimate, e.g.
commercial, sources. Of these, perhaps the most
significant are barbiturates (often found in heroin
samples) and benzodiazepines.
• Both are found under a wide variety of trade names,
but the approach to their analysis is broadly similar,
either when the material is encountered in the
native dosage form, or when it is part of a drug
mixture.

Introduction
• Forensic laboratories cannot limit their search for a cause of death to abused drugs only. Many individuals
die from overdoses of prescription drugs and over-the-counter medications.
• Such deaths are sometimes suicides but many are accidental. In the latter case, they may occur because of
medicinal errors such as taking the wrong drug or the wrong dose of the right drug.
• On other occasions, a patient may suffer some level of organ damage from a primary medical condition and
the organ injury renders him or her incapable of metabolizing a drug in the normal manner.
• This latter type of problem often results in a buildup of the drug in the blood to a point where the drug’s
concentration is greater than the lethal concentration.
• Most forensic chemistry and toxicology laboratories are capable of postmortem identification of a wide
variety of medicinal agents. Increasingly, deaths involve combinations of drugs (prescribed or illicit).
• This trend is sometimes referred to as polypharmacy. For example, a postmortem drug screen might
reveal alcohol, diazepam (Valium®), and zolpidem (Ambien®). Alone, any one drug would not cause death,
but in combination such drugs can become much more dangerous

Sampling of Barbiturates and Benzodiazepines
• When a seizure of pharmaceutical drugs is made, it may comprise a single dose unit, or many tens or hundreds
of thousands of units.
• The number to be analyzed depends upon the legislative system in which the scientist is working, but the
following is recommended by the United Nations Drug Control Program for commercially produced drugs.
• If between 1 and 50 units are seized, then 50%, to a maximum of 20, chosen at random, should be analyzed.
• Of samples containing between 51 and 100 units, 20 should be analyzed.
• For samples of between 101 and 1000 units, 30 should be chosen,
• while for samples greater than 1000 units, the square root, rounded to the nearest integer, should be analyzed.
• Once the materials to be examined have been chosen, it is then a matter of identifying and quantifying (where
necessary) the specific drug present.
• In order to achieve this, a full physical description of the materials should first be carried out. It is sometimes
possible, following this process, to feed the information obtained into relevant databases to identify the drug(s)
present in the dose form.
• The identification process then becomes a simple matter of confirmation. If the dose form is not included in the
databases, however, a full chemical analysis, including drug extraction from the tabletted material, presumptive
testing, thin layer chromatography and a confirmatory technique must be undertaken.

Barbiturates
• Slang names for barbiturates: Yellow Jackets, Reds, Blues, Amy’s and
Rainbows
• Barbiturates are white, crystalline, odorless powder with a faintly bitter taste.
• They are useful in psychiatric disorders, epilepsy and strychnine poisoning.

Barbiturates
• Barbituric acid was discovered in the mid-19th century, with the first medical barbiturate,
barbitone, being synthesized in 1903.
• Phenobarbitone was introduced as a pharmaceutical in 1912.
• Therapeutically, these drugs are used as sedatives, anaesthetics and anticonvulsants.
• Phenobarbitone is also used in the treatment of epilepsy.
• During the course of the mid-20th century, increasing knowledge was gained about the side-
effects and dependence-related problems associated with barbiturate abuse.
• At the time of writing,† many of the barbiturates are controlled at both the national and
international levels.
• Those met with in the forensic science context are diverted from licit sources and may be
encountered mainly as capsules and tablets, injectable solutions and powder forms. They may
also be mixed in with other drugs, for example, heroin.

Barbiturates
• Over 2,500 barbiturates have reportedly been synthesized with more than
50 of these presently marketed for clinical use throughout the world. Twelve
of these are subject to international control under the
• Convention on Psychotropic Substances 1971 as follows:
• Schedule. II: Secobarbital
• Schedule III: Amobarbital, butalbital, cyclobarbital and pentobarbital
• Schedule IV: Allobarbital, barbital, butobarbital, methylphenobarbital, phenobarbital,
secbutabarbital and vinylbital
• In recent years, there has been a significant downturn in prescribing and
therefore the general availability of barbiturates owing to their side-effects
and associated dependency problems. An exception is phenobarbitone which
is still used as an anticonvulsant/anti-epileptic.

