Dr.D.

Ramakrishna
M.Pharm; Ph.D
Pricipal

ENDORSEMENT BY THE PRINCIPAL

This is to certify that the thesis entitled “ ALCOHOL DETERRENT ACTIVITY OF DISULFIRAM AND
ITS ESTIMATION BY RP-HPLC METHOD” submitted to Jawaharlal Nehru Technological
University, Hyderabad in partial fulfilment for the award of the degree of BACHELOR OF
PHARMACY in department of Pharmaceutical Chemistry has been successfully carried out by
SHEEMA SAAMIYA KHATOON (15411R0071), SUMAIYA JALAL (15411R0076), SYEDA ZEBA
SULTANA (15411R0088), TANIA FIRDAUS (15411R0091), AMERA SHEIKH (15411R0099), during
the academic year 2018-2019 under the supervision and guidance of Ms MUBEENA AFRA,
M.Pharm, Assistant Professor, Department of Pharmaceutical Chemistry.

Date:
Place: Hyderabad

Dr.D.Ramakrishna
M.Pharm,Ph.D
PRINCIPLE
Shadan Womens College of Pharmacy
Khairatabad, Hyderabad.
Dr.D.Ramakrishna
M.Pharm;Ph.D.
Principal

ENDORSEMENT BY THE EXTERNAL EXAMINER

This is to certify that the thesis entitled “ALCOHOL DETERRENT ACTIVITY OF DISULFIRAM AND
ITS ESTIMATION BY RP-HPLC METHOD” submitted to Jawaharlal Nehru Technological
University, Hyderabad in partial fulfilment for the award of the degree of BACHELOR OF
PHARMACY in Department of Pharmaceutical Chemistry has been successfully carried out by
SHEEMA SAAMIYA KHATOON (15411R0071), SUMAIYA JALAL (15411R0076), SYEDA ZEBA
SULTANA (15411R0088), TANIA FIRDAUS (15411R0091), AMERA SHEIKH (15411R0099), during
the academic year 2018-2019 under the supervision and guidance of Ms. MUBEENA AFRA,
M.Pharm, Assistant Professor, Department of Pharmaceutical Chemistry.

Date:

INTERNAL EXAMINER EXTERNAL EXAMINER

Dr.D.Ramakrishna
M.Pharm;Ph.D
Principal
CERTIFICATE BY THE GUIDE

This is to certify that the thesis entitled “ALCOHOL DETERRENT ACTIVITY OF DISULFIRAM AND
ITS ESTIMATION BY RP-HPLC METHOD” submitted to Jawaharlal Nehru Technological
University, Hyderabad in partial fulfilment for the award of the degree of BACHELOR OF
PHARMACY in Department of Pharmaceutical Chemistry has been successfully carried out by
SHEEMA SAAMIYA KHATOON (15411R0071), SUMAIYA JALAL (15411R0076), SYEDA ZEBA
SULTANA (15411R0088), TANIA FIRDAUS (15411R0091), AMERA SHEIKH (15411R0099), during
the academic year 2018-2019, under my supervision and guidance.

Date:
Place: Hyderabad

GUIDE
Ms. MUBEENA AFRA
Dept. of Pharmaceutical Chemistry
Shadan Women’s College of Pharmacy,
Khairatabad, Hyderabad.
Dr.D.Ramakrishna
M.Pharm, Ph.D.
Principal

DECLARATION BY THE CANDIDATE

The work presented in this dissertation entitled “ALCOHOL DETERRENT ACTIVITY OF

DISULFIRAM AND ITS ESTIMATION BY RP-HPLC METHOD” was carried out by us under the
supervision of Ms. MUBEENA AFRA, M.Pharm., Asst. Professor, Department of Pharmaceutical
chemistry, Shadan Women’s college of Pharmacy.
We further declare that this work is original and has not been submitted in part or full for any
diploma or degree of this or any other university.

Date:
Place: Hyderabad

SHEEMA SAAMIYA KHATOON (15411R0071)

SUMAIYA JALAL (15411R0076)

SYEDA ZEBA SULTANA (15411R0088)

TANIA FIRDAUS (15411R0091)

AMERA SHEIKH (15411R0099)

 ACKNOWLEDGEMENT
It is by the blessings of the God almighty that we were able to complete our
investigational studies successfully and present this work for which we are eternally
indebted.

It affords us an immense pleasure to acknowledge with gratitude the help and able
guidance rendered to us by a host of people, to whom we owe a substantial measure for
the fulfillment of this project work.

We would like to express our gratitude to our guide Ms. MUBEENA AFRA M.Pharm,
Department of Pharmaceutical Chemistry for her valuable suggestions, scholarly
guidance and constant encouragement throughout my post graduate studies and
dissertation work.

We wish to express gratitude to the Principal DR.D. RAMAKRISHNA M.Pharm,

Ph.D, for his support and encouragement and lending us all the facilities required to
proceed my study.

I consider myself very obliged to have every one’s support and encouragement that made
great efforts towards the pursuit of my education.

I am also thankful to all those who have been of immense help, either directly or
indirectly and whose names have unknowingly missed out, in making this project a
worthy endeavor.

SHEEMA SAAMIYA KHATOON (15411R0071)

SUMAIYA JALAL (15411R0076)
SYEDA ZEBA SULTANA (15411R0088)
TANIA FIRDAUS (15411R0091)
AMERA SHEIKH (15411R0099)
 CONTENTS

I. INTRODUCTION
II. LITERATURE REVIEW

III. AIM & PLAN OF WORK

IV. MATERIALS AND METHODS

V. DRUG PROFILE

VI. RESULTS AND DISCUSSION

VII. SUMMARY & CONCLUSION

VIII. REFERENCE
 ABBREVATIONS

BP British Pharmacopoeia
Cps Centipoises
CRDDS Controlled Release Drug Delivery System
DSC Differential Scanning Calorimetry
F Formulation
FTIR Fourier Transform Infrared Spectroscopy
GIT Gastrointestinal tract
HPMC Hydroxypropylmethylcellulose
IP Indian Pharmacopoeia
MDT Mean dissolution time
MEC Minimum Effective Concentration
MSC Maximum Safe Concentration
Rpm Revolutions per minute
SD Standard Deviation
w/w Weight by weight
β Beta
 INTRODUCTION

Biochemistry is a branch of chemistry which is used in clinical

diagnosis, manufacture of various biological products, treatment of diseases, in

nutrition, agriculture, etc. The importance of biochemistry is immense and

indispensable in our daily life activities.

