Development and Validation of UV Spectrophotometric Method ... · Preparation of Standard Stock...

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Indo American Journal of Pharmaceutical Research, 2013 ISSN NO: 2231-6876

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INDO AMERICAN

JOURNAL OF

PHARMACEUTICAL

RESEARCH

Development and Validation of UV Spectrophotometric Method for Estimation of

Diclofenac Sodium and Eperisone Hydrochloride as API and in Formulated Sustained

Release Granules

Bharat Jhanwar*, Joytosh Banerjee , Atul Kumar, Badri Prakash Nagori Department of Quality Assurance, Lachoo Memorial College of Science and Tech, Pharmacy wing, Sector-A, Shastri

Nagar, Jodhpur (Raj)-342003

Corresponding author

Bharat Jhanwar Lachoo Memorial College of Science and Tech, Sector-A, Shastri Nagar, Jodhpur (Raj) – 342003.

Email: - [email protected]

Copy right © 2013 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical

Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ARTICLE INFO ABSTRACT

Article history Received 20/03/2013

Available online

27/03/2013

Keywords Diclofenac sodium,

Dissolution studies,

Eperisone

Hydrochloride,

Formulation,

Simultaneous

estimation, Sustained

release granules.

The project, for the first time, reports the development of sustained release

granules of Diclofenac sodium and Eperisone hydrochloride. The granules were

subjected to physical tests, dissolution profiling as well as assay. The analytical

method was developed for both the drugs as API and for sustained release granules

on Shimadzu UV-1800 spectrophotometer by simultaneous equation method.

Phosphate buffer of pH 6.8 was selected as a common solvent for estimation of

Diclofenac and Eperisone at 276 nm and 261.4 nm respectively. Beer law was

obeyed in the range of 2-22 g/ml and 2.5-32.5 g/ml with correlation coefficient

of 0.999 and 0.998 respectively. Detection limit and quantification limit were

found to be 0.082 g/ml and 0.22 g/ml for Eperisone and 0.109 g/ml and

0.331 g/ml for Diclofenac respectively. Developed method was found to be

simple, accurate, precise, reproducible and in agreement with all the validation

criteria as per ICH with negligible interference from excipients. Assay results were

found to be 98.76% and 100.09% for Eperisone HCl and Diclofenac Na

respectively. The sustained release granules passed all the physical tests and

demonstrated good dissolution profile.

Please cite this article in press as Bharat Jhanwar et.al. Devlpopment and Validation of UV Spectrophotometric Method for

Estimation of Diclofenac Sodium and Eperisone Hydrochloride as API and in Formulated Sustained Release Granules. Indo American

Journal of Pharm Research.2013:3(3).

http://www.iajpr.com/index.php/en/

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Vol 3, Issue 3, 2013. Bharat Jhanwar et. al. ISSN NO: 2231-6876

Introduction

Eperisone hydrochloride (Figure 1), chemically called 4-ethyl 2-methyl piperidinopropiophenone (EMPP) hydrochloride,

is an antispasmodic agent.1 It has a relatively low incidence of central depression when compared with other anti-

spasmodic drugs, which makes it widely used for the therapeutic treatment of spastic patients to relieve skeletal muscle

stiffness and back pain. It is useful in Diabetical angiopathy and thromboangitis obliterans, raynauds syndrome.2,3

Eperisone Hydrochloride is official in Japanese pharmacopoeia. Diclofenac sodium (Fig. 2) is [o-(2, 6-Dichloroanilino)

phenyl] acetic acid, which has analgesic and antipyretic properties and weak anti-inflammatory activity.4-5

Diclofenac is

official in Indian pharmacopoeia, Japanese pharmacopoeia and British pharmacopoeia. A combination of Eperisone HCl

with Diclofenac sodium as sustained release formulation has been approved by CDSCO in India in 150:100 mg

proportion. Hence, a need was felt to develop approved sustained release formulation for quantitative estimation. The

literature survey revealed that there are some analytical methods reported for Eperisone Hydrochloride and its congeners

like HPTLC, HPLC and their add-ons either individually or in combination with other drug.6-9

Many methods have been

reported in literature for determination of Diclofenac with other drugs.10-12

However, literature did not reveal any reported

methods for the simultaneous estimation of Eperisone HCl and Diclofenac sodium in combined dosage form. Hence, aim

of present work is to formulate sustained release granules of the combination, evaluate it in terms of physical properties

and dissolution profile and develop an analytical technique for estimation of the combination in formulated sustained

release granules.

