High Performance Thin Layer Chromatographic Determination of Diclofenac Sodium and Thiocolchicoside in Fixed Dose Combination

Deshpande P et al.
IRJP 1 (1) 2010 220-224
INTERNATIONAL RESEARCH JOURNAL OF PHARMACY

Available online http://www.irjponline.com
Research Article
HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHIC

DETERMINATION OF DICLOFENAC SODIUM AND THIOCOLCHICOSIDE IN
FIXED DOSE COMBINATION
Gandhi Santosh, Deshpande Padmanabh*, Sengar Mahima
Department of Pharmaceutical Analysis, A.I.S.S.M.S.College of Pharmacy, Kennedy Road, Near R.T.O.,
Pune, Maharastra, India
*
Mr. Padmanabh B. Deshpande, Senior Lecturer, Dept. of Pharm. Analysis, AISSMS College of
Pharmacy, Kennedy road, Near R.T.O., Pune - 411 001. India
E-mail: [email protected]
Article Received on: 12/11/10 Revised on: 30/11/10 Approved for publication: 03/12/10
ABSTRACT
A simple, specific, accurate, and precise high-performance thin-layer chromatographic method for
analysis of Diclofenac Sodium and Thiocolchicoside in fixed dose combination has been developed and
validated. The method use aluminium plates precoated with silica gel 60 F254 as stationary phase and
Toluene: Ethyl acetate: Methanol (5:3:2, v/v/v) as mobile phase. Densitometric evaluation of the
separated bands was performed at 285 nm. The two drugs were satisfactorily resolved with Rf values 0.53
± 0.005 and 0.17 ± 0.002 for Diclofenac Sodium and Thiocolchicoside, respectively. Results were found
to be linear over the concentration range 50–300 ng/ band for both the drugs. Intra-day variation, as RSD
(%), was 0.468 for Diclofenac Sodium and 0.357 for Thiocolchicoside. Interday variation, as RSD (%)
was 0.949 for Diclofenac Sodium and 0.739 for Thiocolchicoside. This method has been successfully
validated and applied for the analysis of drugs in pharmaceutical formulation. The % assay (Mean ± S.D.)
was found to be 100.521 ± 0.833 for Diclofenac Sodium and 101.132 ± 0.612 for Thiocolchicoside.
KEYWORDS: High performance thin layer chromatography, Thiocolchicoside, Diclofenac Sodium
INTRODUCTION
Diclofenac sodium (DIC), [Sodium (o- {(2, 6-dichlorophenyl) amino} phenyl) acetate] is a
synthetic nonsteroidal anti-inflammatory drug (NSAID), has been proved to be safe and efficacious drug
in the treatment of a variety of inflammatory and rheumatoid disorders1. Thiocolchicoside (THIO),
chemically, is (s)-N-[3-(B-D-glucopyranoxyloxy)-5,6,7,9-tetrahydro-1,2-dimethoxy-10-(methylthio)-9-
oxobenzo [a] heptalen-7yl] acetamide. Thiocolchicoside is a muscle relaxant which has been claimed to
possess GABA mimetic and glycinergic actions. It is used in the symptomatic treatment of painful
muscle spasm2.
Literature survey reveals spectrophotometric3 and HPTLC4 5 determination of DIC in combination
with other drugs. HPLC6 and bioanalytical chromatographic methods7 for quantification of DIC are also
reported. For simultaneous determination of THIO with other drugs spectrophotometric8, HPTLC9 and
HPLC methods10,11 are reported.
No reports were found for determination of DIC and THIO by HPTLC method in fixed dose
combination. Aim of present work was to develop simple, economical, rapid, accurate and precise
HPTLC method for determination of these drugs in fixed dose combination. The proposed method is
optimized and validated as per the International Conference on Harmonization (ICH) guidelines12.
IRJP 1 (1) Dec 2010 Page 220-224

