DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR RAPID SIMULTANEOUS ESTIMATION OF CALCIUM PANTOTHENATE AND BIOTIN IN PURE AND TABLET DOSAGE FORM Dissertation submitted to THE TAMILNADU Dr.M.G.R. MEDICAL UNIVERSITY CHENNAI- 600 032 In partial fulfillment of the requirements for the awards of the degree of MASTER OF PHARMACY IN PHARMACEUTICAL ANALYSIS Submitted by R. KIRUTHIKA (REG.NO: 261530352) Under the guidance of Dr. R. RAJAPANDI, M.Pharm., Ph.D., DEPARTMENT OF PHARMACEUTICAL ANALYSIS, ARULMIGU KALASALINGAM COLLEGE OF PHARMACY, ANAND NAGAR, KRISHNANKOIL-626126 OCTOBER - 2017
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DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR RAPID SIMULTANEOUS ESTIMATION OF CALCIUM
PANTOTHENATE AND BIOTIN IN PURE AND TABLET DOSAGE FORM
Dissertation submitted to
THE TAMILNADU Dr.M.G.R. MEDICAL UNIVERSITY CHENNAI- 600 032
In partial fulfillment of the requirements for the awards of the degree of
MASTER OF PHARMACY IN
PHARMACEUTICAL ANALYSIS
Submitted by
R. KIRUTHIKA (REG.NO: 261530352)
Under the guidance of
Dr. R. RAJAPANDI, M.Pharm., Ph.D.,
DEPARTMENT OF PHARMACEUTICAL ANALYSIS,
ARULMIGU KALASALINGAM COLLEGE OF PHARMACY,
ANAND NAGAR, KRISHNANKOIL-626126
OCTOBER - 2017
CERTIFICATE
This is to certify that the investigation described in the dissertation
entitled “DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR RAPID SIMULTANEOUS ESTIMATION OF CALCIUM PANTOTHENATE AND BIOTIN IN PURE AND TABLET DOSAGE FORM” submitted by
Reg. No: 261530352 research work was carried out in the Department of
Pharmaceutical Analysis, Arulmigu Kalasalingam College of Pharmacy,
Anand Nagar, krishnankoil-626126 which is affiliated to The Tamilnadu
Dr.M.G.R. Medical University, Chennai under my supervision and guidance
for the partial fulfillment of degree of MASTER OF PHARMACY in the
department of PHARMACEUTICAL ANALYSIS.
Place: Krishnankoil. Dr. R. RAJAPANDI M. Pharm., Ph.D., Date: Department of pharmaceutical Analysis,
Arulmigu Kalasalingam College of Pharmacy,
Krishnankoil – 626126.
CERTIFICATE
This is certify that the investigation described in the dissertation entitled
“DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR RAPID SIMULTANEOUS ESTIMATION OF CALCIUM PANTOTHENATE AND BIOTIN IN PURE AND TABLET DOSAGE FORM” submitted by
Reg.no: 261530352 research work was carried out in the Department of
Pharmaceutical Analysis, Arulmigu kalasalingam college of pharmacy, Anand
Nagar , Krishnankoil - 626126, which is affiliated to The Tamil Nadu
Dr.M.G.R. Medical University, Chennai, under the supervision and guidance
of Dr. R. RAJAPANDI M.Pharm., Ph.D., for the partial fulfillment of degree of
MASTER OF PHARMACY in the department of PHARMACEUTICAL ANALYSIS.
Place: Krishnankoil. Prof. Dr. N. VENKATESHAN M.Pharm.,Ph.D., Date: Professor & Principal,
Arulmigu Kalasalingam College of Pharmacy,
Krishnankoil - 626126.
EVALUATION CERTIFICATE
This is to certify that dissertation work entitled “DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR RAPID SIMULTANEOUS ESTIMATION OF CALCIUM PANTOTHENATE AND BIOTIN PRESENT IN PURE AND TABLET DOSAGE FORM” submitted by Reg.no: 261530352
was carried out in the Department of Pharmaceutical Analysis, Arulmigu
Kalasalingam College of Pharmacy, Anand Nagar, Krishnankoil -626126,
which is affiliated to The Tamil Nadu Dr.M.G.R. Medical University, Chennai,
under the supervision and guidance of Dr. R. RAJAPANDI M.Pharm., Ph.D., for the partial fulfillment of degree of MASTER OF PHARMACY in the
department of PHARMACEUTICAL ANALYSIS were evaluated by,
Centre : Arulmigu Kalasalingam College of Pharmacy, Krishnankoil.
Date :
Examiners :
1. 2.
ACKNOWLEDGEMENT
I pray our profound gratitude to the almighty god for this invisible help
and blessing for the fulfillment of this work.
I take this privilege and pleasure to acknowledgement the contribution
of many individuals who has been inspirational and supportive throughout my
work under taken and endowed me most precious knowledge to see the
success in my endeavor. My work bears the imprint of this people.
I am grateful to “Kalvivallal” Thiru T. Kalasalingam B.Com., for
providing me required facilities for extending a rich and also I convey my
sincere thanks to “llaiya vallal” Dr. K. Sridharan, Ph.D., Dynamic directors
Dr. S. Shasi Anand, Ph.D., Mr. S. Arjun Kalasalingam, M.S., and
management of our institution for providing me necessary infrastructure.
I expressed my sincere thanks to Dr. N. Venkateshan, M.Pharm., Ph.D., Principal of Arulmigu Kalasalingam College of Pharmacy, Krishnankoil,
for permitting me to do project work in Fourrts India Laboratories Pvt. Limited, Chennai. And for this enthusiastic cooperation and timely advice
and for providing faculties for the completion of my work
I give immense pleasure to express my deepest thanks, heartfelt
indebtedness and respectful guide Dr. R. Rajapandi, M.Pharm, Ph.D., for his
encouragement and guidance during the course of project and special thanks
for providing suggestions during the project. Especially for their patience and
immense acknowledge their constants quest for knowledge and strive for
excellence will always remain a source of inspiration to me.
I respectfully acknowledge to my faculties Dr. J. Amutha Iswarya Devi M.Pharm., Ph.D., Mrs. A. Thenmozhi M.Pharm., Mr. T. Senthil Kumar M.Pharm., for providing suggestions, encouragement during the project.
I express my special thanks to my Industrial Guide, Mr. J. Rajesh M.Sc., PGDQM., Manager and his team Mr. A. Baskar Palraj M.Pharm., Mrs. R. Chitra M.Pharm., Mr. S. Vijay M.Sc., Mr. M. Purushothaman M.Sc.,
Fourrts India Laboratories Pvt. Limited, Chennai for providing me
necessary facilities and constant source of inspiration and has always
encouraged scientific thinking to carry out this dissertation work for providing
much of stimuli in the form of suggestions and guidance of enormous support
for me during my entire project work. And also I would like to express my
respectful thanks to Mr. J. Srinivasan, President of HR, Fourrts India Laboratories Pvt. Limited, Chennai for permitting me to undertake the
research in their company.