 Drug Constitution: Biochemistry gives an idea of the constitution of the

drug, its chances of degradation with varying temperature, etc. How

modification in medicinal chemistry helps improve efficiency, minimize side

effects, etc.

 The half-life: This is a test done on biochemical drugs to know how long a

drug is stable when kept at so and so temperature.

 Drug storage: The storage condition required can be estimated by the

biochemical test. For example, many enzymes, hormones are stored for

dispensing. These get deteriorated over time due to temperature or

oxidation, contamination and due to improper storage.

 Drug metabolism: It also gives an idea of how drug molecules are

metabolized by many biochemical reactions in the presence of enzymes.

This helps to avoid drugs which have a poor metabolism or those with

excessive side effects from being prescribed or dispensed to the patient.

1
DISULFIRAM
Disulfiram (sold under the trade names Antabuse and Antabus) is a drug

used to support the treatment of chronic alcoholism by producing an acute

sensitivity to ethanol (drinking alcohol).

Disulfiram works by inhibiting the enzyme acetaldehyde dehydrogenase, causing

many of the effects of a hangover to be felt immediately following alcohol

consumption. Disulfiram plus alcohol, even small amounts, produce flushing,

throbbing in head and neck, respiratory difficulty, nausea, copious vomiting,

sweating, thirst, chest pain, palpitation, dyspnea, hyperventilation, tachycardia,

hypotension, syncope, marked uneasiness, weakness, vertigo, blurred vision,

and confusion. In severe reactions there may be respiratory depression,

cardiovascular collapse, arrhythmias, myocardial infarction, acute congestive

heart failure, unconsciousness, convulsions, and death. Disulfiram should be

used in conjunction with counseling and support.

In the body, alcohol is converted to acetaldehyde, which is then broken down by

acetaldehyde dehydrogenase. When dehydrogenase enzyme is inhibited,

acetaldehyde builds up, causing the unpleasant effects.

2
Disulfiram has been studied as a possible treatment for cancer and latent HIV

infection. In recent years, several analytical techniques have been evolved in the

identification of drugs.

HPLC METHOD DEVELOPMENT

The term ‘Chromatography’ covers those processes aimed at the separation

of the various species of a mixture based on their distribution characteristics

between a stationary and a mobile phase. HPLC is a very common method for

metabolomics analysis. With the invention of electrospray ionization, HPLC is

coupled to mass spectroscopy. HPLC has lower chromatographic resolution,

requires no derivation for polar molecules and separates molecules in the liquid

phase.

BIOCHEMICAL APPLICATIONS OF HPLC

HPLC has the advantage of much wider range of analytes measurements

with a higher sensitivity than gas chromatographic methods. Relevant to

proteomics, due to the complex structure and nature of

proteins, instrumentation and methods development for sample clean-up, pre-

concentration, fractionation, chromatographic separation and detection becomes

an immediate requirement for the identification of peptides and proteins. Latest

techniques and equipment for separation and detection include nano-HPLC

and multidimensional-HPLC for protein and peptide separation. HPLC is

considered as most reliable and most sensitive technique in genomics used to

3
determine DNA methylation. HPLC finds applications in glycomics and lipidomics

where glycan part is cleaved either enzymatically or chemically from the target

and subjected to analysis. HPLC has a wide application in lipidomics to separate

lipids prior to mass spectrometry. Separation can be achieved by either reverse-

phase (RP) HPLC or normal-phase (NP) HPLC.

 Proteomics

 Lipidomics

 Biopharmaceutical data screening

 Clinical Diagnosis

 Food Technology

 Nano Technology

MODES OF CHROMATOGRAPHY

 Normal Phase Chromatography

 Reversed Phase Chromatography

 Reversed Phase – ion pair Chromatography

 Ion-Exchange Chromatography

 Size Exclusion Chromatography

REVERSED PHASE CHROMATOGRAPHY

The objective was to make less polar or non-polar so that polar solvents can

be used to separate water-soluble polar compounds. Since the ionic nature of the

4
chemically modified silica is now reversed i.e. it is non-polar or the nature of the

phase is reversed. The chromatographic separation carried out with such silica is

referred to as reversed- phase chromatography.

Many chemically bonded stationary phases based on silica are available

commercially. The retention increases as the number of carbon atoms increases.

As a rule, the retention increases with increasing contact area between sample

molecule and stationary phase i.e. with increasing number of water molecules,

which are released during the adsorption of a compound. Branched chain

compounds are eluted more rapidly than their corresponding normal isomers.

Exactly opposite applies in reversed phase system water cannot wet the

non-polar (hydrophobic) alkyl groups such as C18 of ODS phase and therefore

does not interact with the bonded moiety. Hence water is the weakest solvent of

all and gives slowest elution rate. The elution time (retention time) in reversed

phase chromatography increases with increasing amount of water in the mobile

phase.

NORMAL PHASE CHROMATOGRAPHY

In normal phase chromatography, the stationary phase is a polar adsorbent

and the mobile phase is generally a mixture of non-aqueous solvents.

The silica structure is saturated with silanol groups at the end. The adsorption

strengths and hence k’ values (elution series) increase in the following order.

5
Saturated hydrocarbon < olefins < aromatics < organic halogen compounds <

sulphides < esters < aldehydes and ketones < amides < carboxylic acids. If a

molecule has several functional groups, then the most polar one determines the

reaction properties.

The aminopropyl and cyanopropyl phases provide opportunities for specific

interactions between the analyte and the stationary phases and thus offer

additional options for the optimizations of separations.

Chromatographic methods can be classified most practically according to

the stationary and mobile phases, as shown in the table 1:

Table.1.2.1: Classification of Chromatographic Methods

Stationary phase Mobile phase Method

Adsorption column, thin-layer,

ion exchange, High

Solid Liquid
performance liquid

chromatography.

Liquid Liquid Partition, column, thin-layer,

HPLC, paper chromatography.

6
 Gas–Liquid Chromatography
Gas

The modern form of column chromatography has been called high

performance, high pressure, and high-resolution and high-speed liquid

chromatography.

High-Performance Liquid Chromatography (HPLC) is a special branch of

column chromatography in which the mobile phase is forced through the column

at high speed. The essential equipment consists of an eluent, reservoir, a high-

pressure pump, and an injector for introducing the sample, a column containing

the stationary phase, a detector and recorder.

