DEVELOPMENT AND VALIDATION OF STABILITY INDICATING RP UPLC METHOD FOR SIMULTANEOUS ESTIMATION OF THIOCOLCHICOSIDE AND KETOPROFEN IN COMBINED DOSAGE FORM

www.wjpr.net Vol 3, Issue 4, 2014. 1129

DEVELOPMENT AND VALIDATION OF STABILITY-INDICATING

RP-UPLC METHOD FOR SIMULTANEOUS ESTIMATION OF

THIOCOLCHICOSIDE AND KETOPROFEN IN COMBINED DOSAGE

FORM

Balan Paramasivam1*, Kannappan Nagappan2

1

Research Scholar, Faculty of Pharmacy, PRIST University, Thanjavur, Tamil Nadu, India

2

Associate Professor, Department of Pharmacy, Annamalai University, Annamalai Nagar,

Tamil Nadu, India

ABSTRACT

A stability indicating RP-UPLC method was developed and validated

for the simultaneous determination of Thiocolchicoside (TCC) 8mg

and Ketoprofen (KTP) 100mg in tablet dosage form. The

chromatographic separation was Carried out by Thermo Scientific

UPLC Instrument, Accela 1250 Pump, auto sampler with PDA

detector, using Thermo Scientific hypersil gold C18 column, (50 x

2.1mm) particle size 1.9µm using 0.005M KH2PO4 buffer, methanol

and acetonitrile in the ratio of 40:52:08, pH was adjusted to 3 with

ortho phosphoric acid as mobile phase at a flow rate of 250 µl/min

with the detection at 258nm. The run time of TCC and KTP was about

0.59 and 1.01 minutes respectively. The detector response is linear from 4.8-7.2 µg/ml and

64-96 µg/ml concentrations for TCC and KTP respectively. The linear regression equation

was found to be y = 14654x-100.29 (r2 = 0.9999) for TCC and y= 4098.3x +325.38 (r2 =

0.9999) for KTP. The detection limit and quantification limit was 0.19 µg and 0.58 µg for

TCC and 0.4 µg and 1.3 µg for KTP. The percentage of assay for TCC and KTP was about

99.90% and 99.71% respectively. The stability indicating capability was established by

forced degradation experiments. The method was satisfactorily validated as per the ICH

guidelines.

Keywords: Thiocolchicoside; Ketoprofen; Stability indicating; Method development; RP-UPLC; Validation.

Volume 3, Issue 4, 1129-1141. Research Article ISSN 2277 – 7105

Article Received on 15 April 2014,

Revised on 08 May 2014, Accepted on 01 June 2014

*Author for Correspondence Balan Paramasivam

www.wjpr.net Vol 3, Issue 4, 2014. 1130 INTRODUCTION

Thiocolchicoside (TCC) is chemically named as N-[(7S)-3-(β-D-Glucopyranosyloxy)-

1,2-dimethoxy -10- (methylsulfanyl) -9- oxo-5,6,7,9-tetrahydrobenzo[α]heptalen-7-yl] acetamide

(Fig.1). It is a semi synthetic sulfur derivative of colchicoside compound from Gloriosa

superba plant. It is used as muscle relaxant, anti-inflammatory, and analgesic drug and also

used topically for the treatment of muscular spasms and rheumatological disorders (The

Merck Index 14th edition, 2006; Indian Pharmacopoeia, 2010; M. Carta, L. Murru, P. Botta

et al., 2006)

Fig. 1 Chemical Structure of Thiocolchicoside

Ketoprofen (KTP) is chemically named as (RS)-2-(3-benzoylphenyl)-propionic acid (Fig.2).

It is a non steroidal anti-inflammatory drug, used to treat painful conditions such as arthritis,

sprains and strains, gout, menstrual pain, severe toothaches, inflammation, particularly in

rheumatic diseases (The Merck Index.,2006; www.patient.co.uk).

Fig.2 Chemical Structure of Ketoprofen

In Literature review the simple UV spectrophotometer (Wankhede, Sagar B et al., 2010;

Sasmita Kumari Acharjya et al., 2010; M. T. Harde et al., 2012; Chaudhari Bharat G. and

Trivedi Jalpesh B., 2012;) HPLC methods (Rosso A and Zuccaro S., 1998; Sagar

www.wjpr.net Vol 3, Issue 4, 2014. 1131

T.Vetrichelvan., 2014) and HPTLC method (Dnyansing K. Rajput et al., 2013) were reported

for thiocolchicoside and ketoprofen individually and in combination. Extensive literature

survey revealed that no stability indicating RP-UPLC method has been reported for

simultaneous estimation of thiocolchicoside and ketoprofen in combined dosage form.