I also convey my thanks to all the teaching and non-teaching staffs of
our institution for their help during the course.
I am thanking to all my friends who have lent a hand to complete this
dissertation.
Finally, I thank my GOD for giving me a very loving and caring family.
But for their unflinching support constant motivation, immense faith and love. I
wouldn’t have been able to thank my Mother and Father for always being
there for me. Above all it is Almighty who has been pouring his blessings on
me; to him I owe my lifelong indebtedness.
R.KIRUTHIKA.
INDEX
S. No Contents Page No
1 Chapter 1: Introduction 1
2 Chapter 2: Literature Review 13
3 Chapter 3: Research Objective &
Plan of Research Work 23
4 Chapter 4: Drug Profile 25
5 Chapter 5: Materials & Equipments 27
6 Chapter 6: Experimental Works 29
7 Chapter 7: Results & Discussion 55
8 Chapter 8: Summary & Conclusion 91
9 Chapter 9: Glossary 94
10 Chapter10: Bibliography 95
CHAPTER 1: INTRODUCTION
INTRODUCTION
Dept. of Pharmaceutical Analysis Arulmigu Kalasalingam College of Pharmacy Page 1
INTRODUCTION
This thesis deals with the studies carried for the simultaneous
estimation of calcium pantothenate and biotin in tablet dosage form by RP-
HPLC Method.
Modern medicines for human consumption are required to meet exact
standards which relate to their quality, safety and efficacy. The evaluation of
safety and their maintenance in practice is dependent upon the existence of
adequate methods for quality control of products. The purity of standard is a
must therefore, it can strictly define in such a way as to ensure that
successive batches are consistent in composition, irrespective of whether
they came from the same or different manufactures. Here lies the work of the
analytical chemist.
Definition:
Pharmaceutical analysis is branch of pharmaceutical chemistry, which
is define as a process or a sequence of processes to identify and quantify a
substance or drugs the components of a pharmaceutical solution or mixture or
the determination of the structural of chemical compounds used in the
formulation of pharmaceutical products1.
Pharmaceutical Analysis:
Modern methods of pharmaceutical analysis are extremely sensitive,
providing precise and detailed information from small samples of materials. In
general they are readily amenable to automation and also this method is very
rapid, due to this reasons they are now in widespread use in the product
development in the control of manufacturing formulations, as a check on
stability during storage and in monitoring the use of drugs and medicine2, 3.
Role of analytical chemistry in pharmaceutical analysis 4, 5:
Analytical chemistry may be defined as the science and art of
determining the composition of materials in terms of the elements of
composition contained. Its prime importance is to gain about the qualitative
and the quantitative information of the drug substance or chemical species
INTRODUCTION
Dept. of Pharmaceutical Analysis Arulmigu Kalasalingam College of Pharmacy Page 2
i.e., to find out what a substance is composed of and how much. In relation of
this context pharmaceutical analysis plays an important role in the quality
assurance and quality control of drug samples as well as pharmaceutical
formulations. In this connection it should be emphasized that the pharmacists
should be aware and capable of developing new methods for the purpose of
keeping the pharmaceutical industries and quality control laboratories in
viable position. The best way to characterize the quality of the drug is to
determine its purity. There are two possible approaches to reach this goal.
1. Determination of Active Pharmaceutical Ingredient (API).
2. Determination of its impurities with a highly accurate and precise
specific method.
Drugs and pharmaceuticals are chemicals or like substances, which
are of organic, inorganic or other origin. Whatever may be the origin, we use
some property of the medicinal agent to measure them qualitatively or
qualitatively. Pharmaceutical analytical techniques, which are being used, can
be categorized as follows.
Analytical Techniques:
There are three types analytical techniques are used as follows 6:
1. Spectroscopic analysis.
2. Electrochemical analysis.
3. Chromatographic technique.
SPECTROSCOPY:
Measurement based on light and other forms of electromagnetic
radiation are widely used to thought analytical chemistry. The interactions of
radiation and matter are the subject of the science called spectrometry.
Spectroscopic analytical methods are based on the measurement the amount
radiation produce or absorbed by molecular or atomic species of interest 7.
INTRODUCTION
Dept. of Pharmaceutical Analysis Arulmigu Kalasalingam College of Pharmacy Page 3
Spectrophotometric Methods:
The light absorption (or) emission characteristic of drugs is measured
such as UV-Visible, IR, NMR, ESR, fluorescence and mass spectroscopy.
UV-Visible - Changes in electronic energy levels within the
molecule.
Infrared - Changes in the vibrational and rotational movements
of the Molecule.
Microwave - Electron spin resonance or electron paramagnetic
resonance induces changes in the magnetic
properties of unpaired electron.
Radiofrequency - Nuclear magnetic resonance; induces changes in the
magnetic properties of certain atomic nuclei, notably
that of hydrogen and the C13 isotope of carbon 8.
Quantitative spectrophotometric assay of medical substance by using
UV-Visible spectroscopy:
The assay of an absorbing substance may be quickly carried out by
preparing a solution in a transparent solvent and measuring its absorption at a
suitable wavelength of maximum absorption where small errors in setting the
wavelength scale have little effect on the measurement absorbance of
approximately 0.9, around which the accuracy and precision of measurement
are optimal. The concentration of the absorbing substance is then calculated
from the measured absorbance using one of these principal procedures 9.
1. Use of standard absorptivity value.
2. Use of calibration graph.
3. Single or double point standardization.
4. Assay of substances multicomponent samples.
5. Assay as a single components sample.
INTRODUCTION
Dept. of Pharmaceutical Analysis Arulmigu Kalasalingam College of Pharmacy Page 4
6. Assay using absorbance corrected for interference.
7. Assay of after solvent extraction of the sample.
8. Simultaneous equations method.
9. Absorbance ratio method.
10. Geometric correction method 9.
CHROMATOGRAPHY:
Chromatography may be defined as a method of separating mixture of
components into individual components though equilibrium distribution
between two phases. Especially, the technique of chromatography is based
on the difference in the rate at which the components of the mixture move
thorough a porous medium under the influence of some solvent or gas. The
chromatographic method of separation, in general, it involves the following
steps (i.e) adsorption or retention of substance or substance on the stationary
phase. Separated substance by a continuous flow of mobile phase, the
method is being called elution.