The various components of a HPLC system are herewith described.

SYSTEM COMPONENTS:

Solvent delivery system:

7
 The mobile phase is pumped under pressure from one or several

reservoirs and flows through the column at a constant rate. With micro particulate

packing, there is a high-pressure drop across a chromatography column.

The most important component of HPLC in solvent delivery system is the

pump, because its performance directly effects the retention time, reproducibility

and detector sensitivity. Among the several solvent delivery systems

reciprocating pump with twin or triple pistons is widely used, as this system gives

less baseline noise, good flow rate reproducibility etc.

Solvent degassing system:

The constituents of the mobile phase should be degassed and filtered

before use. Several methods are employed to remove the dissolved gases in the

mobile phase. They include heating and stirring, vacuum degassing with an

aspirator, filtration through 0.45 filters, vacuum degassing with an air-soluble

membrane, helium purging ultra-sonication or purging or combination of these

methods.

Sample introduction systems:

Two means for analyte introduction on the column are injection in to a

flowing stream and a stop flow injection. These techniques can be used with a

syringe or an injection valve. Automatic injector is a microprocessor-controlled

version of the manual universal injector. Usually, up to 100 samples can be

loaded in to the auto injector tray.

8
Liquid chromatographic detectors:

The function of the detector in HPLC is to monitor the mobile phase as it

emerges from the column. Generally, there are two types of HPLC detectors,

bulk property detectors and solute property detectors.

1. Bulk property detectors:

These detectors are based on differential measurement of a property,

which is common to both the sample and the mobile phase. Examples of such

detectors are refractive index, conductivity and dielectric constant detectors.

2. Solute property detectors:

Solute property detectors respond to a physical property of the solute,

which is not exhibited by the pure mobile phase. These detectors measure a

property, which is specific to the sample, either with or without the removal of the

mobile phase prior to the detection. UV-Vis and fluorescent detectors are suitable

for gradient elution, because many solvents used in HPLC do not absorb to any

significant extent.

Column and Column-packing materials:

The heart of the system is the column. In order to achieve high efficiency

of separation, the column material packed in such a way that highest numbers of

theoretical plates are possible. Silica (SiO2, H2O) is the most widely used

9
substance for the manufacture of packing materials. In HPLC, generally two

types of columns are used, normal phase columns and reversed phase columns.

The column material (micro-particles, 5-10 μm size) packed in such a

way that highest numbers of theoretical plates are possible. Silica (SiO2 X H2O) is

the most widely used substance for the manufacture of packing materials. In

HPLC, generally two types of columns are used, normal phase columns and

reversed phase columns.

METHOD OPTIMISATION:

Selection of stationary phase / column: Selection of the column is the first and

the most important step in method development. The column is selected

depending on the nature of the solute and the information about the analyte.

Reversed phase mode of chromatography facilitates a wide range of columns

like dimethyl silane (C2), butylsilane (C4), octylsilane (C8), etc. C18 was chosen for

this study since it is most retentive one.

In liquid chromatography, the solute retention is governed by the solute

distribution factor, which reflects the different interactions of the solute –

stationary phase, solute – mobile phase and the mobile phase – stationary

phase. For a given stationary phase, the retention of the given solute depends

directly upon the mobile phase, the nature and the composition of which must be

judiciously selected in order to get appropriate and required solute retention. The

10
mobile must be adapted in terms of elution strength (solute retention) and solvent

selectivity (solute separation) Solvent polarity is the key word in chromatographic

separations since a polar mobile phase will give rise to low solute retention in

normal phase and high solute retention in reverse phase LC.

The following are the parameters, which shall be taken into consideration

while selecting and optimizing the mobile phase.

 Buffer,

 pH of the buffer

 Mobile phase composition.

Selection of detector:

The detector was chosen depending upon some characteristic property of

the analyte like UV absorbance, fluorescence, conductance, oxidation, reduction

etc. characteristics that are to be fulfilled by a detector to be used in HPLC

determination are,

 High sensitivity, facilitating trace analysis

 Negligible baseline noise. To facilitate lower detection

 Low dead volume

 Non destructive to sample

 Inexpensive to purchase and operate

11
 For the greatest sensitivity λmax should be used. Higher wavelengths

give greater selectivity.

METHOD VALIDATION

Method validation can be defined as ICH establishing documented evidence

which provides a high degree of assurance that specific activity will consistently

produce a desired result or product meeting its predetermined specifications and

quality characteristics. For chromatographic methods used in analytical

applications there is more consistency in validation practice with key analytical

parameters.

a) Recovery:

The absolute recovery of analytical method is measured as the response

of a processed spiked matrix standard expressed as a percentage of the

response of pure standard which has not been subjected to sample pre treatment

and indicates whether the method provides a response for the entire amount of

analyte that is present in the sample.

b) Sensitivity:

The method is said to be sensitive if small changes in concentration cause

large changes in response function. The sensitivity of an analytical method is

determined from the slope of the calibration line. The limits of quantification

(LOQ) of bio analytical method are defined as the highest and lowest

12
concentrations. It can be set at  15% for both the upper and lower limit of

quantitation respectively. Any sample concentration that falls outside the

calibration range cannot be interpolated from the calibration line and

extrapolation of the calibration curve is discouraged. If the concentration is over

range, the sample should be diluted in drug-free matrix and re-assayed.

c) Precision:

Precision refers to the reproducibility of measurement within a set, that is,

to the scatter of dispersion of a set about its central value. Standard deviation is

the square root of the sum of squares of deviations of individual results for the

mean, divided by one less than the number of results in the set. The standard

deviation S, is given by

1 n
 x i  x 
2

S= n  1 i1

The square of standard deviation is called variance (S 2). Relative standard

deviation is the standard deviation expressed as a fraction of the mean, i.e., S/x.

It is sometimes multiplied by 100 and expressed as a percent relative standard

deviation. It becomes a more reliable expression of precision.