EXPERIMENTAL Materials

TCC and KTP were obtained from Firstmed Therapeutics Pvt. Ltd, Pondicherry and their

percentage of purity was 97.0 and 99.99 % respectively. Water (HPLC grade) was obtained

from a Milli –Q water purification system, Acetonitrile (HPLC grade), Methanol (HPLC

grade) and KH2PO4 were of analytical grade purchased from Merck. The commercial

combination [LUPIFLEX, TCC (8mg) and KTP (100mg)] was purchased from local drug

store.

Instrumentation

A Thermo Scientific Ultra Performance Liquid Chromatography (UPLC) Instrument, Accela

1250 Pump and auto sampler with PDA detector and Thermo hypersil gold C18 column (50 x

2.1mm) particle size 1.9µm. The UPLC system was operational with CHROMQUEST

software for data processing. Sartorius analytical micro balance, ultra sonicator (DC1500H

MRC), pH meter (MKVI Systronics), micropipettes and micro-pore filtration set etc were

also used.

Chromatographic Conditions

The analysis was carried out on Thermo Scientific hypersil gold C18 Column (50 x 2.1mm,

particle size 1.9µm), using the mobile phase containing 0.005M KH2PO4 buffer, methanol

and acetonitrile in the ratio of 40:52:08, pH adjusted to 3 with ortho phosphoric acid. Then it

was filtered on 0.22 micron membrane filter and sonicated for 15 min. The injection volume

was 10 µl. The flow rate was 250µl/min. The wavelength was selected at 258 nm with PDA

detector for analysis. All determinations were performed at constant column temperature (25

± 20C). The total run time of the analysis was 3 min. The retention time of the TCC and ACF

www.wjpr.net Vol 3, Issue 4, 2014. 1132 Fig.3 UPLC Chromatorgram of Thiocolchicoside with Ketoprofen

Preparation of standard solution

The standard stock solutions were prepared by transferring 16 mg of TCC and 200 mg of

KTP working standards in 25ml volumetric flask and dissolved with methanol upto the mark.

From the standard stock solution 1ml was taken into 100ml flask, further diluted with mobile

phase to get the final concentrations of 6.4 µg/ml of TCC and 80 µg/ml of KTP.

Preparation of sample Solution

Accurately weighed 10 tablets were triturated with a mortar and pestle. An amount equivalent

to 16 mg of TCC and 200 mg of KTP sample was transferred to a 25 ml clean volumetric

flask, diluted with 10 ml methanol, sonicated to dissolve it completely and made the volume

with the same solvent. Further 1ml of the TCC and KTP of above stock solution was taken

into a 100ml volumetric flask, diluted with mobile phase and analyzed under optimized

chromatographic conditions.

Assay

The proposed procedures were successfully applied for the analysis of thiocolchicoside and

ketoprofen in tablet dosage form. The chromatogram was eluted and drug content in each

sample was calculated by comparison with appropriate standard solution of the drug. The

assay results were shown in Table 1.

Table 1 Assay of Thiocolchicoside with Ketoprofen

Formulation Drug Label Claim(mg) Purity (%)

Tablet Thiocolchicoside 8 99.90

www.wjpr.net Vol 3, Issue 4, 2014. 1133 Forced Degradation Studies

It is a process in which the natural degradation rate of a pharmaceutical formulation is

increased by applying the additional stress. UPLC method is used to separate, detect, and

quantify the various drug related degradation substances. The prepared sample (6.4 µg of

TCC and 80 µg of KTP) was treated with various degradation conditions (Blessy M.et.al,

2013; George Ngwa, 2010)such asacidic (0.1N HCL, 60ºC, 24 hours), alkaline (1N NaOH,

60ºC, 24 hours), oxidization (3% H2O2, 60ºC, 24 hours), thermal (60ºC, 24 hours) and

sunlight (24 hours). Both TCC and KTP were found to be highly sensitive to acidic, alkali

and oxidative degradation. The peak area and assay value were dropped in all the above

mentioned conditions. The results of forced degradation studies were given in Table.2 and

shown in Fig.4, Fig.5, Fig.6, Fig.7 and Fig.8.