Barbiturates
• Barbiturates are derivatives of Barbituric Acid. They can be used as hypnotics,
sedatives, anticonvulsants and anesthetics, although they are probably most familiar
as 'sleeping pills’.
• The different properties of the various barbiturates depend upon the side groups
attached to the ring. Barbituric acid was first discovered by the German chemist
Adolf von Baeyer in 1864 by combining Urea with Malonic Acid.
• The medical value of these substances was not realized, however, until 1903 when
two other German chemists, Emil Fischer and Joseph von Mering, discovered that
one of these compounds, diethylbarbituric acid, was very effective in putting dogs to
sleep.
• It is said that Von Mering proposed that the new substance be called 'Veronal',
because the most peaceful place he knew on Earth was the Italian city of
Verona.

Classification of Barbiturates
• effects for 8-12 hrs
• Fatal Dose : 3 – 4 gm
• Example : Phenobarbitone, Mephobarbitone
1. Long Action
• Fatal Dose : 2 - 3 gm
• Example : Amylobarbitone
2. Intermediate
Action
• Fatal Dose : 1-2 gm
• Example : Butobarbitone
3. Short Action
4. Ultra short
Action
• effects for very shorter duration, acts as general anesthetics
• Fatal Dose : 1 gm
• Example : Thiopentone Sodium

Extraction of Barbiturates
• Barbiturates in either the free acid or salt forms are readily soluble in methanol
and thus this is the solvent of choice for extraction in qualitative analysis.
• A known mass of the dose form is dissolved in a volume of methanol to provide the
drug component at a working concentration of between 1 and 20 mg ml−1.
• The extract should be filtered or centrifuged prior to analysis in order to remove
any unwanted particulate materials.
• For quantitative analysis, ethyl acetate can be used as the extraction solvent
– if the drug is in the free acid form – with the extract treated in the
same way as described above.
– If the original material is in the salt form, then the drug can be
converted to the free acid form and extracted if required.

Effects of Barbiturates
Physical Effects Psychological Effects
Sleepiness Mild Euphoria
Nausea Disinhibition
Slurred Speech Memory Impairment
Breathing Disorders Increased Irritability
Reduced Sex Drive Severe Paranoia
Coma SuicidalThoughts

Uses of barbiturates
• Epilepsy/ convulsion, the drug of choice is phenobarbitone
• Anaesthesia- thiopentone is used as an anaesthetic drug
• Used as hypnotic
• Used as anxiolytic
• Used as sedative to induce sleep
• Used in treatment of psycho-somatic disorders

Preparation of barbiturate standard and sample solutions
Barbiturate standard solution
• Weigh an appropriate amount of standard barbiturates into a volumetric flask to obtain a
final concentration of approximately 1 mg/ml of each compound. Dilute to volume with
methanol.
• This stock solution is stable for at least three months when stored at -20°C.
Barbiturate sample solution
• Weigh an appropriate amount of sample obtained by one of the extraction procedure in a
volumetric flask, dissolve in and make up to volume with methanol to produce a final
barbiturate concentration of 1 mg/ml.
• Depending on the provenance of the sample, any undissolved solid particulates can be
removed by filtration

PresumptiveTests for Barbiturates
(a) Solubility
• Place small amounts of the suspect material in each of two test tubes. Add several drops of water to the
first test tube and several drops of ethyl acetate to the second.
• Observe in which solvent the material dissolves. Free acids are soluble in organic solvents such as ethyl
acetate, but are insoluble in water.
• The salt forms of the barbiturates are readily soluble in water, but are insoluble in ethyl acetate. Other
organic solvents such as ether and chloroform may substitute the ethyl acetate.
(b) pH determination
• Place a small amount (ca.10-20 mg) of the suspected barbiturate in a test tube and add 1 ml of water.
• Determine the pH.
• A pH greater than 8.0 indicates that the barbiturate is present as the sodium or calcium salt.

Dille–KoppanyiTest
• Preparation of Reagent:
• A. Cobalt Acetate Solution: 1 gm. of cobalt acetate (tetrahydrate) is dissolved in followed by addition of
0.2 ml. of Acetic Acid.
• B. Isopropyl amine Solution: 5 ml. of isopropyl amine is mixed with 100 ml. of methanol.
• Procedure No. 1: A small amount of extracted material is placed on a spot plate. 3-4 drops of cobalt
acetate solution and 3-4 drops of isopropyl amine solution are added. The appearance of a purple or blue
violet colour indicates the presence of barbiturate.
• Procedure No. 2: The residue of extract of sample is taken in 1 ml. of chloroform. To a portion of
chloroform extract of the sample, 2 drops of freshly prepared 1% cobalt acetate in methanol is added followed
by 1% lithium hydroxide in methanol drop by drop.
• Result- A blue ring at the junction indicates the presence of barbiturates.