Deshpande P et al. IRJP 1 (1) 2010 220-224
MATERIALS AND METHODS

Reagents and Chemicals
Analytically pure samples of DIC and THIO were kindly supplied by Cipla Pvt. Ltd. (Mumbai,
MH) and Aventis Pharma Pvt. Ltd (Goa, India), respectively and were used as such without further
purification. The pharmaceutical dosage form used in this study was a THIOACT-D4 (Sun
Pharmaceuticals Industries Ltd, Mumbai, India) capsule labeled to contain 4 mg of Thiocolchicoside and
50 mg of Diclofenac sodium enteric coated tablet, per capsule were procured from local market.
Instrumentation and Chromatographic Conditions
The samples were applied in the form of bands of width 6 mm with space between bands of 5 mm,
with a 100 µL sample syringe (Hamilton, Bonaduz, Switzerland) on precoated silica gel aluminium plate
60 F254 (10 ×10) with 250 µm thickness (E. MERCK, Darmstadt, Germany) using a CAMAG Linomat 5
sample applicator (Switzerland). The plates were prewashed with methanol and activated at 110 0C for 5
minute, prior to chromatography. The slit dimensions 5 mm × 0.45 mm and scanning speed of 20 mm/sec
were employed in analysis.
The linear ascending development was carried out in 10 cm ×10 cm twin trough glass chamber
(CAMAG, Muttenz, Switzerland) using mobile phase Toluene: Ethylacetate: Methanol (5:3:2, v/v/v). The
optimized chamber saturation time for mobile phase was 15 min. The length of chromatogram run was 9
cm and development time was approximately 30 min. HPTLC plates were dried in a current of air with
the help of a hair dryer. Densitometric scanning was performed on CAMAG thin layer chromatography
scanner 3 at 285 nm for all developments operated by WINCATS software version 1.4.2. The source of
radiation utilized was deuterium lamp emitting a continuous UV spectrum between 200 to 400 nm.
Preparation of Standard Stock Solutions
Standard stock solutions of DIC and THIO were prepared by dissolving 50 mg of drug in 50 mL
methanol separately to get concentration of 1 mg/mL from which 0.5 mL was further diluted to 10 mL to
get working standard stock solution of 50 ng/µL for both the drugs.
Selection of Detection Wavelength
After chromatographic development spots were scanned over the range of 200-400 nm and the
spectra were overlain. It was observed that both drugs showed considerable absorbance at 285 nm. So,
285 nm was selected as the detection wavelength as shown in Figure 1.
Preparation of Calibration Curve
The standard stock solutions of DIC and THIO (50 ng/µL each) were applied by overspotting on
HPTLC plate in range of 2-10 µL with the help of CAMAG 100 µL sample syringe, using Linomat 5
sample applicator. The plate was developed and scanned under above established chromatographic
conditions. Each standard in five replicates was analyzed and peak areas were recorded. Calibration
curves of DIC and THIO were plotted separately of peak area Vs respective concentration of DIC and
THIO.
Analysis of Capsules
Content of twenty capsules were weighed accurately and finely powdered. Two different stock
solutions were prepared for DIC and THIO. A quantity of powder equivalent to 10 mg DIC was weighed
and transferred to a 10 mL volumetric flask containing approximately 5 mL methanol. The mixture was
ultrasonicated for 5 min and diluted to volume with methanol. The solution was filtered using Whatman
no. 41 paper. From stock solution 0.5 mL was taken and diluted to 10 mL with methanol. 2 µL of this
solution was applied to a TLC plate to furnish 100 ng /band for DIC. Similarly a quantity of powder
equivalent to 10 mg THIO was weighed and transferred to a 10 mL volumetric flask containing
approximately 5 mL methanol. The mixture was ultrasonicated for 5 min then diluted to volume with
methanol. The solution was filtered using Whatman no. 41 paper. 2 µL of this stock solution was applied
to a TLC plate to furnish 100 ng / band for THIO. After chromatographic development the peak areas of
the bands were measured at 285 nm and the amount of each drug in each tablet was determined from the
respective calibration plots. The analytical procedure was repeated six times for the homogenous powder
sample.
IRJP 1 (1) Dec 2010 Page 220-224