% Relative standard deviation = S x 100 / x

d) Accuracy:

13
 Accuracy normally refers to the difference between the mean x, of the set

of results and the true or correct value for the quantity measured. For analytical

methods, there are two possible ways of determining the accuracy, absolute

method and comparative method. It is calculated from the expression,

(measured value - true value)

%Bias = X 100
true value

The accuracy of analytical method is then determined at each concentration by

assessing the agreement between the measured and nominal concentrations of

the analytes in the spiked drug – free matrix sampler.

e) Limit of detection (LOD):

The limit of detection (LOD) of an analytical method may be defined as

the concentration, which gives rise to an instrument signal that is significantly

different from the blank. For spectroscopic techniques or other methods that rely

upon a calibration curve for quantitative measurements, the IUPAC approach

employs the standard deviation of the intercept (Sa), which may be related to

LOD and the slope of the calibration curve, b, by

LOD = 3 Sa/ b

f) Limit of Quantitation (LOQ)

The LOQ is the concentration that can be quantitate reliably with a

specified level of accuracy and precision. The LOQ represent the concentration

of analyte that would yield a signal-to-noise ratio of 10.

14
 LOQ = 10 Sa/ b

Where, Sa- the estimate is the standard deviation of the peak area ratio of

analyte to IS (5 injections) of the drugs. b -is slope of the corresponding

calibration curve.

g) Ruggedness

Method Ruggedness is defined as the reproducibility of results when the

method is performed under actual use conditions. This includes different

analysts, laboratories, columns, instruments, source of reagents, chemicals,

solvents etc. Method ruggedness may not be known when a method is first

developed, but insight is obtained during subsequent use of that method.

h) Robustness

The concept of robustness of an analytical procedure has been defined by

the ICH as “a measure of its capacity to remain unaffected by small but

deliberate variations in method parameters”. The robustness of a method is the

ability to remain unaffected by small changes in parameters such as pH of the

mobile phase, temperature, %organic solvent strength and buffer concentration

etc.

i) System suitability

System suitability experiments can be defined as tests to ensure that the

method can generate results of acceptable accuracy and precision. The

15
requirements for system suitability are usually developed after method

development and validation have been completed. (or) The USP (2000) defines

parameters that can be used to determine system suitability prior to analysis.

The criteria selected will be based on the actual performance of the method as

determined during its validation.

16
 Review of Literature

Nikhil P. Kothawade and Suvarna A. Katti

Disulfiram is an alcohol deterrent drug. A simple, precise and specific

spectrophotometric method was developed and validated for estimation of

17
Disulfiram in tablet dosage form. Disulfiram showed maximum absorbance at 216

nm. The drug was derivatized in methanolic solution. Beer Lambart’s law was

obeyed at concentration range of 2-12 ppm. A linearity curve was calibrated in

concentration versus absorbance. The regression equation of curve was

calculated as Y = 0.069 X + 0.087, with correlation coefficient r2 = 0.981.

Accuracy was determined by recovery study and overall percentage recovery

was found to be 96.40%.RSD values of precision were less than 1. The LOD and

LOQ were calculated as 2.233 and 6.768 respectively. The developed method

was validated in terms of linearity, accuracy, precision, limit of detection, limit of

quantification, ruggedness and robustness as per ICH guidelines. The method

can be successfully applied for quality control analysis of Disulfiram in

pharmaceutical formulation.

18
 AIM & PLAN OF
WORK

AIM AND SCOPE OF PRESENT WORK

1. To study the alcohol deterrent activity of Disulfiram.

2. To Estimate Disulfiram simultaneously in tablet dosage forms by RP-HPLC

method.

3. To validate the method according to ICH guidelines.

19
PLAN OF WORK

The experimental work has been planned as follows:

Review of the literature for Disulfiram regarding their physical and chemical

properties, various analytical methods that were conducted for Disulfiram forms

the basis for development of new analytical RP-HPLC method Disulfiram.

DEVELOPMENT OF THE METHOD BY RP- HPLC

1. Selection of the solvent to be used as diluents and mobile phase:

Choosing the suitable solvent in which the drug is soluble and stable.

They must be easily available, economical and of the HPLC grade

2. Selection of Mobile phase:

For the mobile phase, the first variable to be decided is whether an

organic or aqueous eluent should be used. With the RP-HPLC analysis, either an

aqueous eluent or a very polar organic solvent such as methanol or acetonitrile

should be fixed. If the K’ values are too large with an aqueous solvent, organic

solvent should be tried. If the K’ value is too low with organic solvent the

separation should be attempted using a mixture of two solvents with various

properties.

20
  K’-capacity factor is a measurement of the degree where the peak

of the interest is located with respect to void volume, i.e. Elution

time of non-retained components. Generally, the value of K’ is > 2.

 If a buffer is used, the pH as well as ionic strength of the buffer can

be tried.

3. In order to select the wavelength to carry out the analysis, critical examination

of the Ultraviolet absorbance spectra of the drug should be done.

4. A perfect study of the structure of drug and its physicochemical properties; to

select the chromatographic parameters.

5. Selection of method for quantitative chromatographic analysis. Determination

of working concentration range.

6. Validation of the developed method by following ICH guidelines.

21
MATERIALS AND METHODS

22
Instruments:

 HPLC – WATERS Model NO.2695 series Compact System Consisting of

Inertsil-C18 ODS column.

 Electronic balance (SARTORIOUS)

 Digital pH meter (POLOMAN)
 Sonicator (FAST CLEAN)
Chemicals:

 Acetonitrile HPLC Grade

Raw Material:

Disulfiram- Working Standard.

5.1 METHOD DEVELOPMENT FOR HPLC:

The objective of this experiment was to optimize the assay method for
estimation of Disulfiram based on the literature survey made. So here the trials
mentioned describes how the optimization was done.

Trial: 1

Mobile Phase: 100% pure degaussed methanol.

Preparation of Standard Solution:

10mg of Disulfiram drug was weighed and dissolved in 10ml of Mobile

phase and taken in 10ml of volumetric flask and sonicated for 20 minutes to get
1000ppm and 1 ml was taken from this and diluted to 10 ml with mobile phase

Chromatographic Conditions:

Flow rate : 1.0ml/min

Column : Inertsil - C18 ODS column
Detector wavelength : 225nm
Column temp : Ambient
23
 Injection volume : 20µl
Run time : 6min
Retention time : 2.913

Observation: got more peak tailing. The trial 1 chromatogram result was shown
in Fig:1

Trial: 2.

Mobile Phase: methanol and Acetonitrile were mixed in the ratio of 90:10V/V
and sonicated to degas.

Preparation of Standard Solution:

10mg of Disulfiram drug was weighed and dissolved in 10ml of Mobile

phase and taken in 10ml of volumetric flask and sonicated for 20 minutes to get
1000ppm and 1 ml. was taken from this and diluted to 10ml with mobile phase.

