ANALYTICAL METHOD DEVELOPMENT FOR IMPURITIES ...

www.wjpps.com Vol 3, Issue 7, 2014.

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SP. Karuppiah et al. World Journal of Pharmacy and Pharmaceutical Sciences

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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SSJJIIFF IImmppaacctt FFaaccttoorr 22..778866

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)



AS 100 100 Wtspl 100 LC

3) Known impurity Calculation (4, 7-Dichloroquinoline)



AS 100 100 Wtspl 100 LC

4) Known impurity Calculation (Desethyl chloroquine)


----------------- X ---------- X ---------- X ----------- X ------- X ------ - X Wtavg X RRF4

AS 100 100 Wtspl 100 LC

5) Highest Unknown impurity Calculation


----------------- X ---------- X ---------- X ----------- X ------- X ------ - X Wtavg

AS 100 100 Wtspl 100 LC

6) Total Unknown impurity Calculation


----------------- 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.

REFERENCES

1. Snyder. L.R, Kirkland. J.J, Glajch. J.L, Practical HPLC method development, Wiley inter

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Rockville, thirty-six Revision and national formulary thirty-first edition, 2013.

3. Dr.chowdry KPR, Devalo Rao G, Himabindu G, Validation of analytical methods, The

Eastern Pharmacist,1999:39-41:497

4. Sethi, Quantitative Analysis of drugs in Pharmaceutical formulations, CBS publishers

and distributors, NewDelhi, Edition 3,1986:118-156:580-606

5. CRC handbook of chemistry and Physics, Edition 88, 2008

6. Clarke’s isolation and identification of drugs, Pharmaceutical press, 2nd Edition,

1986:201-205.

7. International journal of pharmaceutics,volume 43,Issue3,May1988,Pages215-219.

8. Journal of Chromatography B:Biomedical science and applications, Vol581, Issue1, 2,

October 1998,Pages 83-92


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9. Analytical and semi-preparative High performance liquid chromatographic separation and

assay of Hydroxy chloroquine enantiomers, Dion R.Brocks, Franco M.Pasutto,

Fakhreddin Jamali.

10. Journal of analytical toxicology,volume 18,Issue5,Pages255-260.

11. Journal of Chromatography A, Vol.391,1987,Pages233-242.

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