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Vivek B. Rajendra et al. World Journal of Pharmaceutical research

SPECTROPHOTOMETRIC METHOD FOR THE ESTIMATION OF

CLOPIDOGREL BISULPHATE RESIDUE IN SWAB SAMPLES

Vivek B. Rajendra*, Onkar J. Deshmukh, Pavan Kumar Rawat, Bhushan S. Gulecha,

Shailendrasingh Khushwaha, Shyam V. Ghadlinge

Shreya Life Sciences Pvt. Ltd. Waluj, Aurangabad-431136 Maharashtra, India.

ABSTRACT

A simple, sensitive, rapid, precise, cost effective and reproducible UV

spectrophotometric method has been developed for estimation of

Clopidogrel residue in swab samples to validate cleaning procedure.

The swabbing procedure was optimized in order to obtain suitable

recovery from stainless steel surface using Tex wipe polyurethane

swab stick. A mean recovery of 94.9733% was obtained when swab

dipped in methanol were used. Beers law is valid in concentration

range of 2.5-20 g/ml and UV detection was done at 217nm. The

proposed method was validated in terms of linearity, precision,

accuracy, limit of detection, limit of quantification. The proposedmethod was found to be accurate and precise for routine estimation of

Clopidogrel in quality control laboratories.

KEYWORDS: Clopidogrel, Swab testing, Spectrophotometric,

Cleaning validation.

INTRODUCTION

An important area of concern prior to the manufacture of pharmaceutical products is to assure

proper cleaning of equipments and its associated surfaces. The main rationale for cleaningprocedure validation is to provide documented evidence that the cleaning methods employed

within facility consistently control potential carryover of the product into subsequent product

to a level which is below predetermined levels. The subject of cleaning validation in

pharmaceutical industry is of greater importance and is considered as first step before going

to the next batch.[1-3]

Cleaning validation ensures the safety and purity of the product and

ultimately the quality of process. Now a days cleaning validation becomes regulatory

Volume 1, Issue 3, 850-858. Research Article ISSN 2277 7105

Article Received on11 June 2012,

Revised on 20 June 2012,

Accepted on 28 June 2012

*Correspondence for

Author:

* Vivek B. Rajendra

Shreya Life Sciences Pvt. Ltd.

Waluj, Aurangabad-431136

Maharashtra, India

[email protected]
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requirement. 1963 GMP regulations (part 133.4) and by 1978 cGMP regulations (section

211.6) states that manufacturing equipment must be maintained in clean and orderly manner.4

The main purpose of cleaning validation is to avoid contamination between batches of

different products or to prevent cross contamination. The cleaning validation consist of two

important activities, firstly, development and validation of analytical method for residual

determination at or level below the acceptance limit and secondly, development and

validation of proper cleaning procedure. The FDA has not published specific guidelines to set

acceptance specification or method for determining whether a cleaning process is validated

because of wide variation in the equipment and process used throughout finished and bulk

products. Therefore pharmaceutical companies are expected to establish acceptance criteria

should be practical, achievable, and verifiable and scientifically sound. Important is to

determine sensitivity of analytical method in order to set reasonable limit. Several acceptance

criterias have been reported in literature by industry representative including analytical such

as 100ppm, biological activity levels such as 1/1000 of normal therapeutic dose and

organoleptic levels such as no visible residue.[4-5]

Two types of sampling i.e. direct surface sampling and rinse sampling are reported in

literature. Rinse sampling technique samples a greater surface area but measure removable

rather than residual products (hence indirect method). Direct sampling is done by swab

testing which permit the hot spots and critical sites to be sampled and measure the actual

residues left on the equipment. Swab procedure should define the area to be monitored and

solvents if used.[6-7]

Generally purified water is advisable for cleaning the manufacturing surfaces and drugs

which are insoluble in water or having the light solubility forces pharmaceutical companies to

develop specific cleaning validation program for prevention of cross contamination between

batches of different products.[5]