Chromatographic Conditions:

Flow rate : 1ml/min

Column : Inertsil -C18 ODS column

Detector wavelength : 225nm
Column temp : Ambient
Injection volume : 20µl
Run time : 6min
Retention time : 3.261

Observation: Got more asymmetry. The trial 2 chromatogram result was

shown in Fig.2

Trial: 3.

Mobile Phase: Methanol and Acetonitrile were mixed in the ratio of 80:20 V/V
and sonicated to degas.
24
Preparation of Standard Solution:

10mg of Disulfiram drug was weighed and dissolved in 10ml of Mobile

phase and taken in 10ml of volumetric flask and sonicated for 20 minutes to get
1000ppm and 1 ml. was taken from this and diluted to 10ml with mobile phase.
Chromatographic Conditions:

Flow rate : 1.0ml/min

Column : Inertsil - C18 ODS column

Detector wavelength : 225nm

Column temp : Ambient

Injection volume : 20µl
Run time : 6min
Retention time : 2.925

Observation:

Got peak spliting. The trial 3chromatogram result was shown in Fig:3

5.2 OPTIMIZED METHOD

Mobile Phase: Methanol and Water were taken and sonicated to degas in the
ratio of 60:40.

Preparation of stock solution:

10mg of Disulfiram drug was weighed and dissolved in 10ml of Mobile phase and
taken in 10ml of volumetric flask and sonicated for 20 minutes to get 1000ppm
and 2 ml. was taken from this and diluted to 10ml with mobile phase.

Preparation of working standard solution:

The stock solution equivalent to 20ppm to 70ppm were prepared, sonicated and
filtered through 0.45µ membrane.

25
Optimized chromatographic conditions:

Parameters Method
Stationary phase (column)
Inertsil -ODS C18

Mobile Phase Acetonitrile: Potassium

Dihydrogen Phosphate (70:30).
Flow rate (ml/min) 1.0 ml

Run time (minutes) 6

Column temperature (°C) Ambient

Volume of injection loop (l) 20

Detection wavelength (nm) 225nm

Drug RT (min)
2.922

5.3 METHOD VALIDATION

5.3.1 SYSTEM SUITABILITY:

A Standard solution was prepared by using Disulfiram working

standard as per test method and was injected Five times into the HPLC system.

The system suitability parameters were evaluated from standard

chromatograms by calculating the % RSD from five replicate injections for
Disulfiram, retention times and peak areas.

ACCEPTANCE CRITERIA:

26
1. The % RSD for the retention times of principal peak from 5 replicate injections
of each Standard solution should be not more than 2.0 %

2. The % RSD for the peak area responses of principal peak from 5 replicate
injections of each standard Solution should be not more than 2.0%.

3. The number of theoretical plates (N) for the Disulfiram peaks is NLT 3000.

4. The Tailing factor (T) for the Disulfiram peaks is NMT 2.0

OBSERVATION:

The %RSD for retention times and peak areas were found to be within
the limit. Refer table: 1 As shown in fig 6 – 10.

5.3.2 SPECIFICITY:

Disulfiram identification:

Solutions of standard and sample were prepared as per the test method are
injected into chromatographic system.

ACCEPTENCE CRITERIA:

Chromatogram of standard and sample should be identical with near

Retention time.
OBSERVATION:

The chromatograms of Standard and Sample were same identical with

same retention time. As shown in fig: 11 and 12.

5.3.3 PRECISION:

5.3.3.1Repeatability:

a. System precision: Standard solution prepared as per test method and

injected five times. Fig 13 -17.

27
 b. Method precision: Prepared six sample preparations individually using
single as per test method and injected each solution. Fig 18 – 22.

ACCEPTANCE CRITERIA:

The % relative standard deviation of individual Disulfiram, from the six

units should be not more than 2.0%. The assay of Disulfiram should be not less
than 98% and not more than 102.0%.

OBSERVATION:

Test results are showing that the test method is precise. Refer tables 2
and 3 for system precision and for method precision.

5.3.3.2 Intermediate precision (analyst to analyst variability):

A study was conducted by two analysts as per test method. Fig 23 – 25.

ACCEPTENCE CRITERIA:

The individual assays of Disulfiram should be not less than 98% and not
more than 102% and %RSD of assay should be NMT2.0% by both analysts.

OBSERVATION:

Individual %assays and %RSD of Assay are within limit and passes the
intermediate precision, Refer table: 4

5.3.4 ACCURACY (RECOVERY):

A study of Accuracy was conducted. Drug Assay was performed in

triplicate as per test method with equivalent amount of F Disulfiram into each
volumetric flask for each spike level to get the concentration of Disulfiram
equivalent to 50%, 100%, and 150% of the labeled amount as per the test
method. The average % recovery of Disulfiram was calculated. Fig 26 - 31.

ACCEPTANCE CRITERIA:

28
 The mean % recovery of the Disulfiram at each spike level should be not
less than 98.0% and not more than 102.0%.

OBSERVATION:
Amount found
% Recovery = ---------------------- × 100
Amount added

The recovery results indicating that the test method has an acceptable
level of accuracy. Refer table: 5

5.3.5 LINEARITY OF TEST METHOD:

A Series of solutions are prepared using Disulfiram working standard at

concentration levels from 20ppm to 80 ppm of target concentration. Fig 32 -38.

ACCEPTANCE CRITERIA:
Correlation Coefficient should be not less than 0.9990.
% of y- Intercept should be ±2.0.
% of RSD for level 1 and Level 6 should be not more than 2.0%.

OBSERVATION:

The linear fit of the system was illustrated graphically. The results are presented
in table 6.

5.3.6 RUGGEDNESS OF TEST METHOD:

System to system variability:

System to system variability study was conducted on different HPLC

systems, under similar conditions at different times. Six samples were prepared
and each was analyzed as per test method. Fig 39-44.

29
 Comparison of both the results obtained on two different HPLC systems,
shows that the assay test method is rugged for System to system variability.

ACCEPTANCE CRITERIA:

The % relative standard deviation of Disulfiram from the six sample

preparations should be not more than 2.0%

The % assay of Disulfiram should be between 98.0%-102.0%.

OBSERVATION:

The % RSD was found within the limit. Ref tables: 3 &7.

5.3.7 ROBUSTNESS:
Effect of variation of flow rate:

A study was conducted to determine the effect of variation in flow rate.

Standard solution prepared as per the test method was injected into the HPLC
system using flow rates, 1.0ml/min and 1.2ml/min. The system suitability
parameters were evaluated and found to be within the limits for 1.0ml/min and
1.2ml/min flow. Fig 45 -50.