High Performance Liquid Chromatography (HPLC):
The technique is based on the same mode of separation as that
classical column chromatography that is adsorption, partition, ion exchange
and gel permeation, but it differs from column chromatography in that the
mobile phase is pumped through the packed column under high pressure.
High performance liquid chromatography is a very sensitive analytical
technique most widely used for quantitative and qualitative analysis of
pharmaceuticals. The principle advantage of HPLC compared to classical
column chromatography is improved resolution of the separated substance,
faster separation times and the increased accuracy, precision, and sensitivity.
High-performance liquid chromatography sometimes referred to as high-
pressure liquid chromatography, which is a chromatographic technique used
to separate a mixture of compounds in analytical chemistry with the purpose
of identifying, quantifying and purifying the individual components of the
Dept. of Pharmaceutical Analysis Arulmigu Kalasalingam College of Pharmacy Page 27
MATERIALS AND EQUIPMENTS
Background:
The RP-HPLC method for the estimation of biotin and calcium
pantothenate in solid dosage form has been validated in Fourrts (India)
Laboratories Pvt. Ltd. Analytical method validation for the estimation of biotin
and calcium pantothenate in solid dosage form by RP-HPLC has been
conducted as per the protocol, to ensure that the performance characteristics
of the method meet the requirements for its intended application.
Required instrument and reagent:
Materials:
The following table list materials were used in this study.
S.No Name Grade Supplier Lot No/
B.No Purity Expiry
1 KH2PO4 AR Rankem, RFCL
Ltd, Mumbai. J037C16 100.0% Feb’20
2 KH2PO4 AR Finar Chemical
Ltd, Sanand. 21070305J12 99.5% Oct’17
3 Methanol HPLC Fischer
Chemicals. 1333170916 99.9% Oct’18
4 Methanol HPLC Finar Chemical
Ltd, Sanand. 168071027AP 99.8% Jun’18
5 H3PO4 AR Rankem, RFCL
Ltd, Mumbai. G025J15 88.0% Sep’18
6 Ammonia AR Fischer
Chemicals. 4929/17306-5 25.0% Jun’17
MATERIALS AND EQUIPMENTS
Dept. of Pharmaceutical Analysis Arulmigu Kalasalingam College of Pharmacy Page 28
Working standard:
The following table list standards were used in this study.
S.No Name Grade Lot No/ B.No Purity Expiry
1 Calcium pantothenate BP WS038/09 97.75 23/10/2017
2 Biotin BP WS103/07 99.35 09/02/2018
Instrumentation:
The following table list instruments were used in this study.
S.No Name of the Instrument Ref. Number Make Model
1 Electronic balance I/RD/OEB/EB/01 Adventurer
OHAUS AR2140
2 HPLC I/RD/HPC/02 Shimadzu Prominence
3 HPLC I/RD/HPC/04 Shimadzu LC-2010C HT
Column details:
The following table list column that was used in this study.
Column Ref.
Number I.D. Number Make Specification
1 RD/COL/76 K63402 Nacalai
Tesque,Inc.
Cosmosil ,5C18-MS-II
(250 X 4.6 mm, 5µ)
2 RD/COL/77 K65386 Nacalai
Tesque,Inc.
Cosmosil ,5C18-MS-II
(250 X 4.6 mm, 5µ)
CHAPTER 6: EXPERIMENTAL
WORKS
EXPERIMENTAL WORKS
Dept. of Pharmaceutical Analysis Arulmigu Kalasalingam College of Pharmacy Page 29
EXPERIMENTAL WORKS – (RP- HPLC)
Method development
1. Selection of wavelength:
Biotin and calcium pantothenate working standards were dissolved in
suitable diluent. And the solution were scanned between 200 to 400 nm using
uv-visible spectrophotometer then these were subjected to chromatograph
containing PDA detector to determine the accurate and optimized wavelength.
After reviewing the uv-visible Spectrum, a wavelength of 210 nm was
selected as the optimum wavelength for this method.
2. Selection of Mobile phase:
A mobile phase of water: methanol in the ratio of 1:1 was run through
the HPLC column. But the elution of drug in this mobile phase was poor. So,
mobile phase was changed to mono basic potassium dihydrogen
orthophosphate buffer and methanol in the ratio of 1:1 with pH of 4, again the
elution was poor. So, the concentration of same mobile phase was changed
to 60: 40 (buffer: methanol) and adjusts the pH 2.20 ± 0.05 with 10%
orthophosphoric acid and it was used.
3. Buffer preparation:
Dissolve 4.1 g of mono basic potassium dihydrogen ortho phosphate
in 1000 ml of water. Adjust the pH to 2.20 0.05 with 10 % orthophosphoric
acid. Filter through 0.45 μ membrane filter.
4. Mobile phase preparation:
Measure 600ml of buffer and 400ml of methanol to form the
concentration ratio of 60:40, mix well and degas it.
5. Standard solution (Calcium pantothenate):
Weigh accurately about 100.0 mg of calcium pantothenate working
standard into a clean 100 ml volumetric flask. Dissolve in 25 ml of water and
0.2 ml of ammonia solution. Sonicate for 5 minute, and make up the volume
with the water (Concentration: 1.0 mg/ml of calcium pantothenate).
EXPERIMENTAL WORKS
Dept. of Pharmaceutical Analysis Arulmigu Kalasalingam College of Pharmacy Page 30
6. Standard solution (Biotin):
Weigh accurately about 10.0 mg of biotin working standard into a clean
100 ml volumetric flask. Dissolve in 25 ml of water and 0.2 ml of ammonia
solution. Sonicate for 5 minute, and make up the volume with the water
(Concentration: 0.10 mg/ml of biotin).
7. Standard Solution (Calcium pantothenate+ biotin):
Weigh accurately about 100.0 mg of calcium pantothenate working
standard and 10 mg of biotin working standard into a clean 100 ml volumetric
flask. Dissolve in 25 ml of water and 0.2 ml of ammonia solution. Sonicate for
5 minute, and make up the volume with the water (Concentration: 1.0 mg/ml
of calcium pantothenate and 0.10 mg/ml of biotin).
Chromatographic conditions:
Column : Cosmosil, C18, 250 X 4.6 mm, 5 μm or equivalent
Flow rate : 1.0 ml / min
Detection wavelength : 210 nm
Injection volume : 20 L
Oven temperature : 30°C
Mobile phase : Buffer: methanol (60: 40)
Report:
It shows elution but the peak was not splitted in combined form.
EXPERIMENTAL WORKS
Dept. of Pharmaceutical Analysis Arulmigu Kalasalingam College of Pharmacy Page 31
For improving the elution of biotin and calcium pantothenate in
combination:
The peaks of biotin and calcium pantothenate were not separated in
the combined form. In order to separate peaks either change in ratio of mobile
phase or addition of peak modifier or ion pairing agent has to be used. Here
changing the buffer concentration should be used to its ability to shift peak in
the ratio of 70:30 (buffer: methanol) and thus giving good resolution.