Chemically Clopidogrel is methyl (2S)-2-(2-chlorophenyl)-2-(6,7-dihydro-4H-thieno[3,2-

c]pyridine-5-yl)acetate. Clopidogrel, an antiplatelet agent structurally and pharmacologically

similar to ticlopidine, is used to reduce atherosclerotic events such as myocardial infarction

stroke, and vascular death in patients who have had a recent stoke or have established

peripheral vascular disease. It is practically insoluble in water and slightly soluble in

methanol.[8-10]

Due to its poor solubility and sticky characteristics it is difficult to remove
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clopidogrel residue from manufacturing equipment surfaces. To control the effectiveness of

cleaning, the analytical method should be selective for substance considered and has to

provide sufficient sensitivity since the concentration levels of residue are low. Very few

methods have been reported in literature for estimation of clopidogrel in formulation.

Clopidogrel is soluble in methanol and shows increase in solubility at lower pH. This is the

reason to develop a simple and fast spectrophotometric method using methanol and 0.1N HCl

as solvents for the estimation of the clopidogrel which can be applied in the quality control

laboratories for swab sample analysis. The proposed analytical method has been validated in

terms of linearity, accuracy, precision, limit of detection (LOD) and limit of quantification

(LOQ).[11-13]

MATERIALS AND METHODS

Reagents and ChemicalsClopidogrel working standard was obtained as gift sample from Shreya Life Sciences,

Aurangabad (Standard potency). All other reagents used are analytical grade. Methanol (AR

grade) and 0.1N HCl were used as solvents for swab testing. The sample solution was passed

through 0.45mcm membrane filter. Swab sampling was achieved by using Tip Tx Tm

714

swabs from Tex wipe corporation, (Upper Saddle River, NJ).

Equipments

A Jasco V-630 UV/Visible double beam spectrophotometer (model) with 1 cm matched

quartz cells was used for spectral measurement. Schimadzu AX200 analytical balance was

used for weighing purposes. A tablet compression machine (Karnavati, Mumbai) was

selected for swab testing. An ultrasonic bath was used for extracting drug collected on the

swab.

Preparation of standard stock solution

A Standard solution of Clopidogrel (100g/ml) was prepared by dissolving 10mg

Clopidogrel in 10 ml methanol, ultrasonicating the solution for 10min. 1ml of this solution

was further diluted to10ml with 0.1NHCl to obtain standard stock solution of 100g/ml

concentration.

Preparation of calibration curve

Aliquots of 0.25 to 2 ml portions of the standard solution were transferred to a series of

calibrated 10 ml volumetric flasks, and volume was adjusted with 0.1 N HCl. Solutions were


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scanned in the range of 200-400 nm against blank 0.1 N HCl. The absorption maxima of

solutions were found to be at 217 nm. The absorbance of solutions was measured at 217nm

(Figure 1) against blank (Table I) and calibration curve was constructed (Figure 2). The

optical characteristics were summarized in Table II.

Table-I Calibration curve of Clopidogrel

Sr.No. Concentration

(g/ml)

Absorbance Standard

deviation

1 2.5 0.1478 0.01695

2 5.0 0.2867 0.01364

3 7.5 0.3850 0.00825

4 10.0 0.5398 0.01014

5 12.5 0.6111 0.00699

6 15.0 0.7623 0.01402

7 17.5 0.8658 0.00121

8 20.0 0.9816 0.00416

Fig.1 Absorbance spectrum of Clopidogrel

Fig.2 calibration curve of Clopidogrel


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Table-II Validation parameter

Sr.No. Parameter Result

1 Absorption maxima (nm) 217

2 Linearity Range (g/ml) 2.5-20

3 Standard Regression Equation Y = 0.0484X + 0.0251

4 Correlation Coefficient (R ) 0.997

5 Precision (%) 99.668, 98.563

6 Accuracy (% recovery SD) 94.9733 1.1052

7 LOD (g/ml) 1.711

8 LOQ (g/ml) 5.18

Recovery studies of clopidogrel from clean tip swabs and stainless steel plate

Stainless steel (2 X 2 sq. inch) plate was prepared in house was surface tested. A spiking

solution was prepared by dissolving 10 mg of clopidogrel in 10ml of methanol (AR grade) to

get concentration of1000g/ml. This was further diluted with 0.1N HCl to get 10g/ml. The

sample preparation for controlling the cleaning step of manufacturing process was performed

as follows.