ZWIKKER’STEST
• The residue of extract is taken up in chloroform. To 1 ml. of chloroform
extract, 2–3 drops of 0.5 ml. of 5% pyridine in chloroform is added and
shaken. The color of chloroform layer becomes purple. Then 1 drop of
glacial acetic acid is added. If the color of chloroform layer changes from
purple to weak blue, the presence of non-Thiobarbiturates is indicated.
• If chloroform layer becomes green after adding pyridine in chloroform,
the presence of Thiobarbiturates is indicated.
• Result -This green color changes to light green on adding acetic acid.

TLC of Barbiturates
• In this case, the TLC system most commonly employed uses silica gel plates and a
mobile phase of ethyl acetate/methanol/25% ammonia (85:10:5, by volume).
• The plates are prepared and the chromatogram developed in the standard way.
After development, the plate is removed from the mobile phase, the solvent
front marked, and the plate dried. Visualization of barbiturates is best achieved
by the use of a mercuric chloride–diphenyl carbazone reagent.
• The latter is prepared as two component solutions, i.e. (i) 0.1 g of diphenyl
carbazone in 50 ml of methanol.
• 0.1 g of mercuric chloride in 50 ml of ethanol. These solutions should be freshly
prepared and mixed just before use.
• The presence of barbiturates will give rise to blue–violet spots on a pink
background when using this reagent system.

ConfirmatoryAnalysis of Barbiturates
• Both barbiturates and benzodiazepines can be identified by using
GC–MS methodologies, although each drug class requires a
different pre-treatment routine prior to analysis.

GC–MSAnalysis of Barbiturates
• Due to their highly polar nature, barbiturates require derivatization prior to analysis by GC–MS.
• The derivatization procedure, using 0.2 M trimethylaniline hydroxide in methanol, is, in principle, the
same as that used for other pre-column derivatizations.
• Having derivatized the sample, it can then be analyzed by GC–MS, using the operating conditions.
N-methylation of a generalized barbiturate

GC–MSAnalysis of Barbiturates
System/parameter Description/conditions
Column BP-1: 25 m × 0.22 mm i.d.; df, 0.5 µm
Injection temperature 290◦ C
Column oven temperature programme 200◦ C, no hold; increased to 260◦ C
at 4◦ C min−1
Carrier gas At a flow rate of 1 ml min−1
Detector Mass spectrometric, temperature and
settings as required

Quantification of Barbiturates by HPLC
• Barbiturates can be prepared for HPLC analysis by dissolution of the drug
sample in methanol at a chosen concentration, followed by removal of any
solid particulate material by filtration.
• Such a system can be used to quantify barbiturates with relatively short alkyl
substituents at the C5 position. The analytes will separate, eluting in order
of lipophilicity, e.g. barbitone, butobarbitone, pentobarbitone, etc.

Benzodiazepines
• Benzodiazepines are the pharmaceutical industry’s top-selling family of prescription drugs. Alprazolam, also known as
Xanax, has long been among the industry’s best-selling pills. There are now 94 million prescriptions for various
benzodiazepines in the U.S. alone —nearly one prescription for every three citizens.
• This did not happen overnight. The story of modern benzodiazepines such as Xanax (alprazolam), Ativan (lorazepam) and
Klonopin (clonazepam) begins nearly a century ago, in Germany, where chemists developed a class of sedatives known as
barbiturates. Barbiturates were the first synthetic tranquilizers.
• Their popularity exploded in the United States during the Great Depression of the 1930s, when over-the-counter barbiturates
helped millions of Americans “take the edge off” by reducing brain activity and depressing the central nervous system.
• By the time the U.S. entered World War II in 1941, Americans were consuming more than one billion barbiturates per year.
Doctors and local pharmacists assured customers that the pills were harmless — a safe advance over the opiates previously
used as sleeping aids.
• But these glib assurances were based on promotional materials distributed by the drug makers. The truth was more
complicated than the sales pitch. Although barbiturates did help many people and patients relax and get to sleep, they
also produced tolerance and dependence, and carried a high risk of overdose. Withdrawal could be a tortuous and
sometimes fatal ordeal.
• In 1951, Congress passed a law requiring a doctor’s approval for the purchase of barbiturates. But even before the
clampdown on the over-the-counter sale of barbiturates, pharmaceutical companies were on the hunt for the next generation of
tranquilizers.
• In 1955, a chemist at the Swiss drug firm Hoffmann-La Roche named Leo Sternbach synthesized the first benzodiazepine. The
company called it Librium. It hit the market in 1960.