Table 2 Forced Degradation Study Degration

Parameter

Degration Time

Initial Peak Area Peak Area Degraded Product

% of Recovery

% of Degradation

TCC KTP TCC KTP TCC KTP TCC KTP

Acid Degradation

(0.1N HCL,60°C) 24 Hours 87854 327928 80965 285037 92.2 86.83 7.8 13.17 Alkali Degradation

(0.1N NaOH, 60°C) 24 Hours 87854 327928 73137 320786 83.3 97.74 16.7 2.26 Peroxide

Degradation (3% H2O2, 60°C)

24 Hours 87854 327928 79047 285261 90.02 86.9 9.98 13.1

Thermal Degration

(Oven, 60°C) 24 Hours 87854 327928 74546 268453 84.9 81.78 15.1 18.22 Sunlight 24 Hours 87854 327928 71664 292696 81.62 89.17 18.38 10.83

Fig. 4 Acid Degradation

Minutes

0 1 2 3 4 5

www.wjpr.net Vol 3, Issue 4, 2014. 1134 Fig.5 Alkali Degradation

Fig.6 Peroxide Degradation

Fig.7 Thermal degradation

Minutes

0 1 2 3 4 5

V o lt s -2 0 2 V o lt s -2 0 2 T h io c o lc h ic o s id e K e to p ro fe n Minutes

0 1 2 3 4 5

V o lt s -1 0 1 2 V o lt s -1 0 1 2 T h io c o lc h ic o s id e K e to p ro fe n Minutes

0 1 2 3 4 5

www.wjpr.net Vol 3, Issue 4, 2014. 1135 Fig 8 Sunlight degradation

Method Validation

The developed method was validation as per ICH guidelines (ICH-Guidelines, 1995). The

validation parameters are linearity, accuracy, precision, limit of detection (LOD), limit of

quantitation (LOQ) and robustness.

System Suitability

The six replicated injections were made in the standards solution of both TCC and KTP and

system suitability parameters such as theoretical plates (USP), resolution (USP) and

asymmetry factor were evaluated. The results of the system suitability parameters were given

in Table 3. The 3D chromatogram was shown in Fig.9

Table 3: System Suitability Parameter

Parameters Thiocolchicoside Ketoprofen

Resolution Factor -- 2.6

Theoretical Plates 2281 2543

Asymmetric factor 1.52 1.75

Rt 0.588 1.001

Standard deviation 771 1869

RSD 0.86 0.55

Minutes

0 1 2 3 4 5

V

ol

ts

-5.0 -2.5 0.0 2.5 5.0

V

ol

ts

-5.0 -2.5 0.0 2.5 5.0

T

h

io

c

o

lc

h

ic

o

s

id

e

K

e

to

p

ro

fe

www.wjpr.net Vol 3, Issue 4, 2014. 1136 Fig.9 System Suitability 3D Picture of TCC and KTP

Linearity

Linearity was demonstrated from five different concentration levels for both TCC and ACF,

which were found to be linear in the range of 4.8 µg/ml to7.2 µg/ml and 64 µg/ml to 96

µg/ml respectively. The values were given in Table 4. Correlation coefficient for TCC and

KTP was found to be 0.9999 and 0.9999 respectively. The calibration curves were shown in

the Fig.10 and Fig.11.

Linearity Plot of Thiocolchicoside

y = 14654x - 100.29 R2 = 0.9999

-20000 0 20000 40000 60000 80000 100000 120000

0 1 2 3 4 5 6 7 8

Concentration (µg/ml)

A

rea

www.wjpr.net Vol 3, Issue 4, 2014. 1137

Linearity Plot of Ketoprofen

y = 4098.3x + 325.38 R2 = 0.9999

0 50000 100000 150000 200000 250000 300000 350000 400000 450000

0 10 20 30 40 50 60 70 80 90 100

Concentration (µg/ml)

A

re

a

Fig.11 Linearity Plot for ketoprofen

Table. 4. Linearity of Thiocolchicoside and Ketoprofen

Thiocolchicoside Ketoprofen

Concentration

(µg/ml) Area

Concentration

(µg/ml) Area

4.8 70067 64 263924

5.4 79157 72 295485

6 87089 80 326236

6.6 97185 88 363291

7.2 105516 96 392325

Accuracy (Recovery Studies)

To check the degree of accuracy of the method, the recovery studies were performed by

standard addition method at 80 %, 100 % and 120 %. Known amounts of standard mixture of

TCC and ACF were added to pre-analyzed samples and were subjected to the proposed

UPLC method. Results of recovery studies were given in Table 5.