Heads of fine T x Tm 714 swabs sticks were rinsed with methanol (AR grade). Using

appropriate glass micro syringes 0.4ml ,0.5ml, 0.6ml of above prepared spiking solutions (10

g/ml) were transferred on to three specific areas respectively ( corresponding to 80%, 100%,

120% levels of LOQ) on each recovery plate. The solutions on test surfaces were allowed to

dry. Swab sticks previously placed in 20ml glass test tube containing 5ml methanol (AR

grade) were used for swabbing the stainless steel plate. Swabbing was done first in horizontal

then vertical direction. Finally swab sticks were put again in 20ml test tube containing 10ml

0.1N HCl and ultrasonicated for 20min at an ambient temperature (to extract all the drug

present in the swab). This process was repeated thrice. Finally absorbance of these sample

solutions was measured at detection wavelength of 217nm.

Method for cleaning the instrument

Tablet compression machine was cleaned with dry cloth. To remove the traces of residue of

drug, machine was then cleaned with 2% SLS solutions twice and then cleaned (wiped) with


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methanol (AR grade) with sterile cotton plug.

Methods for swab testing

Critical sites were selected and marked with area shown in table III each swab was dipped in

5ml methanol. Swabs were taken in selected area using separate swab for different area

carefully. Swabbing is done first in horizontal then in vertical direction. Then swabs were

dipped in 10ml 0.1N HCl contained in 20ml glass test tube. Each sampled glass test tube was

wrapped and ultrasonicated for 20min to extract the entire drug present in the swab in to the

solvent. Extracted clopidogrel sample solutions were filtered using membrane filter (0.45

m) and analyse using UV-visible spectrophotometer at 217nm.

TableIII Critical sites and areas selected for UV reading

Critical sites selected Area used for swab

testing

Drug content (g)

Turret 2cm X 2cm Not detected

Upper Punch (12.5mm) 1cm X 1cm Not detected

Lower Punch (12.5mm) 1cm X 1cm Not detected

Die 1cm X 1cm 3.471

Upper camp tract 2cm X 2cm Not detected

Hopper2cm X 2cm 2.417

Feeder 2cm X 2cm 4.710

Platform 2cm X 2cm 14.690

RESULTS AND DISCUSSION

Development and Validation of Analytical method

Spectrophotometric method for the determination of clopidogrel was developed and validated

by determining the linearity, precision, accuracy, LOD, LOQ. Detection wavelength of

217nm was selected for analysis because of drug sufficient absorption, lesser interference and

low quantities of clopidogrel may be detected correctly.

Linearity

Clopidogrel exhibits its maximum absorption at 217 nm and obeyed Beer's law in the range

of 2.5-20 g/ml. linear regression of absorbance Vs concentration yielded equation


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Y = 0.0484X + 0.0251. The correlation coefficient (R2=0.998) obtained for regression line

showed excellent linearity relationship between absorbance and concentration of clopidogrel

shown in figure 2.

Precision

Intra-day precision was evaluated by analyzing six test samples of Clopidogrel. The

intermediate precision (inter-day precision) of the method was determined by evaluating the

samples of Clopidogrel on different days and by two different analysts in the same

laboratory. The assay and relative standard deviation (RSD) values are 99.668%, 0.8554 and

98.563%, 1.0603 respectively (Table-IV).