Benzodiazepines
• Despite industry assurances about safety, stories of dependence, social dislocation and difficult withdrawal began to tarnish Valium’s
reputation during the 1970s. Once again, this spurred the industry to find a new generation of tranquilizers with new names.
• In 1975, La-Roche began marketing clonazepam (brand name, Klonopin); two years later, Wyeth Pharmaceuticals released
lorazepam (brand name, Ativan).
• Both drugs were marketed to doctors and the public as “different” from Valium — safer, faster-acting, requiring much lower dosages,
and carrying less risk. But the new drugs’ similarities with Valium were more important than the differences accentuated by the drug
makers, who now eschewed the old label “tranquilizer” altogether, in favor of the new umbrella-term “anxiolytic.” This new rubric
falsely suggested an entirely new class of drugs, with a fundamentally different neurochemistry. Their lower dosages, meanwhile,
masked the new drugs’ dramatically increased potency: 1 milligram of clonazepam (Klonopin) and alprazolam (Xanax) equals roughly
20 milligrams of Valium.
• In 1982 Malcolm Lader, a British psychopharmacologist who directed a research group at the Institute of Psychiatry,
researched benzodiazepine-related brain changes. His research discoveries led him to become a benzodiazepine-harmed
patient advocate. Unfortunately, for unknown reasons, the documentation surrounding his research was sealed and labeled
secret by the U.K. government, not to be opened until the year 2014.
• In 2020, after FDA reporting program caught their attention, the FDA investigated and updated their benzodiazepine warnings to
include the risks of abuse, addiction, physical dependence, and withdrawal reactions. They did not announce their findings to
physicians, research safe cessation or offer any potential funding or help for protracted syndromes.
Today, the number of prescriptions continues to rise, as does the size of the benzodiazepine-injured community. Nearly four
decades after Senate hearings on the dangers of benzodiazepines, research into these drugs and physician knowledge remain
woefully inadequate. The need for action grows more urgent with every passing day.

Introduction • The abuse or misuse of Benzodiazepines is internationally
widespread which means that any forensic laboratory may
encounter a range of these compounds. In general,
benzodiazepines encountered in the illicit market are diverted
from legitimated sources.
• The benzodiazepines, specifically aprazolam and diazepam, are
among the most often diverted and abused psychotropic
substances.
• In a few cases, combination products such as chlordiazepoxide-
amitriptyline and chlordiazepoxide-clidinium bromide also appear
on the illicit market.
• Analysts should be aware of the particular benzodiazepines
commonly available in their area as well as the characteristics and
methodologies for their identification and analysis. As
benzodiazepine derivatives end up on the illicit market from
diversion from legitimate sources, reference should be made to
national pharmacopoeias and drug tablet and capsule
identification guides for preliminary screening information.

Benzodiazepines
• Benzodiazepines, which have the generalized
structure as shown in figure, were introduced to
replace the barbiturates as tranquilizers,
anxiolytics, anticonvulsants and muscle relaxants.
• All those encountered in the forensic science
context have been diverted from licit sources.
• The majority appear as tablets and capsules,
although powders and injectable solutions may
also be encountered.

Benzodiazepines
• Benzodiazepines are formulated largely as capsules and tablets. However,
some are available in other pharmaceutical form such as injectable solutions.
• Diazepam, the most traded and widely available benzodiazepine, can be
found as capsules, tablets, aqueous or polyethyleneglycol solutions for
injection, syrups and suppositories.
• They are commonly present as the free base or as the hydrochloride, mesilate
or other salt.
• However, some also have carboxylic acid functionalities and may be
presented as potassium salts, e.g. clorazepate.
• All the benzodiazepines are generally soluble in methanol.

