analytical method development for impurities
TRANSCRIPT
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ANALYTICAL METHOD DEVELOPMENT FOR IMPURITIES
PRESENT IN HYDROXY CHLOROQUINE SULPHATE FROM
ANTIMALARIAL TABLET FORMULATION
SP. Karuppiah*, K. Anver Basha
Department of chemistry, Sathyabama University, Chennai-600 117,Tamilnadu,India.
ABSTRACT
The study reveals that stability indicating analytical method
development for the estimation of Impurities present in Hydroxy
chloroquine Sulphate (HCS) from anti malarial tablet formulation.The
developed method is specific,linear,accurate,robust and can be used for
regular analysis. The known impurities are identified and estimated
quantitatively as i) Hydroxy Chloroquine –O-Sulphate (HCOS) ii)4,7-
Dichloroquinoline (4,7 DCQ) iii)Desethyl Hydroxy Chloroquine
(DEHC) iv) Desethyl Chloroquine (DEC) by RP-HPLC method.The
Relative Response Factor(RRF) and Relative Retention Time (RRT)
has established for the quantification of impurities without using known impurities every
time. Gradient elution has set up in proper proportion with buffer: methanol (90:10) and
acetonitrile (100%) in two different channels using Inertsil C8-3 (150 X 4.6), 5µm column in
a detection wavelength of 380 nm. The range is covered from 50% to 150% of working
concentration of 2ppm which is the range of Hydroxy chloroquine sulphate tablets 200 mg.
The recovery study of each impurity is carried out and the result obtained is well within
acceptance criterion of 90% to 110%.
Keywords: Hydroxy chloroquine sulphate, impurity profiling, Hydroxy Chloroquine –O-
Sulphate, 4,7-Dichloroquinoline, Desethyl Hydroxy Chloroquine, Desethyl Chloroquine.
INTRODUCTION
The chemical name of Hydroxychloroquine Sulphate is (RS)-2-N-[4-(7-chloro-4-
quinolylamino) Pentyl ] - N-ethylaminoethanol sulphate.(C18H26ClN3O, H2SO4) having
molecular weight 434.0 which is white or almost white crystalline powder.(Figure-1)
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VVoolluummee 33,, IIssssuuee 77,, 11555566--11556677.. RReesseeaarrcchh AArrttiiccllee IISSSSNN 2278 – 4357
Article Received on
05 May 2014,
Revised on 25 May
2014,
Accepted on 25 June 2014
*Correspondence for Author
SP. Karuppiah*
Research Scholar, Sathyabama
University, Rajiv Gandhi Road,
JPR nagar,Chennai-600 117.
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Figure1 : Chemical structure of Figure2 : Chemical structure of
Hydroxy Chloroquine sulphate Hydroxy Chloroquine-O- sulphate
Figure3 : Chemical structure of Figure4 : Chemical structure of
4,7-Dichloroquinoline Desethyl hydroxyl chloroquine
Figure5 : Chemical structure of
Desethyl chloroquine
The chemical name of Hydroxy Chloroquine –O-Sulphate is ( 2-[[4-[(7-Chloro-4-
quinolinyl)amino]pentyl]ethylamino]ethanol 1-(Hydrogen Sulfate); 2-[[4-[(7-Chloro-4-
quinolyl)amino]pentyl]ethylamino]ethanol Sulfate (C18H26CLN3O4S) having molecular
weight 415.93.(Figure-2)
The chemical name of 4,7-dichloroquinoline is ( 4-[(7-chloroquinolin-4-yl)amino]-2-
[(diethylamino)methyl]phenol}, (C9H5Cl2N) having molecular weight 198.05. (Figure-3)
The chemical name of desethyl hydoxychloroquine is [4-[(7-Chloro-4-quinolyl) amino]
pentyl]amino]ethanol (C16H22ClN3O) having molecular weight 307.82. (Figure-4)
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The chemical name of desethylchloroquine is N4-(7-Chloro-4-quinolinyl)-N1-ethyl-1,4-
pentanediamine (C16H22ClN3) having molecular weight 291.82.(Figure-5)
The above four metabolites of hydroxy chloroquine sulphate is separated each other by a
single HPLC method to determine quantitatively the amount present in tablet formulation.
Specificity by selectivity is performed to prove the method is specific and stability indicating.
MATERIALS AND METHODS
The materials used for this study is formulated tablets, active pharmaceutical ingredient,
placebo used for making tablets, working /reference standards, Impurity standards, chemicals,
solvents and laboratory glassware.
RESULTS AND DISCUSSION
Table No.1: Drug Product Specification
Product Name Batch Number Manufacturer
Plaquebaf(Hydroxychloroquine
Sulphate tablets 200 mg)
FD/HCQS/054 In-house
Plaquenil(Hydroxychloroquine
Sulphate tablets 200 mg)
IW0015 Sanofi, Fawdon
manufacturing centre.