Recovery Studies
To check the accuracy of the method, recovery studies were carried out by addition of standard
drug solution to pre-analyzed sample solution at three different levels 50, 100 and 150 %.
Precision
To study intra-day variation, six mixed standard solutions containing DIC (100 ng/band) and
THIO (100 ng/band) were prepared and analyzed on the same day to record any intra-day variation in the
results. To study inter-day variation, analysis of three mixed standard solutions of the same concentration
was performed on three different days.
Specificity and Selectivity
The specificity of the method was ascertained by analyzing standard drug and sample. The spots
for both drugs were confirmed by comparing the Rf and spectra of the sample spots with that of standard
drugs.
LOD and LOQ
LOD and LOQ were calculated as 3.3 σ /S and 10 σ /S, respectively, where σ is the standard
deviation of the response (y-intercept) and S is the slope of the calibration plot.
Robustness Studies
The robustness of the method was studied, during method development, by small but deliberate
variations in chamber saturation period (± 10 %), development distance (± 10 %), time from application
to development (0, 10, 20, 30 min), time from development to scanning (0, 30, 60, 90 min). One factor at
a time was changed to study the effect on the peak area of the drugs (Concentration level 100 ng/band for
DIC and 100 ng/band for THIO).
RESULTS AND DISCUSSION

Different mobile phases containing various ratios of Toluene, Methanol, n- Hexane, Ethyl acetate and
Isopropyl alcohol were examined (data not shown). Finally the mobile phase containing Toluene–ethyl
acetate-methanol (5:3:2, v/v/v) was selected as optimal for obtaining well defined and resolved peaks.
The optimum wavelength for detection and quantitation used was 285 nm. The retention factors for DIC
and THIO were found to be 0.53 ± 0.005 and 0.17 ± 0.002, respectively. Densitogram of mixed standard
solution of DIC and THIO is shown in Figure 2.
The standard calibration curves were found to be linear over a concentration range of 50-300
ng/band for both drugs with correlation coefficients of 0.992 ± 0.172 for DIC and 0.995 ± 0.376 for
THIO. For DIC, the recovery study results ranged from 100.615 to 101.422 % with % RSD values
ranging from 0.415 to 0.719. For THIO, the recovery results ranged from 100.782 to 101.088 % with %
RSD values ranging from 0.413 to 0.761. Results of recovery studies are reported in Table 1.
The proposed method was also evaluated by the assay of commercially available tablets
containing DIC and THIO. Six replicate determinations were performed on the accurately weighed
amounts of tablets. The % assay was found to be 101.126 ± 0.439 and 100.915 ± 0.155 for DIC and THIO
(mean ± % RSD) respectively. Intra-day variation, as RSD (%), was found to be 0.468 for DIC and 0.357
for THIO, respectively. Inter-day variation, as RSD (%), was found to be 0.949 for DIC and 0.739 for
THIO, respectively. The spectra acquired for DIC and THIO extracted from the tablet were also
compared with those acquired from DIC and THIO standards; correlation was good indicates specificity
of the method. Robustness of the method checked after deliberate alterations of the analytical parameters
showed that areas of peaks of interest remained unaffected by small changes in the operational parameters
(% RSD < 2). Summary of validation parameters of proposed RP-HPLC method is given in Table 2.
CONCLUSION
The validated HPTLC method employed here proved to be simple, fast, accurate, precise and
robust, thus can be used for routine analysis of DIC and THIO in combined tablet dosage form.
IRJP 1 (1) Dec 2010 Page 220-224