Table 5 Accuracy (Recovery) Data

Parameters TCC KTP

% Recovery % RSD % Recovery % RSD

80% 99.94 0.2 100.08 0.28

100% 99.97 0.29 100.13 0.58

120% 100.31 0.59 100.07 0.23

Precision

Intra day precision (Repeatability) and Inter day precision (Reproducibility) was evaluated by

www.wjpr.net Vol 3, Issue 4, 2014. 1138

from a single lot formulation. Percentage relative standard deviation (%RSD) was calculated.

The results for precision were given in Table 6.

Table 6 Precision

Drug Intra day assay Inter day assay % Obtained % RSD % Obtained % RSD

TCC 100.73 0.18 100.62 0.15

KTP 99.87 0.29 99.85 0.33

Robustness

To evaluate the robustness of the developed RP-UPLC, small deliberate variations in the

parameters of optimized method were done. The effect of ±2 % change in flow rate, ±2 nm

wavelength and 2 different analysts on the retention time and area were studied. The results

of robustness were tabulated in Table 7.

Table 7: Results of Robustness

Factor Level Retention time Area (µv 2

sec)

TCC KTP TCC KTP

Standard 250µl / min 0.588 1.001 88047 319306

Flow rate 225µl / min 0.663 1.123 84452 314819

275µl / min 0.533 0.893 65668 249941

Wavelength 256nm 0.59 0.998 50504 253243

260nm 0.999 0.999 125943 308988

Detection Limit (DL) and Quantitation Limit (QL)

The detection limit (DL) is the lowest concentration of analyte in a sample that can be

detected. The quantification limit (QL) is defined as the lowest concentration of the substance

that can be quantified with acceptable precision and accuracy. The detection limit and

quantitation limit were calculated as DL = 3.3 x σ /slope and QL = 10.0 x σ / Slope, here σ is

residual variance due to regression. The DL and QL values were given in Table 8.

Table 8 Detection Limit and Quantitation Limit

Sensitivity TCC KTP

DL 0.19µg 0.4µg

www.wjpr.net Vol 3, Issue 4, 2014. 1139 RESULTS AND DISCUSSION

To develop a suitable UPLC method for analysis of the drugs in pharmaceutical formulation,

in the beginning various tests were carried out to select the optimum conditions. After many

number of trials various proportions of solvents including buffers and acetonitrile were used

for this study. The goal of this study was to develop a rapid UPLC method for the analysis of

TCC and KTP in a finished combined tablet dosage form using a 0.005M KH2PO4 buffer,

methanol and acetonitrile in the ratio of 40:52:08, pH was adjusted to 3 with ortho phosphoric

acid at a flow rate of 250 µl/min, hypersil C18 (50 x 2.1 mm), particle size 1.9µm column

with the PDA detection at 258 nm. The retention time was found to be 0.59 min and 1.01 min

for TCC and KTP respectively. The linear regression equations were y = 14654x-100.29 (r2 =

0.9999) for TCC and y= 4098.3x +325.38 (r2 = 0.9999) for KTP. The developed method was

found to be accurate and precise; the RSD values were less than 1. The stability indicating

capability was established by forced degradation experiments. The percentage of assay for

TCC and KTP was about 99.90 % and 99.71 % respectively.

CONCLUSION

The developed method was simple, rapid, and accurate for simultaneous determination of

thiocolchicoside and ketoprofen in combined tablet dosage form. This method shows accurate

precise and sensitive and less time consuming and also economical. Thus, the proposed

method can be easily adopted for routine analysis of thiocolchicoside and ketoprofen in

combined dosage forms.

ACKNOWLEDGEMENTS

The authors are very much grateful to PRIST University, Thanjavur, India and Ideal

Analytical & Research Institution, Puducherry, India for providing all the facilities to perform

the research work.

REFERENCES

1. Blessy M.et.al, Development of forced degradation and stability indicating studies of

drugs – A review, J. Pharm. Anal.(2013) http://dx.doi.org/10.1016/j.jpha.2013.09.003

2. Chaudhari Bharat G.* and Trivedi Jalpesh B; Simultaneous Spectrophotometric

Estimation of .