Buffer preparation:
Dissolve 4.1 g of mono basic potassium dihydrogen ortho phosphate in
1000 ml of water. Adjust the pH to 2.20 0.05 with 10 % orthophosphoric
acid. Filter through 0.45 μ membrane filter.
Mobile phase preparation:
Measure 700ml of buffer and 300ml of methanol to form the
concentration ratio of 70:30, mix well and degas it.
Standard solution (Calcium pantothenate):
Weigh accurately about 100.0 mg of calcium pantothenate working
standard into a clean 100 ml volumetric flask. Dissolve in 25 ml of water and
0.2 ml of ammonia solution. Sonicate for 5 minute, and make up the volume
with the water (Concentration: 1.0 mg/ml of calcium pantothenate).
Standard solution (Biotin):
Weigh accurately about 10.0 mg of biotin working standard into a clean
100 ml volumetric flask. Dissolve in 25 ml of water and 0.2 ml of ammonia
solution. Sonicate for 5 minute, and make up the volume with the water
(Concentration: 0.10 mg/ml of biotin).
Standard solution (Calcium pantothenate+ biotin):
Weigh accurately about 100.0 mg of calcium pantothenate working
standard and 10 mg of Biotin working standard into a clean 100 ml volumetric
flask. Dissolve in 25 ml of water and 0.2 ml of ammonia solution. Sonicate for
5 minute, and make up the volume with the water (Concentration: 1.0 mg/ml
of calcium pantothenate and 0.10 mg/ml of biotin).
EXPERIMENTAL WORKS
Dept. of Pharmaceutical Analysis Arulmigu Kalasalingam College of Pharmacy Page 32
Chromatographic conditions:
Column : Cosmosil, C18, 250 X 4.6 mm, 5 μm or equivalent
Flow rate : 1.0 ml / min
Detection wavelength : 210 nm
Injection volume : 20 L
Oven temperature : 30°C
Mobile phase : Buffer: methanol (70: 30)
Report:
Elution of both biotin and calcium pantothenate was found to be
extremely good when change in concentration of mobile phase was used.
Selection of phase:
On the basis of RP-HPLC mode and number of carbon atom present in
molecule C18 bonded stationary phase was tried. Cosmosil, C18, 250 X 4.6
mm, 5 μm or equivalent, for this stationary phase was selected as it gives
good result in case of system suitability parameter i.e. resolution, USP
tangent, USP tailing.
Selection of other parameter:
Other parameter like mobile phase, flow rate, column, temperature and
wavelength of detector can be selected on the basis of chemical properties of
components present in the sample, sensitivity and system suitability
requirement of the analytical method.
EXPERIMENTAL WORKS
Dept. of Pharmaceutical Analysis Arulmigu Kalasalingam College of Pharmacy Page 33
OPTIMIZED METHOD
Buffer preparation:
Dissolve 4.1 g of mono basic potassium dihydrogen ortho phosphate in
1000 ml of water. Adjust the pH to 2.20 0.05 with 10 % orthophosphoric
acid. Filter through 0.45μ membrane filter.
Mobile phase preparation:
Measure 700ml of buffer and 300ml of methanol to form the
concentration ratio of 70:30, mix well and degas it.
Standard solution (Calcium pantothenate):
Weigh accurately about 100.0 mg of calcium pantothenate working
standard into a clean 100 ml volumetric flask. Dissolve in 25 ml of water and
0.2 ml of ammonia solution. Sonicate for 5 minute, and make up the volume
with the water (Concentration: 1.0 mg/ml of calcium pantothenate).
Standard solution (Biotin):
Weigh accurately about 10.0 mg of biotin working standard into a clean
100 ml volumetric flask. Dissolve in 25 ml of water and 0.2 ml of ammonia
solution. Sonicate for 5 minute, and make up the volume with the water
(Concentration: 0.10 mg/ml of biotin).
Standard solution (Calcium pantothenate+ biotin):
Weigh accurately about 100.0 mg of calcium pantothenate working
standard and 10 mg of biotin working standard into a clean 100 ml volumetric
flask. Dissolve in 25 ml of water and 0.2 ml of ammonia solution. Sonicate for
5 minute, and make up the volume with the water (Concentration: 1.0 mg/ml
of calcium pantothenate and 0.10 mg/ml of biotin).
EXPERIMENTAL WORKS
Dept. of Pharmaceutical Analysis Arulmigu Kalasalingam College of Pharmacy Page 34
Chromatographic conditions:
Column : Cosmosil, C18, 250 X 4.6 mm, 5 μm or equivalent
Flow rate : 1.0 ml / min
Detection wavelength : 210 nm
Injection volume : 20 L
Oven temperature : 30°C
Mobile phase : Buffer: methanol (70: 30)
Calculation:
% Assay of calcium pantothenate:
Area of sample X Std wt (mg) X 100 X Purity of std. X Avg wt. of tablet (mg) X 100 = ---------------------------------------------------------------------------------------- Area of std X 100 X Sample wt (mg) X 100 X 100 = --------------- % of calcium pantothenate
% Assay of biotin:
Area of sample X Std wt (mg) X 100 X Purity of std. X Avg wt. of tablet (mg) X 100 = ------------------------------------------------------------------------------------------------- Area of std X 100 X Sample wt (mg) X 100 X 10 = --------------- % of biotin
Parameters used for assay validation:
The validation of the assay procedure was carried out using the following
parameters.
1. Specificity:
Analyze the placebo, calcium pantothenate and biotin separately. A
solution of placebo was spiked with the calcium pantothenate and biotin at its
working concentration. The solution was analyzed as per the RP-HPLC
method described.
EXPERIMENTAL WORKS
Dept. of Pharmaceutical Analysis Arulmigu Kalasalingam College of Pharmacy Page 35
(i) Placebo solution:
Weigh accurately about 430 mg of powdered placebo into a clean
100 ml volumetric flask. Add 25 ml of water and 0.2 ml of ammonia solution.
Sonicate for 5 minute, and make up the volume with the water and filter
(Concentration: 4.30 mg/ml of placebo).
(ii) Standard solution (Calcium pantothenate):
Weigh accurately about 100.0 mg of calcium pantothenate working
standard into a clean 100 ml volumetric flask. Dissolve in 25 ml of water and
0.2 ml of ammonia solution. Sonicate for 5 minute, and make up the volume
with the water (Concentration: 1.0 mg/ml of calcium pantothenate).