Figure 1: Eperisone hydrochloride

Figure 2: Diclofenac sodium

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Material and Methods

Equipments

Shimadzu UV-1800, connected to computer having UV- Probe software was employed with matching pair of 1 cm quartz

cuvette (Shimadzu Corporation, Kyoto, Japan). The spectral bandwidth is 0.5 nm. All weights were measured on Digital

balance GR-100 (A&D comp Ltd.).

Chemicals

Eperisone hydrochloride was kindly provided by Sun Pharma, Silvassa. Diclofenac sodium was kindly provided by

Anantha Drugs Ltd, Rajasthan. The purity of reference standards were more than 98.5%w/w. Starch, lactose, magnesium

stearate HPMC(LV-50) and talc were obtained from Loba chem. and Merck Ltd, Maharashtra. Distilled water was

obtained from in house laboratory. Chemicals and solvents used were of A.R. grade.

Preparation of buffer solution

Phosphate buffer of pH 6.8 was prepared according to I.P. 27.2 gm of potassium dihydrogen phosphate was dissolved in

1000 ml. 250 ml of this solution was transferred to a 1000 ml volumetric flask. 112 ml of 0.2M sodium hydroxide solution

was transferred. The volume was made upto 1000ml with distilled water to get a final solution of pH 6.8.

Preparation of Standard Stock Solution

Accurately weighed Eperisone and Diclofenac (50 mg) were separately transferred to 100 ml volumetric flask and

dissolved in phosphate buffer (6.8). Volumes were made up to the mark with phosphate buffer. Further 10 ml of each

resulted solution was transferred to a 100 ml volumetric flask separately and volume was made up to the mark to produce

final working standard stock solutions containing 50μg/ml of EPR and 50μg/ml DIC.

Simultaneous Equation Method

Working standard solutions having 10μg/ml of DIC and 15 μg/ml of EPR were scanned individually in range of

200-400 nm to determine there λmax. The λmax of DIC (Figure 3) and EPR (Figure 4) were found to be 261.4 nm

and 276 nm respectively.

Figure 3: Spectrum scan of Diclofenac Na (λmax276)

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Figure 4: Spectrum scan of Eperisone HCl (λmax261.4)

Standard solutions having concentration 2.5 to 32.5 g/ml for DIC were obtained by transferring 0.5, 1.0, 1.5,

2.0, 2.5, 3.0, 3.5, 4.0, 4.5 5.0, 5.5, 6.0, 6.5 ml of standard stock solution to 10 ml volumetric flasks. Standard

solutions of EPR in the concentration range of 2 to 22 g/ml were obtained by transferring 0.4, 0.8, 1.2, 1.6,

2.0, 2.4, 2.8, 3.2, 3.6, 4.0 and 4.5 ml of the stock solution to 10 ml volumetric flasks. The volume in each

volumetric flask was made up with phosphate buffer (6.8). The absorbance of these dilutions were measured at

λmax of each other against phosphate buffer as blank and absorptivity coefficients E(1%1cm) were calculated

using Beer Lambert law for each concentration. Calibration curves were plotted at each wavelength (Figure 5-

8). Linear equations generated by calibration curves are as follows

DIC at 276.0 nm, Y = 0.03018x - 0.00042, DIC at 261.4 nm, Y = 0.02418x - 0.00217

EPR at 276.0 nm, Y = 0.02950x + 0.01543, EPR at 261.4 nm, Y = 0.04836x + 0.00305

The concentration of two drugs in mixture was calculated by using following equations.

…………..1

and

…………..2

Where, Cx and Cy are the concentrations of DIC and EPR respectively in mixture and in sample solutions. A1 and A2 are

the absorbance of sample at 276 nm (λ1) and 261.4 nm (λ2) respectively, ax1 and ax2 are the absorptivity of DIC at 276 nm

and 261.4 nm, ay1 and ay2 are the absorptivity of EPR at 276nm and 261.4nm.

Formulation of granules

The formulation of sustained release granules of diclofenac sodium and eperisone hydrochloride was prepared in the ratio

of 1:1.5. The quantities of the drug as well the excipients used are shown in table 1.