Classification of Benzodiazepines
1. Short acting Benzodiazepines
• Short-acting compounds have an average half-life of 1–12 hours.
• They have few residual effects if taken before sleep time, rebound insomnia may occur upon discontinuation, and they
might cause daytime withdrawal symptoms such as next day rebound anxiety with prolonged usage.
• Examples are Brotizolam, midazolam, and Triazolam.
2. Intermediate acting Benzodiazepines
• Intermediate-acting compounds have an average half-life of 12–40 hours.
• They may have some residual effects in the first half of the day if used as a hypnotic. Rebound insomnia, however, is
more common upon withdrawal of intermediate-acting benzodiazepines than longer-acting benzodiazepines.
• Examples are alprazolam, estazolam, flunitrazepam, clonazepam, lormetazepam, lorazepam, nitrazepam, and
temazepam
3. Long acting Benzodiazepines
• Long-acting compounds have a half-life of 40–250 hours.
• They have a risk of accumulation in the elderly and in individuals with severely impaired liver function, but they have
a reduced severity of rebound effects and withdrawal.
• Examples are diazepam, clorazepate, chlordiazepoxide, and flurazepam.

Use of Benzodiazepines
Benzodiazepines are suggested for relaxation, calmness, and relief from
anxiety and tension.
Medically, Benzodiazepines are used to treat following ailments:
• Seizure disorders
• Anxiety disorders
• Movement disorders
• Insomnia
• Mania
• For inducing skeletal muscle relaxation
• Pre-anaesthetic medication
• Treatment of alcohol withdrawal.

Signs and Symptoms
Benzodiazepines are one of the harmless drugs when their over-dosage occurs
alone. Cases have been reported when as many as 70 tablets of any of them are
unlikely to produce anything more than mild effect in most adults.
Various benzodiazepines have active metabolites that justifies for their prolonged
sedative effects. Benzodiazepines potentiate the effects of other CNS depressants,
particularly alcohol, tricyclic antidepressants and barbiturates. Flurazepam is most
likely to produce significant CNS depression. The signs and symptoms that are
produced are:
(i) Drowsiness (ii) Dizziness (iii) Ataxia and slurred speech (iv)
Respiratory depression (v) Hypotension and coma
Long-term use of benzodiazepines is associated with the development of tolerance.
Tolerance to certain benzodiazepines occurs most often in those who have used for 6
months or more.

Withdrawal Symptoms of Benzodiazepines
 Insomnia
 Gastric problems
 Tremors
 Agitation
 Fearfulness
 Muscle spasms
• However, fewer recurrent effects comprises of irritability, excessive perspiration,
depersonalization, derealization, allergic reaction to stimuli, depression, suicidal
behavior, psychosis, seizures, and delirium tremens.
• Severe symptoms typically occur as a result of abrupt or over-rapid withdrawal.
• Abrupt withdrawal can be dangerous; therefore, a gradual reduction procedure is
suggested.

Fatal Dose
• It is ambiguous for most benzodiazepines. Even consumption of up to 2000
mg diazepam has not resulted in death, or for that matter, even serious
morbidity.
• However, several cases of fatality due to triazolam and flunitrazepam
overdose have been stated.
• In general, benzodiazepine metabolism appears to be inhibited by
ethanol when given concurrently. Clinically, associated administration of
high doses of ethanol and benzodiazepines act to synergistically reduce
respiration

Preparation of benzodiazepines standard and sample
solutions
Benzodiazepines standard solution
• Weigh an appropriate amount of the benzodiazepine standard into a volumetric flask,
dissolve in and make up to volume with 50 per cent aqueous methanol to produce a solution
of concentration 1mg/ml.
Benzodiazepines sample solution
• Weigh an appropriate amount of the sample obtained by one of the extraction methods into a
volumetric flask, dissolve in and make up to volume with 50 per cent v/v aqueous methanol
to produce a final benzodiazepine concentration of approximately 1mg/ml.
• Depending on the provenance of the sample, any undissolved particulates can be removed by
filtration.

Extraction of Benzodiazepines
• Benzodiazepines may conveniently be extracted into methanol for
both qualitative and quantitative analyses.
• The dose form should be triturated in methanol and as with
barbiturates any solid material removed by centrifugation or
filtration prior to analysis of the drug in solution.