ACCEPTANCE CRITERIA:

The Tailing Factor of Disulfiram standards should be NMT 2.0 for

Variation in Flow.

OBSERVATION:

The tailing factor for MF was found to be within the limits. As shown in
table 10.

5.3.8 LIMIT OF DETECTION AND QUANTITATION (LOD and LOQ):

From the linearity data calculate the limit of detection and quantitation, using the
following formula.

30
 LOD= 3.3 σ
S

σ = standard deviation of the response

S = slope of the calibration curve of the analyte.

LOQ = 10 σ
S

σ = standard deviation of the response

S = slope of the calibration curve of the analyte.

31
DRUG PROFILE

32
 DISULFIRAM

Synonym: 1,1'-dithiobis(N,N-diethylthioformamide)

Chemical Structure:

Drug Bank Code: DB00822

IUPAC: N,N-diethyl[(diethylcarbamothioyl)disulfanyl]carbothioamide

Molecular Framework: Aliphatic acyclic compounds

Molecular formula: C10H20N2S4

Molecular weight: 296.539 g/mol

Monoisotopic: 296.05093141

Smiles: CCN(CC)C(=S)SSC(=S)N(CC)CC

Physicochemical Properties

Appearance: White crystals or fine white powder.

Solubility: Soluble in water

Melting Point: 71.5 °C

Boiling point: 117 °C at 1.70E+01 mm Hg

pKa: Strongest Acidic - 3.88

Strongest Basic - -4.16

logP: 3.88

Indication: For the treatment and management of chronic alcoholism.

33
Pharmacology and Biochemistry

Pharmacodynamics:

Disulfiram produces a sensitivity to alcohol which results in a highly unpleasant

reaction when the patient under treatment ingests even small amounts of

alcohol. Disulfiram blocks the oxidation of alcohol at the acetaldehyde stage

during alcohol metabolism following disulfiram intake, the concentration of

acetaldehyde occurring in the blood may be 5 to 10 times higher than that found

during metabolism of the same amount of alcohol alone. Accumulation of

acetaldehyde in the blood produces a complex of highly unpleasant symptoms

referred to hereinafter as the disulfiram-alcohol reaction. This reaction, which is

proportional to the dosage of both disulfiram and alcohol, will persist if alcohol is

being metabolized. Disulfiram does not appear to influence the rate of alcohol

elimination from the body. The longer a patient remains on therapy, the more

exquisitely sensitive he becomes to alcohol.

Mechanism of action:

Disulfiram blocks the oxidation of alcohol at the acetaldehyde stage during

alcohol metabolism following disulfiram intake causing an accumulation of

acetaldehyde in the blood producing highly unpleasant symptoms. Disulfiram

blocks the oxidation of alcohol through its irreversible inactivation of aldehyde

dehydrogenase, which acts in the second step of ethanol utilization. In addition,

disulfiram competitively binds and inhibits the peripheral benzodiazepine

34
 receptor, which may indicate some value in the treatment of the symptoms of

alcohol withdrawal, however this activity has not been extensively studied.

Human Metabolite Information:

Description

A carbamate derivative used as an alcohol deterrent. It is a relatively nontoxic

substance when administered alone, but markedly alters the intermediary

metabolism of alcohol. When alcohol is ingested after administration of

disulfiram, blood acetaldehyde concentrations are increased, followed by

flushing, systemic vasodilation, respiratory difficulties, nausea, hypotension, and

other symptoms. It acts by inhibiting aldehyde dehydrogenase. 

Cellular Locations: Cytoplasm and Membrane.

Absorption : Disulfiram is absorbed slowly from the gastrointestinal tract (80 to

90% of oral dose).

Metabolism: Hepatic.
Half-life: Approximately 5 hours following single dose.

35
&

36
 RESULTS AND DISCUSSION

Method Development:

Fig1: Chromatogram of Trial 1

0.060

2.913
0.050

0.040

0.030
AU

0.020

0.010

0.000

-0.010
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00
Minutes

Inference : got more peak tailing .

S.NO Name of the peak Retention time(min)

1. DISULFIRAM 2.913

Fig 2: Chromatogram of Trial 2:

3.261

0.08

0.06
AU

0.04

0.02

0.00
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

37
Inference: Got more asymmetry.

S.NO Name of the peak Retention time(min)

1 DISULFIRAM 3.261

Fig 3: Chromatogram of Trial3

2.925
0.08

0.06

0.04
AU

0.02

0.00

-0.02

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

Inference: Got peak spliting.

S.NO Name of the peak Retention time(min)

1. DISULFIRAM 2.925

6.2 OPTIMIZED METHOD

Fig 4: Chromatogram of standard
Disulfiram - 2.922

0.40

0.30
AU

0.20

0.10

0.00

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

38
Inference: Got chromatogram at Rt of 2.922 for standard

S.NO Name of the peak Retention time(min)

1 DISULFIRAM 2.922

Fig5:Chromatogram of sample

Disulfiram - 2.921
0.40

0.30
AU

0.20

0.10

0.00

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

Inference: Got same peak with same Rt 2.921 as of standard.

S.NO Name of the peak Retention time(min)

1. DISULFIRAM 2.921

6.3 VALIDATION DATA

6.3.1 SYSTEM SUITABILITY:

TABLE-1: Data of System Suitability

USP Tailing
Injection RT Peak Area USP Plate count

1 2.921 2102936.24 11003 1.126

39
 2 2.920 2102846.85 11040 1.126

3 2.922 2103011.54 11020 1.130

4 2.923 2102942.85 11038 1.127

5 2.921 2103038.45 11060 1.128

Mean 2.9214 2102955.18 11032 1.128

SD 0.00114 74.7604 ------- -------

% RSD 0.03928 0.00355 ------- -------

Fig : 6-10 Chromatograms of system suitability(standards 1-5)

