Stability Indicating HPLC Method for Simultaneous Determination of Mephenesin and Diclofenac Diethylamine
S. V. MULGUND, M. S. PHOUJDAR, S. V. LONDHE, P. S. MALLADE, T. S. KULKARNI,
Department of Pharmaceutical Chemistry, Sinhgad College of Pharmacy, Vadgaon (Bk),
Running title: Stability Indicating HPLC Method for Mephenesin and Diclofenac
. *Address for correspondence E-mail: [email protected]
A simple, specific, accurate and stability-indicating reversed phase high performance liquid chromatographic method was developed for the simultaneous determination of mephenesin and diclofenac diethylamine, using a Spheri-5-RP-18 column and a mobile phase composed of methanol: water (70:30, v/v), pH 3.0 adjusted with o-phosphoric acid. The retention times of mephenesin and diclofenac diethylamine were found to be 3.9 min and 14.5 min, respectively. Linearity was established for mephenesin and diclofenac diethylamine in the range of 50-300 μg/ml and 10-60 μg/ml, respectively. The percentage recoveries of mephenesin and diclofenac diethylamine were found to be in the range of 99.06-100.60% and 98.95-99.98%, respectively. Both the drugs were subjected to acid, alkali and neutral hydrolysis, oxidation, dry heat, photolytic and UV degradation. The degradation studies indicated, Mephenesin to be susceptible to neutral hydrolysis, while diclofenac diethylamine showed degradation in acid, H2O2, photolytic and in
presence of UV radiation. The degradation products of diclofenac diethylamine in acidic and photolytic conditions were well resolved from the pure drug with significant differences in their retention time values. This method can be successfully employed for simultaneous quantitative analysis of Mephenesin and Diclofenac diethylamine in bulk drugs and formulations. Keywords: Mephenesin, diclofenac diethylamine, stress testing, degradation products, stability indicating method, HPLC.
Mephenesin (MEP), 3-(2-methylphenoxy)-1,2-propanediol (fig. 1), is a white crystalline
powder, almost odorless, slightly soluble in water but freely soluble in alcohol, chloroform and
solvent ether. MEP is centrally acting muscle relaxant and a topical analgesic. It is official in Indian
Pharmacopoeia, which recommends a titrimetric method for its analysis. Diclofenac diethylamine
(DDEA), diethylammonium2-[(2,6-dichloroanilino)phenyl]acetate (fig. 2) is a white to light beige
crystalline powder, sparingly soluble in water and acetone, freely soluble in ethanol and methanol.
It is commonly used as an analgesic and antiinflammatory agent. DDEA is official in British
Pharmacopoeia, which recommends HPLC and HPTLC methods for its analysis.
MEP and DDEA combination gel is a recently introduced topical analgesic anti-
inflammatory combination in Indian market. Literature survey reveals that many analytical methods
are reported for determination of MEP[3-6] and DDEA[7-17] individually. However, no method is
reported for simultaneous estimation of these two drugs by reverse phase HPLC.
The International Conference on Harmonization (ICH) guideline entitled “Stability testing
of new drug substances and products” requires that stress testing be carried out to elucidate the
inherent stability characteristics of the active substance. An ideal stability-indicating method is
one that resolves the drug and its degradation products efficiently. Consequently, the
implementation of an analytical methodology to determine MEP and DDEA simultaneously, in
presence of its degradation products is rather a challenge for pharmaceutical analyst. Therefore, it
was thought necessary to study the stability of MEP and DDEA under acidic, alkaline, oxidative,
UV and photolytic conditions. This paper reports validated stability-indicating HPLC method for
simultaneous determination of MEP and DDEA in presence of their degradation products. The
proposed method is simple, accurate, reproducible, stability-indicating and suitable for routine
determination of MEP and DDEA in combined dosage form. The method was validated in
MATERIALS AND METHODS:
MEP and DDEA of pharmaceutical grade were kindly supplied as gift samples by Nulife
Pharmaceuticals, Pune, India, and were certified to contain 99.65% (w/w) and 99.35% (w/w)
respectively, on dried basis. Methanol and water used were of HPLC grade and were purchased
from Spectrochem Pvt. Ltd. Mumbai, India. The gel formulation (Systaflam Gel, Systopic
Laboratories Pvt. Ltd., Bangalore, India) containing 5 % w/w of MEP and 1.16 %w/w of DDEA
was procured from local market and used for analysis of marketed formulation. The liquid
chromatographic system was of Perkin Elmer (USA), series 200, which consisted of following
components: a gradient pump, variable wavelength programmable UV/Vis detector, a manual
injection facility with 20 μl fixed loop. The chromatographic analysis was performed using Total
ChromNavigator version 6.3 software on a Spheri-5-RP-18 column (250×4.6 mm, 5 µm particle