PresumptiveAnalysis of Benzodiazepines
ZIMMERMANTEST
Preparation of reagent:
Solution 1: 2,4-Dinitrobenzene (1 % w/v) in Methanol
Solution 2: 15 % Potassium Hydroxide aqueous solution
Procedure:
On a micro-test plate, place appropriate quantity of the sample to be tested, add one drop of
Solution 1 followed by one drop of Solution 2, and mix.
Result:
Red-purple to pink colour indicates the probable presence of a benzodiazepine

VITALI – MORIN’STEST
Solution 1: Conc. Nitric Acid
Solution 2: Acetone
Solution 3: 0.1 N Ethanolic Potassium Hydroxide
Procedure:
On a micro-test plate, place adequate quantity of the sample to be tested, add Solution 1
followed by Solution 2 and further followed by Solution 3, and mix.
Result:
Yellow- Orange indicates the possible presence of benzodiazepines.

MARQUISTEST
1 volume of Formalin is added to 9 volume of concentrated Sulphuric Acid
Procedure:
On a micro-test plate, place adequate quantity of the sample to be tested, add
the reagent and agitate.
Result:
Yellow- Orange indicates the possible presence of benzodiazepines.

FORMALDEHYDE- SULPHURICACIDTEST
4 parts of Sulphuric Acid is added with 6 parts of Formalin.
Procedure:
On a micro-test plate, place adequate quantity of the sample to be tested, add the
reagent and agitate.
Result:
Red/ Pink/ Blue/ violet/ Red-violet/ Blue- violet indicates the possible presence of
benzodiazepines

HYDROCHLORICACIDTEST
0.2 N Hydrochloric Acid
Procedure:
On a micro-test plate, place adequate quantity of the sample to be tested,
add the reagent and agitate.
Result:
Appearance of Yellow Color points toward the possible existence of
Benzodiazepines.

TLCAnalysis for Benzodiazepines

InstrumentalAnalysis for Benzodiazepines
GasChromatography
• Gas Chromatography can be recommended as a suitable method for the
analysis of most benzodiazepines, several of them, particularly the 3-
hydroxyderivatives, undergo thermal degradation and rearrangements.
• Chlordiazepoxide, cloxazolam, lormetazepam, haloxazolam, oxazolam, ethyl
loflazepate and temazepam yield multiple peaks.

GasChromatography
• Benzodiazepine derivatives may be studied by analyzing the samples with the
help of Mass spectrometry (GC-MS).
• The value of principal peaks at m/z can be compared either with the standard
value given in the literatures or with using the standard sample of
Benzodiazepine derivatives.
• Identification is accomplished by comparing the Retention Time and Mass
Spectrum of the analyte with that of a reference standard

GasChromatography
SYSTEM Conditions
Column BP-1: 25 m × 0.22 mm i.d.; df, 0.25 μm
Injection Temperature 275°C
Column Oven Temperature 250°C
Carrier Gas He, at a flow rate of 1 ml/ min
Split Ratio 20:1
Detector Mass spectrometric, temperature and
settings as required

HIGH PERFORMANCE LIQUIDCHROMATOGRAPHY (HPLC)
• The analysis of forensic samples by HPLC has become popular due to
development of HPLC in attaining performance by using high performance
column, new detector, optimum methods etc.
• The HPLC method allows quantitative determination of nonvolatile and
thermally labile compounds without derivatization and much clean-up of
samples.

HIGH PERFORMANCE LIQUIDCHROMATOGRAPHY (HPLC)
SYSTEM Conditions
Column Spherisorb ODS-2: 25 cm × 4.6 mm i.d.;
5 μm particle size
Mobile Phase MeOH/water/0.1 M phosphate buffer
at pH 7.25 (55:25:20), or (70:10:20)
Flow Rate 1.5 ml/ min
Injection volume 5–10 μl
Detection UV, 240 nm

Quantification of Benzodiazepines by HPLC
• The basic principles behind the quantification of benzodiazepines are the
same as those applicable to barbiturates.
• The compounds elute in order of increasing lipophilicity and, owing to the
lack of any good chromophoric groups (ef. the barbiturates), a UV detection
wavelength of 240 nm is also used for such materials.

Questions….
1. Why is the Rf value of phenobarbitone less than cyclobarbitone ?

Questions….
2. Why is it necessary to increase the proportion of acetonitrile in
the mobile phase as the alkyl chain-length of the C5 substituents
increases in Barbiturates?

Questions….
3. Why is an isocratic mobile phase system preferred to a gradient
system in the HPLC analysis of barbiturates?
 The method transfer is easily possible.
 There is no need to re-equilibrate with the initial mobile phase
composition between consecutive injections.
 It is a simple way of separation as compared to gradient elution.
But, This will separate a limited number of molecules.

Analysis of Narcotic and Psychotropic Substances

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