Table No.2: Chemicals & Reagents Specification
Chemicals Name Reagent Grade Manufacturer
Mono basic sodium phosphate
anhydrous
Analytical reagent Ranchem
Di basic potassium phosphate Analytical reagent Ranchem
Methanol HPLC Qualigen
Acetonitrile HPLC Qualigen
Triethylamine HPLC Ranchem
Water HPLC Ranchem
Table No.3: Drug Substance & Impurities Specification
Name of the standards Category Manufacturer
Hydroxychloroquine Sulphate API IPCA Laboratories
Hydroxychloroquine-O-Sulphate Impurity Analytica chemie Inc.,
4,7-Dichloroquinoline Impurity Analytica chemie Inc.,
Desethyl Hydroxy-chloroquine Impurity Analytica chemie Inc.,
Desethyl Chloroquine Impurity Analytica chemie Inc.,
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Table No.4: Drug Products Formula
Name of the Ingredients Quantity per tablet in mg Grade
DRY MIX
Hydroxychloroquine Sulphate 200.00 Pharmacopeial
Lactose monohydrate 75.00 Pharmacopeial
Maize Starch 15.25 Pharmacopeial
BINDER
Polyvinylpyrrolidone (K-30) 0.775 Pharmacopeial
Methocel E-5 (Hypromellose 5Cps) 2.325 Pharmacopeial
Purified Water 20.00 Pharmacopeial
LUBRICATION
Maize Starch 12.00 Pharmacopeial
Magnesium stearate 4.650 Pharmacopeial
Coating
Opadry White 04F58804
(Hypromellose 15 Cps, Macrogol
6000, Titanium dioxide)
10.00 Pharmacopeial
Total Coated tablet weight 340.00
Analytical Methods
The method established for the separation of all the impurities present in Hydroxy
chloroquine sulphate tablets 200 mg is by using the analytical column Inertsil C8-3,150 X
4.6mm, 5µm at 30°C column oven condition and 320 nm UV absorbance with 1 ml flow rate
of mobile phase –A & mobile phase –B in gradient elution.
i) Gradient Programme
TIME(MINS) MOBILE PHASE-A IN % MOBILE PHASE-B IN %
0 88 12
25 84 16
32 84 16
50 50 50
52 50 50
57 40 60
65 40 60
67 88 12
80 88 12
ii) Mobile phase Preparation
Transfer about 4 g of disodium hydrogen phosphate anhydrous, 2 g of potassium dihydrogen
phosphate and 0.5ml triethylamine in to beaker containing 1000ml water. Filter the solution
through 0.45µ membrane filter. Prepare Mobile phase A by mixing filtered and degassed
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mixture of buffer and methanol in the ratio of 90:10(%V/V).Use 100% acetonitrile as Mobile
phase B.The diluent is prepared by mixing water and methanol in the ratio of 50:50(%V/V) .
iii) Analytical Procedure
The diluted standard solution is prepared by dissolving known quantity of Hydroxy
chloroquine sulphate in diluent to get 2 ppm concentration.The resolution solution is
prepared by dissolving 10 ppm each of hydroxy chloroquine sulphate and hydroxyl
chloroquine-o- sulphate.To check the interference due to placebo 2ppm solution of placebo is
prepared. Finally 20 tablets fine powder is used to prepare 2ppm concentration of sample
solution.
Inject 20 microlitre each of Blank, Placebo, Resolution solution, Standard preparation and
sample preparation in to the chromatograph and record the response for all the peaks. The
system suitability is evaluated as % RSD of peak area responses of Hydroxychloroquine
sulphate from five replicate injections of standard solution should not be more than 5.0%,
% RSD of peak RT responses of Hydroxychloroquine sulphate from five replicate injections
of standard solution should not be more than 1.0%,the tailing factor for standard peak is
NMT 2.0,The theoretical plates for standard peak is NLT 2000 and The resolution between
Hydroxychloroquine and O-sulphate is NLT 3.0.
Figure5 : Blank Chromatogram
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Figure6 : 4,7-Dichloroquinoline Chromatogram
Figure7 : Desethyl chloroquine Chromatogram
Figure8 : Hydroxychloroquine-O- sulphate Chromatogram
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Figure9 : Resolution solution Chromatogram
Figure10 : sample spiked Chromatogram
Figure11 : sample Unspiked Chromatogram
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Figure12 : Hydroxy chloroquine standard Chromatogram
Figure13 : Peak Identification Solution Chromatogram
Calculate the content of each known impurity,unknown impurity and total impurities by
taking the expression that divide the area of each known impurity present is test preparation
by mean area of diluted standard preparation and is multiplied by respective concentration.