(iii) Standard solution (Biotin):
Weigh accurately about 10.0 mg of biotin working standard into a
clean 100ml volumetric flask. Dissolve in 25 ml of water and 0.2 ml of
ammonia solution. Sonicate for 5 minute, and make up the volume with the
water (Concentration: 0.10 mg/ml of biotin).
(iv) Standard solution (Calcium pantothenate + biotin):
Weigh accurately about 100.0 mg of calcium pantothenate working
standard and 10 mg of biotin working standard into a clean 100 ml volumetric
flask. Dissolve in 25 ml of water and 0.2 ml of ammonia solution. Sonicate for
5 minute, and make up the volume with the water (Concentration: 1.0 mg/ml
of calcium pantothenate and 0.10 mg/ml of biotin).
(v) Standard + placebo solution:
Weigh accurately about 430.0 mg of placebo, 100.0 mg of calcium
pantothenate working standard and 10 mg of biotin working standard into a
clean 100 ml volumetric flask. Dissolve in 25 ml of water and 0.2 ml of
ammonia solution. Sonicate for 5 minute, and make up the volume with the
water (Concentration: 4.30 mg/ml of Placebo, 1.0 mg/ml of calcium
pantothenate and 0.10 mg/ml of biotin).
EXPERIMENTAL WORKS
Dept. of Pharmaceutical Analysis Arulmigu Kalasalingam College of Pharmacy Page 36
Acceptance criteria:
The placebo chromatogram should not show any peak at the retention
time of calcium pantothenate and biotin.
2. System precision:
A standard solution of 1.00 mg/ml of calcium pantothenate, and 0.10
mg/ml of biotin was prepared and analyzed as per the method.
(i) Standard solution:
Weigh accurately about 100.0 mg of calcium pantothenate working
standard and 10 mg of biotin working standard into a clean 100 ml volumetric
flask. Dissolve in 25 ml of water and 0.2 ml of ammonia solution. Sonicate for
5 minute, and make up the volume with the water (Concentration: 1.0 mg/ml
of calcium pantothenate and 0.10 mg/ml of biotin).
Procedure:
Inject the standard solution (6 injections). Ensure the following system
suitability criteria. Table 6.1 expresses the criteria for system precision.
Table 6.1: Acceptance criteria
System suitability parameter Acceptance criteria
Tailing factor NMT 2.0
Column efficiency NLT 2000 theoretical plates
Relative standard deviation NMT 2.0%
3. Linearity and Range:
(a) Linearity of Calcium pantothenate:
The linearity of the RP-HPLC method was demonstrated for
calcium pantothenate ranging from 0.4960 mg/ml to 1.4940 mg/ml, which is
equivalent to 50% to 150% of the calcium pantothenate working strength. Five
standard solutions at the concentrations within the mentioned range were
prepared and analyzed as per the method.
EXPERIMENTAL WORKS
Dept. of Pharmaceutical Analysis Arulmigu Kalasalingam College of Pharmacy Page 37
(b) Linearity of biotin:
The linearity of the RP-HPLC method was demonstrated for biotin
solutions ranging from 0.0520 mg/ml to 0.1500 mg/ml, which is equivalent to
50% to 150% of the biotin working strength. Five standard solutions at the
concentrations within the mentioned range were prepared and analyzed as
per the method.
Solutions:
(i) Level - I (50%):
Weigh accurately about 50.0 mg of calcium pantothenate working
standard and 5.0 mg of biotin working standard into a clean 100 ml volumetric
flask. Dissolve in 25 ml of water and 0.2 ml of ammonia solution. Sonicate for
5 minute, and make up the volume with the water (Concentration: 0.50 mg/ml
of calcium pantothenate and 0.05 mg/ml of biotin).
(ii) Level - II (80%):
Weigh accurately about 80.0 mg of calcium pantothenate working
standard and 8.0 mg of biotin working standard into a clean 100 ml volumetric
flask. Dissolve in 25 ml of water and 0.2 ml of ammonia solution. Sonicate for
5 minute, and make up the volume with the water (Concentration: 0.80 mg/ml
of calcium pantothenate and 0.08 mg/ml of biotin).
(iii) Level - III (100%):
Weigh accurately about 100.0 mg of calcium pantothenate working
standard and 10.0 mg of biotin working standard into a clean 100 ml
volumetric flask. Dissolve in 25 ml of water and 0.2 ml of ammonia solution.
Sonicate for 5 minute, and make up the volume with the water (Concentration:
1.00 mg/ml of calcium pantothenate and 0.10 mg/ml of biotin).
(iv) Level – IV (120%):
Weigh accurately about 120.0 mg of calcium pantothenate working
standard and 12.0 mg of biotin working standard into a clean 100 ml
volumetric flask. Dissolve in 25 ml of water and 0.2 ml of ammonia solution.
Sonicate for 5 minute, and make up the volume with the water (Concentration:
1.20 mg/ml of calcium pantothenate and 0.12 mg/ml of biotin).
EXPERIMENTAL WORKS
Dept. of Pharmaceutical Analysis Arulmigu Kalasalingam College of Pharmacy Page 38
(v) Level – V (150%):
Weigh accurately about 150.0 mg of calcium pantothenate working
standard and 15.0 mg of biotin working standard into a clean 100 ml
volumetric flask. Dissolve in 25 ml of water and 0.2 ml of ammonia solution.
Sonicate for 5 minute, and make up the volume with the water (Concentration:
1.50 mg/ml of calcium pantothenate and 0.15 mg/ml of biotin).
Procedure:
Prepare the above solutions ranging from 50% to 150% and inject each
level in duplicate.
Perform the Correlation co-efficient by covering at least five points and
report the linearity as the range for determining the assay.
Acceptance criteria:
The plot of concentration versus peak area should be linear with a
correlation co- efficient not less than 0.995.
4. Accuracy:
The accuracy of the method was determined by using three solutions
containing placebo spiked with calcium pantothenate and biotin at
approximately 50%, 100% and 150% of its working strength. Each level was
analyzed.
Solutions:
(i) Level - I (50%) – 1:
Weigh accurately about 430 mg of placebo, 50.0 mg of calcium
pantothenate working standard and 5.0 mg of biotin working standard into a
clean 100 ml volumetric flask. Dissolve in 25 ml of water and 0.2 ml of
ammonia solution. Sonicate for 5 minute, and make up the volume with the
water (Concentration: 4.30 mg/ml of placebo, 0.50 mg/ml of calcium
pantothenate and 0.05 mg/ml of biotin).
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Dept. of Pharmaceutical Analysis Arulmigu Kalasalingam College of Pharmacy Page 39
(ii) Level - I (50%) – 2:
Weigh accurately about 430 mg of placebo, 50.0 mg of calcium
pantothenate working standard and 5.0 mg of biotin working standard into a
clean 100 ml volumetric flask. Dissolve in 25 ml of water and 0.2 ml of
ammonia solution. Sonicate for 5 minute, and make up the volume with the
water (Concentration: 4.30 mg/ml of placebo, 0.50 mg/ml of calcium
pantothenate and 0.05 mg/ml of biotin).