3. Thiocolchicoside and dexketoprofen trometamol in Pharmaceutical dosage form;

www.wjpr.net Vol 3, Issue 4, 2014. 1140

4. Dnyansing K. Rajput, Atul A. Shirkhedkar, Jyoti K. Rajput, Harun M. Patel,and Sanjay

5. J.Surana. Stability Studies of Thiocolchicoside in Bulk and Capsules Using RP-HPTLC /

Densitometry. Journal of Analytical Methods in Chemistry. Volume 2013 (2013), Article

ID 142628, 7 pages http://dx.doi.org/10.1155/2013/142628

6. G. Abirami,* and T. Vetrichelvan; A New RP-HPLC Method for Simultaneous

Estimation of

7. Thiocolchicoside and Ketoprofen in tablet dosage form; World Journal of Pharmacy and

Pharmaceutical Sciences, 2014, Volume 3, Issue 2, 2564-2575

8. George Ngwa, Forced Degradation as an Integral part of HPLC Stability – Indicating

Method

9. Development, Drug Delivery Technology, June 2010, Vol 10, No.5

10.Gupta KR et al., Development and Validation of Stability- Indicating RP-HPLC and

Assay Method for Determination of Thiocolchicoside in Capsule. American Journal of

PharmTech Research 2012. Vol.2, Issue. 1; page No. 589-602

11.Hilmi I, Bayram BO. Validation of analytical procedure for the determination of

Tiocolchicoside drug by HPLC.Salon1A & B (Intercontinental at the Plaza), 2007;281.

12.http://www.rxlist.com/orudis-drug.htm

13.http://www.patient.co.uk/medicine/ketoprofen-for-pain-and-inflammation

14.ICH-Guidelines Q2A, Validation of Analytical Procedures: Definition and terminology

Geneva, Switzerland 1995. 68-76.

15.ICH of Technical requirements for the registration of Pharmaceutical for Human Use

(ICH) Q2b.Validation Analyticla Procedures Methodology (1996)

16.M. T. Harde*, S. B. Jadhav, D. L. Dharam, P. D. Chaudhari: Development and Validation

of UV-Visible Spectrophotometric Methods for Simultaneous Estimation of

Thiocolchicoside and Dexketoprofen in Bulk and Tablet Dosage Form; International

Journal of Pharmaceutical Sciences and Drug Research 2012; 4(2): 160-163

17.M. Carta, L. Murru, P. Botta et al., “The muscle relaxant thiocolchicoside is an antagonist

of GABAA receptor function in the central nervous system,” Neuropharmacology, 2006,

vol. 51, no. 4,pp. 805–815

18.Ministry of Health & Family Welfare, “Indian Pharmacopoeia,”in Indian

Pharmacopoeial Convention, vol. 3, pp. 2213–2215,Ministry of Health & Family

Welfare, Ghaziabad, India, 6th edition, 2010.

19.Rosso A, Zuccaro S. Determination of alkaloids from the colchicines family by

www.wjpr.net Vol 3, Issue 4, 2014. 1141

20.Sasmita Kumari Acharjya, Priyambada Mallick, Pnakini Panda and M. Mathrusri

annapurna,Spectrophotometric methods for the determination of thiocolchicoside in bulk

and pharmaceutical dosage forms; J Pharm Educ Res. June 2010,Vol. 1, Issue.1

21.Sagar B.Wankhede*, Somnath S. Zambare, Nitin R.Dixit and Sohan S. Chitlange

RP-HPLC Method for Simultaneous Estimation of Thiocolchicoside and Ketoprofen in

Combined Dosage Form; Der Pharmacia Lettre, 2010, Vol.2, Issue.3; Page 315-320

22.The Merck Index: An Encyclopedia of chemicals, Drugs and Biological. Whitehouse

station, NJ, USA, 14th Edn, 2006; 1603.

23.The Merck Index: An Encyclopedia of chemicals, Drugs and Biological. Whitehouse

station, NJ, USA, 14th Edn, 2006; 918.

24.Venkatachalam T and Lalitha K.G; Reverse – Phase HPLC Method for Simultaneous

Analysis of Thiocolchicoside and Ketoprofen in tablet formulations; International Journal

of Pharmacy and Pharmaceutical Sciences 2013 Vol 5, Issue 4, 679-682

25.Wankhede, Sagar B, Zambare, Somnath S, Chitlange, Sohan S. Estimation of

Thiocolchicoside And Ketoprofen in pharmaceutical dosage form by Spectrophotometric