Table No.5: The RRT & RRF Impurities
Name of the Impurity RRT RRF
Hydroxychloroquine sulphate 1.00
Desethyl Hydroxy-chloroquine 0.55 0.99
Desethyl chloroquine 0.77 0.56
Hydroxychloroquine-O-sulphate 1.25 1.09
4, 7-Dichloroquinoline 2.02 0.84
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1) Known impurity Calculation (Desethyl Hydroxy-chloroquine)
AT1 Wtstd 1 50 p 100
----------------- X ---------- X ---------- X ---------- X ------- X ------ - X Wtavg X RRF1
AS 100 100 Wtspl 100 LC
2) Known impurity Calculation (Hydroxychloroquine-O-sulphate)
AT2 Wtstd 1 50 p 100
----------------- X ---------- X ---------- X ---------- X ------- X ------ - X Wtavg X RRF2
AS 100 100 Wtspl 100 LC
3) Known impurity Calculation (4, 7-Dichloroquinoline)
AT3 Wtstd 1 50 p 100
----------------- X ---------- X ---------- X ---------- X ------- X ------ - X Wtavg X RRF3
AS 100 100 Wtspl 100 LC
4) Known impurity Calculation (Desethyl chloroquine)
AT4 Wtstd 1 50 p 100
----------------- X ---------- X ---------- X ----------- X ------- X ------ - X Wtavg X RRF4
AS 100 100 Wtspl 100 LC
5) Highest Unknown impurity Calculation
AT5 Wtstd 1 50 p 100
----------------- X ---------- X ---------- X ----------- X ------- X ------ - X Wtavg
AS 100 100 Wtspl 100 LC
6) Total Unknown impurity Calculation
AT6 Wtstd 1 50 p 100
----------------- X ---------- X ---------- X ---------- X ------- X ------ - X Wtavg
AS 100 100 Wtspl 100 LC
AT1 = peak area due to Desethyl Hydroxy-chloroquine obtained with RS test
preparation
AT2 = peak area due to Hydroxychloroquine-O-sulphate obtained with RS test
preparation
AT3 = peak area due to 4, 7-Dichloroquinoline obtained with RS test preparation
AT4 = peak area due to Desethyl chloroquine obtained with RS test preparation
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AT5 = peak area due to Highest Unknown impurity peak obtained with RS test
preparation
AT5 = peak area due to Highest Unknown impurity peaks obtained with RS test
preparation
AS = Mean peak area due to Hydroxychloroquine Sulphate obtained with diluted
standard Preparation
Wtstd =Weight of Hydroxychloroquine Sulphate working standard in mg
Wtspl =Weight of sample taken in mg
Wtavg = Average weight of capsule in mg.
L.C. =Label claim in mg per tablet
P =Potency of Hydroxychloroquine Sulphate working standard in percentage on
as is basis
RRF1 =0.99
RRF2 =1.09
RRF3 =0.84
RRF4 =0.56
Total impurities = sum of all known impurities and unknown impurities
The impurities present in hydroxy chloroquine tablets have separated quantitatively to
determine content estimation in percentage by HPLC. The method precision value obtained
for six sets of sample preparation of the same batch is well within the acceptance criteria of
90% to 110% of label claim. The resolution between closely eluting impurity peaks are
increased by optimizing the mobile phase gradient programme.
Table No.6: Method precision value
Name of the Impurity SET1
%
SET2
%
SET3
%
SET4
%
SET5
%
SET6
%
Limits
%
Desethyl Hydroxy-
chloroquine 0.11 0.12 0.11 0.13 0.12 0.14 0.50
Desethyl chloroquine 0.06 0.06 0.07 0.08 0.05 0.07 0.15
Hydroxychloroquine-O-
sulphate 0.04 0.05 0.06 0.04 0.05 0.04 0.50
4, 7-Dichloroquinoline 0.02 0.03 0.02 0.03 0.02 0.02 0.10
Total unknown
Impurities 0.21 0.23 0.21 0.24 0.21 0.22 0.50
Total Impurities 0.44 0.49 0.47 0.54 0.45 0.47 1.0
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Specificity of the product has performed by selectivity to prove the method is specific. The
peak purity value of each impurity peak as well as main peak is passing and the system
suitability parameters are well within the acceptance criteria. The method is linear in the
range of 0.50 mcg to 3.0 mcg. The RRT and RRF has determined for this impurity separation
to eliminate the use of impurity standard for each analysis by applying the factors in
calculation part. The developed method is specific, linear, accurate and robust over a set of
validation parameters as per ICH guidelines. The method is suitable for the determination of
related substances estimation of Hydroxy chloroquine sulphate tablets.
ACKNOWLEDGEMENTS
Dr.K.Anver Basha, research guide, Sathyabama university for his expert guidance and
assistance.
Department of chemistry, Sathyabama University, Chennai-600 117,Tamilnadu,India,for
providing all the facilitation to complete this project.
IPCA Laboratories LTD., Chemical Research & Development Centre,142 AB, Kandivli
Industrial Estate, Kandivli (West),Mumbai 400 067, Maharashtra, for providing Active
pharmaceutical ingredients used for this development.
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