ACKNOWLEDGEMENTS
The authors express their gratitude to Cipla Pharmaceuticals Pvt. Ltd. (Mumbai, MH) and Aventis
Pharmaceuticals Ltd (Goa, India) for the gift samples of pure drug. Special thanks to Dr. (Mrs.) A. R.
Madgulkar, Principal, AISSMS College of Pharmacy for providing facilities and his constant support.
REFERENCES
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2. http://en.wikipedia.org/wiki/Thiocolchicoside accessed on 15/04/ 09.
3. Fernández de Córdova ML, Barrales PO, Díaz AM. Sensitive and selective determination of
diclofenac sodium in pharmaceutical preparations by solid phase ultraviolet absorptiometry.
Analytica Chimica Acta. 1998; 369: 263-268.
4. Dighe VV, Sane RT, Menon SN, Tambe HN, Pillai S, Gokarn VN. Simultaneous determination
of diclofenac sodium and paracetamol in pharmaceutical preparation and in bulk drug powder by
high performance thin layer chromatography. J. Planar Chromatogr. 2006; 19: 443-448.
5. Shah HJ, Rathod SI, Shah SA, Savale SS, Shishoo CJ. Sensitive high performance thin layer
chromatography method for monitoring dissolution profiles of diclofenac from different tablets
containing combined diclofenac and acetaminophen. J. Planar Chromatogr. 2003; 16: 36-44.
6. Vora A, Damle M, Bhat L, Godge R. Simultaneous determination of diclofenac sodium and
rabeprazole sodium in bulk and pharmaceutical dosage form by LC. Chromatographia. 2007; 66:
941-943.
7. Kulhmann O, Stoldt G, Struck HG, Krauss GJ. Simultaneous determination of diclofenac and
oxybuprocaine in human aqueous humor with HPLC and electrochemical detection. J. Pharm.
Biomed. Anal. 1998; 17: 1351-1356.
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on a capillary electrophoresis microchip. Analytica Chimica Acta. 2006; 572: 205–211.
9. El-Ragehy NA, Ellaithy MM, El-Ghobashy MA. Determination of thiocolchicoside in its binary
mixtures (thiocolchicoside/glafenine and thiocolchicoside/floctafenine) by TLC/densitometry. II
Farmaco. 2003; 58: 463-468.
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Table 1: Recovery studies of DIC and THIO

Amount Amount Total amount
Drug taken added found % Recovery % RSDa
(ng/band) (ng/band) (ng/band)
100 50 152.010 101.341 0.531
DIC 100 100 201.230 100.615 0.418
100 150 253.554 101.422 0.729
100 50 151.173 100.782 0.495
THIO 100 100 202.176 101.088 0.418
100 150 252.190 100.876 0.768
a
Average of three determinations
IRJP 1 (1) Dec 2010 Page 220-224

Table 2: Summary of validation parameters of RP-HPLC method

Parameter DIC THIO
Detection Wavelength (nm) 285
Beer’s Law Limit (ng/band) 50-300 50-300
Correlation Coefficient (r) 0.992 0.995
a
Linear Regression Equation (y = mx + c)
Intercept (c) 302.3 324.6
Slope (m) 10.95 12.13
LOD (ng/band) 11.673 7.089
LOQ (ng/band) 38.52 23.393
a
With respect to y = mx + c, where y is the peak area and x is the concentration (ng/band)
Figure 1: In situ overlain spectrum of DIC and THIO measured from 200 to 400 nm.
Figure 2: Representative densitogram obtained fro mixed standard solution of DIC (50 ng/band, Rf
= 0.53 ± 0.005) and THIO (50 ng/band, Rf = 0.17 ± 0.002)
Source of support: Nil, Conflict of interest: None Declared
IRJP 1 (1) Dec 2010 Page 220-224

High Performance Thin Layer Chromatographic Determination of Diclofenac Sodium and Thiocolchicoside in Fixed Dose Combination

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Deshpande P et al.

IRJP 1 (1) 2010 220-224

INTERNATIONAL RESEARCH JOURNAL OF PHARMACY

HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHIC

KEYWORDS: High performance thin layer chromatography, Thiocolchicoside, Diclofenac Sodium

IRJP 1 (1) Dec 2010 Page 220-224

MATERIALS AND METHODS

IRJP 1 (1) Dec 2010 Page 220-224

RESULTS AND DISCUSSION

IRJP 1 (1) Dec 2010 Page 220-224

Table 1: Recovery studies of DIC and THIO

IRJP 1 (1) Dec 2010 Page 220-224

Table 2: Summary of validation parameters of RP-HPLC method

Source of support: Nil, Conflict of interest: None Declared

IRJP 1 (1) Dec 2010 Page 220-224

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