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Table 1: Formulation of sustained release granules

S. no. Ingredients used in formula Quantity used (mg)

Batch 1 Batch 2

1 Eperisone Hydrochloride 150 150

2 Diclofenac Sodium 100 100

3 Hydroxy Propyl Methyl Cellulose (E 50-LV) 100 120

4 Starch paste (10%) Q.S. Q.S.

5 Lactose 84 65

7 Talc 11 10

8 Magnesium Stearate 4 3

Preparation of granules

Formulation of granules was done in two batches using wet granulation technique. Drug and polymer (H.P.M.C.) were

accurately weighed and sifted through sieve 40#. Binder was prepared using starch in water to generate starch paste.

Afterward cohesive mass was passed through sieve 22# and dried for 20-25 min. Dried granules were passed through

sieve 20# and mixed with other suitable excipients already sifted through sieve 60#. Dried granules with excipients were

packed in suitable air tight container for further evaluation.

Evaluation of granules

The granules were evaluated for various physical parameters like angle of repose, bulk density, tapped density, Carr’s

compressibility index, Hausner’s index and Loss on drying. Bulk density depends upon the geometry of particle and

cohesive property of it. These parameters in the prescribed limits show flow characteristics of the formulation. The results

are shown in table 2.

Table 2: Measurement of physical properties of granules

Parameters Batch-1 (Bt-1) Batch-2 (Bt-2)

Observations Mean Observations Mean

Angle of repose 19.6 20.1 19.8 19.8 21.1 21.2 21.1 21.1

Bulk Density 0.37 0.37 0.37 0.37 0.38 0.38 0.383 0.38

Tapped Density 0.45 0.44 0.45 0.45 0.48 0.46 0.478 0.48

Car’s index 18.4 17.3 17.18 17.6 20.3 20.1 19.8 20.3

Hausner’s index 1.22 1.21 1.207 1.21 1.25 1.25 1.258 1.25

L.O.D. (%Loss) 3.6 3.3

LOD: - Loss on drying

Method Validation

The developed methods have been validated in terms of linearity, range, specificity, accuracy, precision, assay, LOD and

LOQ as per ICH Q2(R1) guidelines (ICH, 2005).13

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Linearity

Appropriate aliquots of DIC and EPR standard stock solution were transferred to volumetric flask of 10 ml capacity. The

volume was adjusted to the mark with phosphate buffer. All absorbance were measured at 276 nm and 261.4 nm.

Calibration curves were constructed by plotting average absorbance (n=6) versus concentrations for both drugs. Straight

line equations were obtained from these calibration curves. Optical and regression parameters are given in Table 3.

Range

Range of an analytical method is defined as the interval between upper and lower levels.

Table 3: Optical and regression characteristics

Parameters Eperisone HCl Diclofenac sodium

261.4 nm 276 nm 276 nm 261.4 nm

Beer’s law limit 2.0-22.0 2.0-22.0 2.5-32.5 2.5-32.5

Absorptivity coefficients 48.40 31.45 31.17 24.02

Molar absorptivity 14.3 x 103 9.3 x 10

3 9.9 x 10

3 7.6 x 10

3

Sandell’s sensitivity 0.02066 0.03179 0.03208 0.04163

Slope (m)

Intercept (c)

0.04836 0.02950 0.03018 0.02418

+0.00305 +0.01543 -0.00042 -0.00217

Correlation coefficient (r2)

0.9996 0.9985 0.9989 0.9993

Table 4: Value of different type of range

S. No Range Diclofenac Na Eperisone HCl

1 Linearity 2.5-32.5µg/ml 2.0-22.0 µg/ml

2 Working 0.331-32.5 µg/ml 0.2476- 22.0 µg/ml

3 Target concentration 17.5 12.0

4 Target range 14.0, 17.5, 21.0 9.6, 12.0, 14.4

Working range: It begins from limit of quantitation to the maximum concentration used for the development of the

analytical method.

Linearity range: It is the interval in which the response is directly proportional to the concentration between the

upper and lower levels.

Target concentration: It is defined as the concentration, which is equal to the midpoint of linearity range.

Target range: It is that concentration which is 80%, 100% and 120% of the target concentration.

The various ranges have been reported in table 4.

Specificity

Commonly used excipients (lactose, starch, magnesium stearate and talc) were added into a pre-weighed quantity of

standard drug synthetic mixture (1:1.5) and then absorbance was measured before and after addition of excipients.