(iii) Level - I (50%) – 3:
Weigh accurately about 430 mg of placebo, 50.0 mg of calcium
pantothenate working standard and 5.0 mg of biotin working standard into a
clean 100 ml volumetric flask. Dissolve in 25 ml of water and 0.2 ml of
ammonia solution. Sonicate for 5 minute, and make up the volume with the
water (Concentration: 4.30 mg/ml of placebo, 0.50 mg/ml of calcium
pantothenate and 0.05 mg/ml of biotin).
(iv) Level - II (100%) – 1:
Weigh accurately about 430 mg of placebo, 100.0 mg of calcium
pantothenate working standard and 10.0 mg of biotin working standard into a
clean 100 ml volumetric flask. Dissolve in 25 ml of water and 0.2 ml of
ammonia solution. Sonicate for 5 minute, and make up the volume with the
water (Concentration: 4.30 mg/ml of placebo, 1.00 mg/ml of calcium
pantothenate and 0.10 mg/ml of biotin).
(v) Level – II (100%) – 2:
Weigh accurately about 430 mg of placebo, 100.0 mg of calcium
pantothenate working standard and 10.0 mg of biotin working standard into a
clean 100 ml volumetric flask. Dissolve in 25 ml of water and 0.2 ml of
ammonia solution. Sonicate for 5 minute, and make up the volume with the
water (Concentration: 4.30 mg/ml of placebo, 1.00 mg/ml of calcium
pantothenate and 0.10 mg/ml of biotin).
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Dept. of Pharmaceutical Analysis Arulmigu Kalasalingam College of Pharmacy Page 40
(vi) Level – II (100%) – 3:
Weigh accurately about 430 mg of placebo, 100.0 mg of calcium
pantothenate working standard and 10.0 mg of Biotin working standard into a
clean 100 ml volumetric flask. Dissolve in 25 ml of water and 0.2 ml of
ammonia solution. Sonicate for 5 minute, and make up the volume with the
water (Concentration: 4.30 mg/ml of placebo, 1.00 mg/ml of calcium
pantothenate and 0.10 mg/ml of biotin).
(vii) Level – III (150%) – 1:
Weigh accurately about 430 mg of placebo, 150.0 mg of calcium
pantothenate working standard and 15.0 mg of biotin working standard into a
clean 100 ml volumetric flask. Dissolve in 25 ml of water and 0.2 ml of
ammonia solution. Sonicate for 5 minute, and make up the volume with the
water (Concentration: 4.30 mg/ml of placebo, 1.50 mg/ml of calcium
pantothenate and 0.15 mg/ml of biotin).
(viii) Level – III (150%) – 2:
Weigh accurately about 430 mg of placebo, 150.0 mg of calcium
pantothenate working standard and 15.0 mg of biotin working standard into a
clean 100 ml volumetric flask. Dissolve in 25 ml of water and 0.2 ml of
ammonia solution. Sonicate for 5 minute, and make up the volume with the
water (Concentration: 4.30 mg/ml of placebo, 1.50 mg/ml of calcium
pantothenate and 0.15 mg/ml of biotin).
(ix) Level – III (150%) – 3:
Weigh accurately about 430 mg of placebo, 150.0 mg of calcium
pantothenate working standard and 15.0 mg of biotin working standard into a
clean 100 ml volumetric flask. Dissolve in 25 ml of water and 0.2 ml of
ammonia solution. Sonicate for 5 minute, and make up the volume with the
water (Concentration: 4.30 mg/ml of placebo, 1.50 mg/ml of calcium
pantothenate and 0.15 mg/ml of biotin).
EXPERIMENTAL WORKS
Dept. of Pharmaceutical Analysis Arulmigu Kalasalingam College of Pharmacy Page 41
Procedure
Prepare the above solutions in the range of from 50%, 100% and 150%
and inject each solution in duplicate as per the test method.
Calculate the recovery in each level by calculating the measured
concentration against theoretical concentration.
Acceptance criteria:
The recovery should be in the range of 98.0-102.0%
5. Method precision:
The method precision was performed by analyzing a sample solution of
Hairgro tablets (B.No: RD16047) as per the test method (six replicate sample
preparation).
Solutions:
(i) Standard solution:
Weigh accurately about 430 mg of placebo, 100.0 mg of calcium
pantothenate working standard and 10.0 mg of biotin working standard into a
clean 100 ml volumetric flask. Dissolve in 25 ml of water and 0.2 ml of
ammonia solution. Sonicate for 5 minute, and make up the volume with the
water (Concentration: 4.30 mg/ml of placebo, 1.00 mg/ml of calcium
pantothenate and 0.10 mg/ml of biotin).
(ii) Test solution - 1:
Weigh and finely powder 20 tablets. Weigh accurately about 540
mg of powdered tablets into a clean 100 ml volumetric flask. Add 25 ml of
water and 0.2 ml of ammonia solution. Sonicate for 5 minute, and make up
the volume with the water and filter (Concentration: Equivalent to 1.00 mg/ml
of calcium pantothenate and 0.10 mg/ml of biotin).
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Dept. of Pharmaceutical Analysis Arulmigu Kalasalingam College of Pharmacy Page 42
(iii) Test solution - 2:
Weigh and finely powder 20 tablets. Weigh accurately about 540
mg of powdered tablets into a clean 100 ml volumetric flask. Add 25 ml of
water and 0.2 ml of ammonia solution. Sonicate for 5 minute, and make up
the volume with the water and filter (Concentration: Equivalent to 1.00 mg/ml
of calcium pantothenate and 0.10 mg/ml of biotin).
(iv) Test solution - 3:
Weigh and finely powder 20 tablets. Weigh accurately about 540
mg of powdered tablets into a clean 100 ml volumetric flask. Add 25 ml of
water and 0.2 ml of ammonia solution. Sonicate for 5 minute, and make up
the volume with the water and filter (Concentration: Equivalent to 1.00 mg/ml
of calcium pantothenate and 0.10 mg/ml of biotin).
(v) Test solution – 4:
Weigh and finely powder 20 tablets. Weigh accurately about 540
mg of powdered tablets into a clean 100 ml volumetric flask. Add 25 ml of
water and 0.2 ml of ammonia solution. Sonicate for 5 minute, and make up
the volume with the water and filter (Concentration: Equivalent to 1.00 mg/ml
of calcium pantothenate and 0.10 mg/ml of biotin).