Disulfiram - 2.921

0.40

0.30
AU

0.20

0.10

0.00

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

Inference: System suitability Chromatogram for standard – 1

40
 Disulfiram - 2.920
0.40

0.30
AU

0.20

0.10

0.00

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

Inference : System sutabilty Chromatogram for standard – 2

Disulfiram - 2.922
0.40

0.30
AU

0.20

0.10

0.00

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

Inference: System suitability Chromatogram for standard - 3

Disulfiram - 2.923

0.40

0.30
AU

0.20

0.10

0.00

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

Inference: System suitability Chromatogram for standard - 4

41
 Disulfiram - 2.921
0.40

0.30
AU

0.20

0.10

0.00

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

Inference: System suitability Chromatogram for standard - 5

6.3.2: SPECIFICITY:
Fig 11: Chromatogram of standard
Disulfiram - 2.921

0.40

0.30
AU

0.20

0.10

0.00

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

Inference: Got a peak for standard at Rt of 2.921

Fig 12: Chromatogram of Blank

0.0000

-0.0002

-0.0004
AU

-0.0006

-0.0008

-0.0010

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

42
6.3.2: PRECISION:

6.3.2.1Repeatability:

(a)System precision:

TABLE-2 Data of Repeatability (System precision)

Injection Peak Areas of Disulfiram % Assay

1 2102965.54 100.22

2 2102912.84 100.22

Concentration
3 2102886.52 100.22
40ppm

4 2103045.69 100.23

5 2102946.46 100.22

Mean 2102951.41 100.22

Statistical
SD 60.8501 0.00290
Analysis
% RSD 0.00289 0.00290

Fig13-17 Chromatograms of system precision

Disulfiram - 2.922

0.40

0.30
AU

0.20

0.10

0.00

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

43
Inference: Chromatogram for system precision (standard - 1)

Disulfiram - 2.921
0.40

0.30
AU

0.20

0.10

0.00

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

Inference: Chromatogram for system precision (standard - 2)

Disulfiram - 2.920

0.40

0.30
AU

0.20

0.10

0.00

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

Inference: Chromatogram for system precision (standard - 3)

Disulfiram - 2.920

0.40

0.30
AU

0.20

0.10

0.00

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

Inference: Chromatogram for system precision (standard - 4)

44
 Disulfiram - 2.921
0.40

0.30
AU

0.20

0.10

0.00

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

Inference: Chromatogram for system precision (standard - 5)

(b)Method precision:
TABLE-3: Data of Repeatability (Method precision)
Peak Areas of
Injection
Disulfiram %Assay

1 2102877.32 100.22

Concentration 2 2102956.23 100.22

40ppm 2102997.12 100.23

3

4 2103022.22 100.23

5 2103075.84 100.23

6 2103124.45 100.23

Mean 2103008.86 100.23

Statistical
SD 87.4487 0.00418
Analysis
% RSD 0.00415 0.00417

45
 Fig 18-22: Chromatograms of Repeatability

Disulfiram - 2.921
0.40

0.30
AU

0.20

0.10

0.00

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

Inference: Chromatogram for Repeatability (standard - 1)

Disulfiram - 2.920

0.40

0.30
AU

0.20

0.10

0.00

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

Inference: Chromatogram for Repeatability (standard - 2)

Disulfiram - 2.922

0.40

0.30
AU

0.20

0.10

0.00

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

Inference: Chromatogram for Repeatability (standard - 3)

46
 Disulfiram - 2.923
0.40

0.30
AU

0.20

0.10

0.00

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

Inference: Chromatogram for Repeatability (standard - 4)

Disulfiram - 2.921
0.40

0.30
AU

0.20

0.10

0.00

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

Inference: Chromatogram for Repeatability (standard - 5)

6.3.4 ACCURACY (RECOVERY)

TABLE-5: Data of Accuracy

Concentration Area Amount Amount

Statistical Analysis
added found % Recovery
% of spiked
of % Recovery
level (ppm) (ppm)

50% 1051468.23
20 19.98 99.92 MEAN 100.12
Sample 1

50% 1051386.32 20 19.98 99.92

47
Sample 2

50% 1051424.65
20 20.10 100.52 %RSD 0.3493
Sample 3

100 % 2102943.87
40 40.09 100.22 MEAN 100.33
Sample 1

100 % 2102999.91
40 40.09 100.23
Sample 2

100% 2103055.26
40 40.21 100.53 %RSD 0.17665
Sample 3

150% 3154498.65
60 60.19 100.33 MEAN 100.39
Sample 1

150% 3154326.45
60 60.19 100.32 %RSD 0.1163
Sample 2

150% 3154421.36
60 60.31 100.53
Sample 3

Fig 26-27: Chromatograms for accuracy (50%)

48
 0.20

Disulfiram - 2.921
AU 0.15

0.10

0.05

0.00

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

Inference: Chromatogram for standard 1

Disulfiram - 2.922
0.20

0.15
AU

0.10

0.05

0.00

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

Inference: Chromatogram for standard 2

Fig 28-29: Chromatograms for accuracy (100%)

Disulfiram - 2.922

0.40

0.30
AU

0.20

0.10

0.00

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

Inference: Chromatogram for standard 1

49
 Disulfiram - 2.921
0.40

0.30
AU

0.20

0.10

0.00

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

Inference: Chromatogram for standard 2

Fig 30-31: Chromatograms for Accuracy (150%)

0.60

Disulfiram - 2.920
0.50

0.40

0.30
AU

0.20

0.10

0.00

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

Inference: Chromatogram for standard

0.60
Disulfiram - 2.923

0.50

0.40
AU

0.30

0.20

0.10

0.00

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

Inference: Chromatogram for standard 2

6.3.5 LINEARITY:

TABLE 6: Data of Linearity

50
 Concentration Average Statistical Analysis

(ppm)
Area

0 0 Slope 52296

20 1051491.45 y-Intercept 6338

30 1577236.65 Correlation Coefficient 0.999

40 2102982.87

50 2628727.42

60 3154473.86

70 3649285.27

Fig: 32 Linearity Plot (Concentration Vs Response)

4000000

3500000 f(x) = 52295.6413934426 x + 6338.83196721319

R² = 0.999942409805364
3000000

2500000

2000000 Series2
Linear (Series2)
1500000

1000000

500000

0
0 10 20 30 40 50 60 70 80

51
 Fig 33: Chromatograms for 20 ppm:

Disulfiram - 2.922
0.20

0.15
AU

0.10

0.05

0.00

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

Inference: Chromatogram for 20 ppm standard 1

Fig 34-35: chromatograms for 30ppm, 40 ppm

0.30 Disulfiram - 2.922

0.25

0.20
AU

0.15

0.10

0.05

0.00

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

Inference: Chromatogram for 30 ppm standard 1

Disulfiram - 2.921

0.40

0.30
AU

0.20

0.10

0.00

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

Inference: Chromatogram for 40 ppm standard 1

52
6.3.6 Ruggedness:

a) System to System variability:

For system 1 Refer: Table3

TABLE: 7 Data of system to system variability (sample)

System-2
S.NO: Peak area Assay % of

Disulfiram

1 2102837.37 100.22

2 2102956.99 100.22

3 2102890.92 100.23

4 2103072.69 100.23

5 2103127.56 100.23

6 2102954.32 100.22

Mean 2102973.30 100.22

%RSD 0.00519 0.00520

Chromatograms of system to system variability Fig 39-44.