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Calculations were done to determine the quantity of the drugs and the interference of additives on absorbance as shown in

table 5.

Accuracy

Accuracy was determined from recovery of the method by spiking of standard drug mixture of DIC and EPR (1:1.5) to the

pre-analyzed granules mixture (12:8) preparation at 3 different concentration levels 20%, 40% and 60% of ratio of drug in

formulated granules. Spiking above that level produced absorbance beyond the beer lamberts range. Each concentration

was analyzed 9 times and average recoveries are shown in table 6.

Table 5: Specificity study for the synthetic mixture

S.No. Diclofenac sodium : Eperisone HCl % Interference

Diclofenac Na (276nm) Eperisone HCl (261.4nm)

1. 2.5:3.75 0.3846 0.806

2. 5.0:7.5 -0.196 0.132

3. 7.5:11.25 0.119 0.1776

4. 10.0:15 0.2979 0.465

5. 12.5:18.75 0.080 0.2668

6. 15.0:22.5 0.1980 0.2665

7. 17.5:26.25 0.1141 0.1888

Mean 0.142 0.145

Table 6: Data for accuracy or recovery studies

Preanalyzed granules mixture

EPR:DIC=12 : 8

Spiking concentration

EPR:DIC=1.5:1

Mean percent

recovery

%RSD

or %CV

Eperisone HCl (EPR) 2.5, 5.0 and 7.5 99.44 0.361 0.363

Diclofenac sodium (DIC) 1.68, 3.3 and 5.0 99.91 0.433 0.434

EPR: - Eperisone, DIC: - Diclofenac

Precision in mixture

Repeatability

It indicates the precision under the same operating conditions over a short interval of time and inter-assay

precision. Repeatability was performed for six times with single concentration ratio (12:8) in formulated

granules (EPR: DIC).

Intermediate Precision: In intra-day study concentration of drugs were calculated on the same day at an

interval of two hour. In inter day study the drug contents were calculated on three different days at 80%, 100%

and 120% of ratio (12:8) in formulated granules. The results of statistical analysis as are given in table 7.

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Limit of Detection and Quantitation

LOD and LOQ were determined from the linearity data. This helps to determine sensitivity of the method.

Assay

About 450 mg of formulated granules were weighed (unit batch) and were finely triturated. Proper dilutions were done up

to 8 g/ml and 12 g/ml of DIC and EPR respectively and absorbance were measured (n=6). Percent weight claim of

formulated sustained release granules of DIC and EPR was determined by using equation 1 and 2. The results are given in

table 8.

Figure 5: Standard curve of Eperisone HCl at 261.4 nm.

Figure 6: Standard curve of Eperisone HCl at 276nm

Figure 7: Standard curve of Diclofenac at 276 nm

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Figure 8: Standard curve of Diclofenac sodium at 261.4nm

Table 7: Precision studies values and results

Concentration levels

in ratio 1.5:1

Repeatability (12:8) Reproducibility

EPR 12 g/ml DIC

8 g/ml

Intra-day Inter-day

EPR DIC EPR DIC

9.6 : 6.4

(80%)

---------- ----------- 9.6

0.115

6.5

0.022

9.6

0.047

6.48

0.032

12 : 8

(100%)

12.06

0.047

8.04

0.0340

12.07

0.113

8.02

0.109

12.07

0.081

8.06

0.035

14.4 : 9.6

(120%)

---------- ----------- 14.4

0.026

9.67

0.041

14.42

0.054

9.58

0.042

Mean % RSD 0.3897 0.4228 0.71 1.05 0.513 0.461

EPR: - Eperisone, DIC: - Diclofenac, RSD: - Relative standard deviation

Table 8: Result of assay using developed method

Approved

formulation &

ratio (S.R.)

Actual Claim

weight (mg) in

prepared formula

Mean Concentration

found

( g/ml) *

Mean Amt. found per

450 (mg) *

Percent amount

found per 450 mg

(Assay)

EPR 150 153.6 12.05 151.7 98.76

DIC 100 102.4 8.02 102.5 100.09

* Average of six determination, SR: - Sustained release, mg: - milligrams

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In Vitro Release (Dissolution studies)

In vitro release was determined by using USP-II single paddle type apparatus. It was operated at 50 rpm in 1000 ml of

phosphate buffer (6.8) for 9 hours at 37 0.5◦C. The results are shown in table 9. Higuchi curve, Korsemeyer peppas curve

and First order release curve were used to represent the dissolution profile as shown in figure 9 to 14.