(vi) Test solution - 5:
Weigh and finely powder 20 tablets. Weigh accurately about 540
mg of powdered tablets into a clean 100 ml volumetric flask. Add 25 ml of
water and 0.2 ml of ammonia solution. Sonicate for 5 minute, and make up
the volume with the water and filter (Concentration: Equivalent to 1.00 mg/ml
of calcium pantothenate and 0.10 mg/ml of biotin).
(vii) Test solution - 6:
Weigh and finely powder 20 tablets. Weigh accurately about 540
mg of powdered tablets into a clean 100 ml volumetric flask. Add 25 ml of
water and 0.2 ml of ammonia solution. Sonicate for 5 minute, and make up
the volume with the water and filter. (Concentration: Equivalent to 1.00 mg/ml
of calcium pantothenate and 0.10 mg/ml of biotin)
EXPERIMENTAL WORKS
Dept. of Pharmaceutical Analysis Arulmigu Kalasalingam College of Pharmacy Page 43
Procedure:
Prepare the test solution of Hairgro tablets as per the test method and
inject each solution. Calculate the precision of the method by calculating %
assay of each solution against standard solution. Report the % RSD of all
individual assay values.
Acceptance criteria:
The percentage relative standard deviation for the assay values should
be less than 2.0.
6. Ruggedness (Intermediate precision)
The ruggedness of the method was performed by analyzing a sample
solution of Hairgro tablets (B.No:RD16047) as per the test method (six
replicate sample preparation) and injected each solution in duplicate using
different instrument, column, reagent, and analyst on different days. The
results of set I were compared with the results of set II.
Table 6.2
Parameter Set I Set II
Instrument to instrument Instrument – 1 Instrument – 2
Column to column Column – 1 Column – 2
Reagent to reagent Reagent – 1 Reagent – 2
Analyst to analyst Analyst – 1 Analyst – 2
Day to day Day – 1 Day – 2
Solutions:
(i) Standard solution:
Weigh accurately about 430 mg of placebo, 100.0 mg of calcium
pantothenate working standard and 10.0 mg of biotin working standard into a
clean 100 ml volumetric flask. Dissolve in 25 ml of water and 0.2 ml of
EXPERIMENTAL WORKS
Dept. of Pharmaceutical Analysis Arulmigu Kalasalingam College of Pharmacy Page 44
ammonia solution. Sonicate for 5 minute, and make up the volume with the
water (Concentration: 4.30 mg/ml of placebo, 1.00 mg/ml of calcium
pantothenate and 0.10 mg/ml of biotin).
(ii) Test solution - 1:
Weigh and finely powder 20 tablets. Weigh accurately about 540
mg of powdered tablets into a clean 100 ml volumetric flask. Add 25 ml of
water and 0.2 ml of ammonia solution. Sonicate for 5 minute, and make up
the volume with the water and filter (Concentration: Equivalent to 1.00 mg/ml
of calcium pantothenate and 0.10 mg/ml of biotin).
(iii) Test solution - 2:
Weigh and finely powder 20 tablets. Weigh accurately about 540
mg of powdered tablets into a clean 100 ml volumetric flask. Add 25 ml of
water and 0.2 ml of ammonia solution. Sonicate for 5 minute, and make up
the volume with the water and filter (Concentration: Equivalent to 1.00 mg/ml
of calcium pantothenate and 0.10 mg/ml of biotin).
(iv) Test solution - 3:
Weigh and finely powder 20 tablets. Weigh accurately about 540
mg of powdered tablets into a clean 100 ml volumetric flask. Add 25 ml of
water and 0.2 ml of ammonia solution. Sonicate for 5 minute, and make up
the volume with the water and filter (Concentration: Equivalent to 1.00 mg/ml
of calcium pantothenate and 0.10 mg/ml of biotin).
(v) Test solution – 4:
Weigh and finely powder 20 tablets. Weigh accurately about 540
mg of powdered tablets into a clean 100 ml volumetric flask. Add 25 ml of
water and 0.2 ml of ammonia solution. Sonicate for 5 minute, and make up
the volume with the water and filter (Concentration: Equivalent to 1.00 mg/ml
of calcium pantothenate and 0.10 mg/ml of biotin).
EXPERIMENTAL WORKS
Dept. of Pharmaceutical Analysis Arulmigu Kalasalingam College of Pharmacy Page 45
(vi) Test solution - 5:
Weigh and finely powder 20 tablets. Weigh accurately about 540
mg of powdered tablets into a clean 100 ml volumetric flask. Add 25 ml of
water and 0.2 ml of ammonia solution. Sonicate for 5 minute, and make up
the volume with the water and filter (Concentration: Equivalent to 1.00 mg/ml
of calcium pantothenate and 0.10 mg/ml of biotin).
(vii) Test solution - 6:
Weigh and finely powder 20 tablets. Weigh accurately about 540
mg of powdered tablets into a clean 100 ml volumetric flask. Add 25 ml of
water and 0.2 ml of ammonia solution. Sonicate for 5 minute, and make up
the volume with the water and filter (Concentration: Equivalent to 1.00 mg/ml
of calcium pantothenate and 0.10 mg/ml of biotin).
Procedure:
Prepare the test solution of Hairgro tablets by different analyst with
different reagent on different day as per the test method. Inject each solution
with different instrument using different column, different reagent, different
analyst and different days.
Calculate the ruggedness of the method by calculating % assay of
each solution against standard solution.
Report the overall % RSD of all individual assay values in set-I and
set–II.
Acceptance criteria:
The overall % RSD should not be more than 2.0%.
7. Robustness
The following table (Table 6.3) shows the parameters of the method that
were altered to test the robustness of the method. The robustness of the
method is to be determined by analyzing the standard solution six times with
varying RP-HPLC conditions as described below. Table 6.4 expresses the
criteria for robustness
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Table 6.3: Parameters altered for robustness test
Parameter Actual Low High
Flow rate 1.00 ml/min 0.90 ml/min 1.10 ml/min
Mobile Phase ratio 70 : 30 72 : 28 68 : 32
Buffer pH 2.20 2.10 2.30
Column oven temperature 30°C 28°C 32°C
Table 6.4: Acceptance criteria
System suitability parameter Acceptance criteria
Tailing factor NMT 2.0
Column efficiency NLT 2000 theoretical plates
Relative standard deviation NMT 2.0%
7.1 Chromatographic conditions (Actual):
Column : Cosmosil, C18, 250 X 4.6 mm, 5 μm or equivalent
Flow rate : 1.0 ml / min
Detection wavelength : 210 nm
Injection volume : 20 L
Oven temperature : 30°C
Mobile phase : Buffer: methanol (70: 30)
Preparation of buffer:
Dissolve 4.1 g of mono basic potassium dihydrogen ortho phosphate in
1000 ml of water. Adjust the pH to 2.20 0.05 with 10 % orthophosphoric
acid. Filter through 0.45 μ membrane filter.