53
 Disulfiram - 2.922
0.40

0.30
AU

0.20

0.10

0.00

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

Infe

rence: Chromatogram of system to system variability std- 1

Disulfiram - 2.921
0.40

0.30
AU

0.20

0.10

0.00

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

Inference: Chromatogram of system to system variability std- 2

Disulfiram - 2.920

0.40

0.30
AU

0.20

0.10

0.00

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

Inference: Chromatogram of system to system variability std- 3

54
 Disulfiram - 2.920
0.40

0.30
AU

0.20

0.10

0.00

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

Inference: Chromatogram of system to system variability std- 4

Disulfiram - 2.921
0.40

0.30
AU

0.20

0.10

0.00

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

Inference: Chromatogram of system to system variability std- 5

Disulfiram - 2.920

0.40

0.30
AU

0.20

0.10

0.00

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

Inference: Chromatogram of system to system variability std- 6

6.3.7 Robustness:

TABLE: 10 Data for Effect of variation in flow rate:

Flow Std Area Tailing Flow Std Area Tailing Flow Std Area Tailing

0.8 factor factor 1.2 ml factor

55
 ml 1432628.65 1.108 1.0 ml 2102946.64 1.112 2843631.12 1.125

1432568.45 1.112 2102928.28 1.114 2843589.98 1.123

1432670.79 1.114 2103048.82 1.112 2843652.32 1.124

1432521.56 1.120 2103010.01 1.113 2843628.62 1.124

1432689.98 1.119 2102989.56 1.114 2843672.56 1.123

Avg 1432615.88 1.114 Avg 2102984.66 1.113 Avg 2843634.92 1.123

SD 70.3826 0.0049 SD 48.4957 0.001 SD 30.7947 0.0008

%RS 0.00491 0.4467 %RSD 0.00230 0.0898 %RSD 0.0010 0.0744

Fig 45-46: Chromatograms of robustness

a) Effect of variation of flow rate (for 0.8 ml/min flow)

Disulfiram - 3.263

0.40

0.30
AU

0.20

0.10

0.00

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

Inference: Chromatogram for robustness standard - 1

56
 Disulfiram - 3.260
0.40

0.30
AU

0.20

0.10

0.00

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

Inference: Chromatogram for robustness standard - 2

Fig 47-48: chromatograms for 1ml/min

Disulfiram - 2.920
0.40

0.30
AU

0.20

0.10

0.00

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

Inference: Chromatogram for robustness standard - 1

Disulfiram - 2.922

0.40

0.30
AU

0.20

0.10

0.00

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

Inference: Chromatogram for robustness standard - 2

57
 Fig 49-50: Chromatograms for 1.2ml/min

0.40

Disulfiram - 2.651
0.30
AU

0.20

0.10

0.00

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

Inference: Chromatogram for robustness standard - 1

0.40
Disulfiram - 2.649

0.30
AU

0.20

0.10

0.00

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Minutes

Inference: Chromatogram for robustness standard - 2

6.3.8 LIMIT OF DETECTION AND LIMIT OF QUANTITATION (LOD and LOQ):

From the linearity plot the LOD and LOQ are calculated:
LOD = 3.3 σ
S
3.3 × 74.7604
= ------------------ = 0.00471
52296

LOQ = 10 σ
S

10 × 74.7604
= ----------------- = 0.01429
52296

58
Summary
&

59
 Summary and conclusion

Disulfiram is being used successfully for over 6 decades now; it is the

oldest molecule used in alcohol dependence pharmacotherapy, and disulfiram

has stood the test of time. It is relatively safe at 125mg without its deterrent effect

being hampered. Moreover, it’s safe and effective. Since usage of disulfiram

needs continued and re-peated consultation with the treating psychiatrist and

active decision-making by the patient, it is likely that such consultations give a

chance to both to improve on the abstinence attempts of the patient.

It has a good safety record and its pharmacokinetics have been extensively

studied. The psychological aspects of super-vised disulfiram therapy along with

regular consultations with the health care provider contribute to a good clinical

outcome with this therapy.

Disulfiram was estimated in tablet dosage form by studying different parameters.

First, maximum absorbance was found to be at 225nm, and the peak purity was

excellent. Injection volume was selected to be 20µl which gave a good peak

area. The column used for study was Inertsil C18 chosen good peak shape.

Ambient temperature was found to be suitable for the nature of drug solution.

The flow rate was fixed at 1.0ml/min because of good peak area and satisfactory

retention time. Different pH and ratios of mobile phase were studied, mobile

phase with ratio of Methanol and Water (60:40) was fixed due to good

symmetrical peak. So, this mobile phase was used for the proposed study.

60
Methanol was selected because of maximum extraction sonication time was fixed

to be 6min at which all the drug particles were completely soluble and showed

good recovery. Run time was selected to be 6min because analyze gave peak

around 2.922 and to reduce the total run time.

The present recovery was found to be 98.0-102 was linear and precise over the

same range. Both system and method precision were found to be accurate and

well within range. Detection limit was found to be 0.00471. The analytical

method was found linearity over the range of 20-70ppm of the target

concentration. The analytical passed both robustness and ruggedness tests. On

both cases, relative standard deviation was well satisfactory.

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 References

1. Blessy M. et al, A review – development and stability indicating studies of

drugs, Journal of pharmaceutical analysis, Jan-Sept 2013

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II,479,1479,1598

3. United states pharmacopoeia 34, (2007) NF-29, United state pharmacopoeial

convention, volume – II,2187

4. Kishanta Pradhan, Uma shankar Mishra, Subasini Pattnaik, “Method

development, validation and stability study of Irbesartan in bulk and
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5. Human Metabolome Database (HMDB), Wishart Research Group, University

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and validation of UV spectrophotometric method for determination of Nebivolol
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pharmaceutical and clinical research. vol-5, suppl 4,2012.

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Geneva, Switzerland: ICH; 2005.

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