Figure 9: Higuchi curve for Diclofenac sodium

Figure 10: Higuchi curve for Eperisone hydrochloride

Figure 11:Korsmeyer Peppas curve for Eperisone hydrochloride

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Figure 12: Korsmeyer Peppas curve for Diclofenac sodium

Figure 13: First order release curve for Diclofenac sodium

Figure 14: First order release curve for Eperisone HCl

Results and discussions

Linearity: Beer law was obeyed in the range of 2-22 g/ml and 2.5-32.5 g/ml respectively for Eperisone

hydrochloride and Diclofenac sodium at 261.4 and 276.0 nm with coefficient of correlation 0.999 and 0.998, 0.999 and

0.998 respectively. The data are shown in table 3.

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Specificity

Developed method has negligible excipients interference of less than 0.5%. The data are shown in table 5.

Accuracy

Accuracy was determined using recovery studies on formulated sustained release formulation. Recovery was found to be

greater than 98% which showed good accuracy of method for estimation as show in table 6.

Precision

Precision was determined by studying the repeatability and intermediate precision. Results indicate good repeatability and

intermediate precision. Percentage RSD was found to be less than 2 as shown in table 7.

Sensitivity

The LOD values were found to be 0.0820 g/ml and 0.1092 g/ml. LOQ values were found to be 0.220 g/ml and 0.331

g/ml for EPR and DIC respectively. Low values of LOD and LOQ indicated good sensitivity of proposed methods.

Assay

Percentage assay of the drugs in formulated sustained release granules was found to be 98.76% and 100.09% for EPR and

DIC with standard deviation less than 2 respectively. The data are shown in table 8.

Dissolution studies

Batch I showed 98% release whereas batch II showed more sustained release of 95% after 9 hours that could be extended

up to 10 hours. Data of In-vitro dissolution profiling are given in table 9-10. Korsmeyer Peppas kinetic model was found

to exhibit good correlation coefficient of 0.99 for both drugs.

Table 9: - Dissolution profile of drug from sustained release granules

Percent drug dissolved/1000ml of phosphate buffer (6.8)

Time in

hours

Batch I Batch II

Diclofenac Na Eperisone HCl Diclofenac Na Eperisone HCl

1 24.6 22.6 23.2 21.5

2 41.1 33.8 32.5 28.7

3 56.4 51.6 42.7 38.3

4 69.2 60.2 51.7 45.7

5 77.1 76 61.7 54.3

6 84.6 82.1 71.6 67.9

7 93.6 88 83.6 73.8

8 96.7 94 87.2 81.8

9 98.4 98 95.1 91

HCl: - Hydrochloride, Na: - Sodium

Table 10: Correlation coefficients of kinetic models

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Kinetic Models Correlation Coefficient (r2 )

Batch –I (100 mg HPMC) Batch –II (120 mg HPMC)

DIC EPR DIC EPR

First order 0.952 0.93 0.904 0.914

Higuchi Square root 0.97 0.96 0.974 0.985

Korsmeyer Peppas 0.98 0.988 0.991 0.997

HPMC:- hydroxy propyl methyl cellulose and EPR: - Eperisone, DIC: - Diclofenac

Conclusion

Sustained release granules of Eperisone HCl and Diclofenac Na were successfully formulated and evaluated for physical

properties and dissolution profile. A simple and economic U.V. spectrophotometric method has also been successfully

developed estimation the same and was found to be specific, accurate and precise as well as having good reproducibility.

It followed all the parameters for validation as per ICH (Q2R1) guidelines. Hence, the developed method can be used as

routine analysis of any marketed sustained release dosage form of the given combination.

Acknowledgments

The authors are to M/s Sun Pharmaceuticals Ltd, Silvassa (Dadar) and M/s Anantha Drugs Ltd, Ganganagar (Raj.) for

standard drugs. The author is highly grateful to Prof (Dr) Javed Akhtar (Department of QA, JNU, Rajasthan), for his

constant guidance and support. The author would also like to thank Mr. Sourabh Jain and Mr. Shekhar Choudhary for

estending their support in the completion of the project.

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