Table-IV Determination of Precision

Sample

Number

Assay of Clopidogrel as % of labelled amount

Analyst-I (Intra-day precision) Analyst-II (Inter-day precision)

1 99.842 97.360

2 98.190 97.124

3 100.323 99.493

4 100.559 99.431

5 99.860 98.732

6 99.239 99.239

Mean 99.668 98.563

Std. deviation 0.8554 1.0603

Accuracy

Accuracy of the procedure was determined by comparing the analytical amount determined

Vs known amount spiked at 80,100,120% of LOQ conc. with 3 measurements (n=3) for each

concentration level investigated. Accuracy defined as mean % recovery of 94.9733% shown

in Table-V indicates spectrophotometric method developed for clopidogrel could be

considered accurate within concentration range investigated.

Limit of Detection (LOD) and Limit of Quantification (LOQ)

The LOD and LOQ of clopidogrel were determined by using standard deviation of the

response and slope approach as defined in International Conference on Harmonization (ICH)

guidelines.12-13

The LOD and LOQ was found to be 1.711g/ml and 5.18 g/ml respectively.


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Table-V Determination of Accuracy

Concentration

(g/ml)

Level of addition % Recovery Avg. % recovery

4 80% of LOQ conc. 95.9762

94.9733 1.10525 100% of LOQ conc. 95.1502

6 120% of LOQ conc. 93.7876

Selectivity

During sample preparation some potential contaminant substance may extract from swab tip

which could interfere with quantitation of Clopidogrel. The selectivity was studied bycomparing swab blank solution and standard solution (10g/ml) of Clopidogrel.

CONCLUSION

The developed method was found to be simple, sensitive, accurate, precise, reproducible and

can be used for the routine determination of residual Clopidogrel (swab samples) in quality

control laboratories. The method requires inexpensive chemicals and can give rapid results as

compared to other analytical technique.

REFERENCES

1.Galatowitsch S. The importance of cleaning validation. Clean Rooms. 2000: 14(6):19-22.

2.Agalloco J. Points to consider in validation of equipment cleaning procedure.

J.Parentral.Sci.Tech. 1992: 42(5):163.

3.Le Blanc DA. Validated cleaning technologies for pharmaceutical manufacturing. Inter

Pharm Press. Denver. 2000.

4.Guide to inspection of validation of cleaning processes. Reference material for FDA

investigation and personnel, Food and Drug Administration, Washington DC. 1993.

5.Dutt R., Kapoor R.P., Dutt V., Singh G. and Kumar G. Spectrophotometric method for the

determination of valsartan residue in swab samples. Indian Drugs. 2007: 44(8):591-596.

6.Cleaning validation in active pharmaceutical ingredient manufacturing plant. 1999. Actice

Pharmaceutical ingredients committee.


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7.Engineer M., In., Cleaning validation mini monograph #1., Klenzaids Biocles Devices (P)

LTD.Mumbai, Klenzaids Academy.

8.Anandakumar K. et al. RP-HPLC analysis of Aspirin and Clopidogrel Bisulphate in

combination. Ind.J.Ph.Sci. 2007: 69(4):597-599.

9.Belal F. et al. Stability-Indicating Micellar Liquid Chromatographic Method for the

Determination of Clopidogrel. Application to Tablets and Content Uniformity Testing.

Journal of Liquid Chromatography & Related Technologies. 2009: 32(20): 2993 - 3008

10.Zaazaa HE. et al. Spectrophotometric and spectrodensitometric determination of

Clopidogrel Bisulfate with kinetic study of its alkaline degradation. Talanta. 2009: 78(3):

874-884.

11.U.S. Pharmacopoeia 26 and National Formulary 21. 2003. 26th

Revision. United States

Pharmacopoeial convention Rockville, MD: 2439.

12.ICH Q2A. Guidelines on validation of analytical procedure: Definitions and

Terminology Federal Register. 1995: 60:11260.

13.ICH Q2B. Guidelines on validation of analytical procedure: Methodology Federal

Register. 1996: 60:27464.

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