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Standard solution:
Weigh accurately about 100.0 mg of calcium pantothenate working
standard and 10 mg of Biotin working standard into a clean 100 ml volumetric
flask. Dissolve in 25 ml of water and 0.2 ml of ammonia solution. Sonicate for
5 minute, and make up the volume with the water.
Test Solution:
Weigh and finely powder 20 tablets. Weigh accurately about 540 mg of
powdered tablets into a clean 100 ml volumetric flask. Add 25 ml of water and
0.2 ml of ammonia solution. Sonicate for 5 minute, and make up the volume
with the water and filter.
7.2 Chromatographic conditions (Flow-Low):
Column : Cosmosil, C18, 250 X 4.6 mm, 5 μm or Equivalent
Flow rate : 0.9 ml / min
Detection wavelength : 210 nm
Injection volume : 20 L
Oven temperature : 30°C
Mobile phase : Buffer: methanol (70: 30)
Preparation of buffer:
Dissolve 4.1 g of mono basic potassium dihydrogen ortho phosphate in
1000 ml of water. Adjust the pH to 2.20 0.05 with 10 % orthophosphoric
acid. Filter through 0.45 μ membrane filter.
Standard solution:
Weigh accurately about 100.0 mg of calcium pantothenate working
standard and 10 mg of biotin working standard into a clean 100 ml volumetric
flask. Dissolve in 25 ml of water and 0.2 ml of ammonia solution. Sonicate for
5 minute, and make up the volume with the water.
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Test solution:
Weigh and finely powder 20 tablets. Weigh accurately about 540 mg of
powdered tablets into a clean 100 ml volumetric flask. Add 25 ml of water and
0.2 ml of ammonia solution. Sonicate for 5 minute, and make up the volume
with the water and filter.
7.3 Chromatographic conditions (Flow-High):
Column : Cosmosil, C18, 250 X 4.6 mm, 5 μm or equivalent
Flow rate : 1.1 ml / min
Detection wavelength : 210 nm
Injection volume : 20 L
Oven temperature : 30°C
Mobile phase : Buffer: methanol (70: 30)
Preparation of buffer:
Dissolve 4.1 g of mono basic potassium dihydrogen ortho phosphate in
1000 ml of water. Adjust the pH to 2.20 0.05 with 10 % orthophosphoric
acid. Filter through 0.45 μ membrane filter.
Standard solution:
Weigh accurately about 100.0 mg of calcium pantothenate working
standard and 10 mg of biotin working standard into a clean 100 ml volumetric
flask. Dissolve in 25 ml of water and 0.2 ml of ammonia solution. Sonicate for
5 minute, and make up the volume with the water.
Test solution:
Weigh and finely powder 20 tablets. Weigh accurately about 540 mg of
powdered tablets into a clean 100 ml volumetric flask. Add 25 ml of water and
0.2 ml of ammonia solution. Sonicate for 5 minute, and make up the volume
with the water and filter.
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7.4 Chromatographic conditions (Mobile Phase – Ratio - Low):
Column : Cosmosil, C18, 250 X 4.6 mm, 5 μm or equivalent
Flow rate : 1.0 ml / min
Detection wavelength : 210 nm
Injection volume : 20 L
Oven temperature : 30°C
Mobile phase : Buffer: methanol (72: 28)
Preparation of buffer:
Dissolve 4.1 g of mono basic potassium dihydrogen ortho phosphate in
1000 ml of water. Adjust the pH to 2.20 0.05 with 10 % orthophosphoric
acid. Filter through 0.45 μ membrane filter.
Standard solution:
Weigh accurately about 100.0 mg of calcium pantothenate working
standard and 10 mg of biotin working standard into a clean 100 ml volumetric
flask. Dissolve in 25 ml of water and 0.2 ml of ammonia solution. Sonicate for
5 minute, and make up the volume with the water.
Test solution:
Weigh and finely powder 20 tablets. Weigh accurately about 540 mg of
powdered tablets into a clean 100 ml volumetric flask. Add 25 ml of water and
0.2 ml of ammonia solution. Sonicate for 5 minute, and make up the volume
with the water and filter.
7.5 Chromatographic conditions (Mobile Phase – Ratio - High):
Column : Cosmosil, C18, 250 X 4.6 mm, 5 μm or equivalent
Flow rate : 1.0 ml / min
Detection wavelength : 210 nm
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Injection volume : 20 L
Oven temperature : 30°C
Mobile phase : Buffer: methanol (68: 32)
Preparation of buffer:
Dissolve 4.1 g of mono basic potassium dihydrogen ortho phosphate in
1000 ml of water. Adjust the pH to 2.20 0.05 with 10 % orthophosphoric
acid. Filter through 0.45 μ membrane filter.
Standard solution:
Weigh accurately about 100.0 mg of calcium pantothenate working
standard and 10 mg of biotin working standard into a clean 100 ml volumetric
flask. Dissolve in 25 ml of water and 0.2 ml of ammonia solution. Sonicate for
5 minute, and make up the volume with the water.
Test solution:
Weigh and finely powder 20 tablets. Weigh accurately about 540 mg of
powdered tablets into a clean 100 ml volumetric flask. Add 25 ml of water and
0.2 ml of ammonia solution. Sonicate for 5 minute, and make up the volume
with the water and filter.
7.6 Chromatographic conditions (Buffer pH - Low):
Column : Cosmosil, C18, 250 X 4.6 mm, 5 μm or equivalent
Flow rate : 1.0 ml / min
Detection wavelength : 210 nm
Injection volume : 20 L
Oven temperature : 30°C
Mobile phase : Buffer: methanol (70: 30)
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Dept. of Pharmaceutical Analysis Arulmigu Kalasalingam College of Pharmacy Page 51
Preparation of buffer:
Dissolve 4.1 g of mono basic potassium dihydrogen ortho phosphate in
1000 ml of water. Adjust the pH to 2.10 0.05 with 10 % orthophosphoric
acid. Filter through 0.45 μ membrane filter.
Standard solution:
Weigh accurately about 100.0 mg of calcium pantothenate working
standard and 10 mg of biotin working standard into a clean 100 ml volumetric
flask. Dissolve in 25 ml of water and 0.2 ml of ammonia solution. Sonicate for
5 minute, and make up the volume with the water.
Test solution
Weigh and finely powder 20 tablets. Weigh accurately about 540 mg of
powdered tablets into a clean 100 ml volumetric flask. Add 25 ml of water and
0.2 ml of ammonia solution. Sonicate for 5 minute, and make up the volume