size). In addition, an electronic balance (Shimadzu AX200), a pH meter (Systronics model EQMK
VI), a sonicator (Spectra Lab, model UCB 40), a hot air oven (Labhosp), UV chamber (Labhosp)
Preparation of Mobile Phase and Stock Solutions:
Seven hundred millilitres of methanol and 300 ml of water were mixed and pH of mixture
was adjusted to 3.0 with o-phosphoric acid. This mixture was sonicated for 10 min and filtered
through 0.22 µm membrane filter and used as mobile phase. Stock solutions were prepared by
weighing 10 mg each of MEP and DDEA. The weighed drugs were transferred to two separate 10
ml volumetric flasks. Volumes were made up to the mark with methanol to obtain a solution
containing 1000 μg/ml of MEP and DDEA. The solutions were further diluted with the same
solvent to obtain final concentrations of 100 µg/ml of each drug. The HPLC analysis was
performed on reversed-phase high-performance liquid chromatographic system with isocratic
elution mode using a mobile phase of methanol:water (70:30, v/v) pH 3.0 adjusted with o-
phosphoric acid on Spheri-5-RP-18 column (250×4.6 mm, 5 µm particle size) with 1 ml/min flow
Calibration curves for MEP and DDEA:
Gel formulation contains MEP and DDEA in a ratio of 5:1. Appropriate aliquots of MEP
and DDEA stock solutions were taken in different 10 ml volumetric flasks and diluted up to the
mark with mobile phase to obtain final concentrations of 50-300 μg/ml and 10-60 μg/ml of MEP
and DDEA, respectively. The solutions were injected using a 20 μl fixed loop system and
chromatograms were recorded. Calibration curves were constructed by plotting average peak areas
versus concentrations and regression equations were computed for both the drugs (Table 1).
Analysis of Marketed Formulations:
About 1000 mg of gel containing 50 mg of MEP and 11.6 mg of DDEA was accurately
weighed and transferred into a 100 ml volumetric flask containing 50 ml methanol, sonicated until
the gel get dissolved and diluted up to the mark with same solvent to get final concentrations of 500
μg/ml and 116 μg/ml of MEP and DDEA, respectively. The above solution was filtered using
Whatman filter paper No 1. Appropriate volume of the aliquot was transferred to a 10 ml
volumetric flask and the volume was made upto the mark with mobile phase to obtain a solution
containing 50 μg/ml of MEP and 11.6 μg/ml of DDEA. A 20 μl volume of above sample solution
was injected into HPLC and peak areas were measured under optimized chromatographic
The method of analysis was validated as per the recommendations of ICH and USP for
the parameters like accuracy, linearity, precision, detection limit, quantitation limit and robustness.
The accuracy of the method was determined by calculating percentage recovery of MEP and
DDEA. For both the drugs, recovery studies were carried out by applying the method to drug
sample to which known amount of MEP and DDEA corresponding to 80, 100 and 120% of label
claim had been added (standard addition method). At each level of the amount six determinations
were performed and the results obtained were compared.
Intraday and interday precision study of MEP and DDEA was carried out by estimating the
corresponding responses 3 times on the same day and on 3 different days for the concentration of
50 μg/ml and 10 μg/ml of MEP and DDEA, respectively. The limit of detection (LOD) and limit of
quantitation (LOQ) were calculated using following formulae: LOD= 3.3(SD)/S and LOQ= 10
(SD)/S, where SD=standard deviation of response (peak area) and S= average of the slope of the
System suitability tests are an integral part of chromatographic method which are used to
verify reproducibility of the chromatographic system. To ascertain its effectiveness, certain system
suitability test parameters were checked by repetitively injecting the drug solution at the
concentration level 50 μg/ml and 10 μg/ml for MEP and DDEA, respectively to check the
reproducibility of the system and the results are shown in Table 2.
For robustness evaluation of HPLC method a few parameters like flow rate, percentage of
methanol in the mobile phase and pH of mobile phase were deliberately changed. One factor was
changed at one time to estimate the effect. Each factor selected was changed at three levels (-1, 0,
+1) with respect to optimized parameters. Robustness of the method was done at the concentration
level 50 μg/ml and 10 μg/ml for MEP and DDEA respectively.
Forced degradation studies:
Forced degradation studies of both the drugs were carried out under conditions of
hydrolysis, dry heat, oxidation, UV light and photolysis. MEP and DDEA were weighed (100 mg
each) and transferred into two 50 ml volumetric flasks and diluted up to the mark with methanol to
give 2000 μg/ml concentration of each drug. These stock solutions were used for forced
Forced degradation in basic media was performed by taking 10 ml of stock solution of MEP
and DDEA each in separate round bottom flasks. Then 10 ml of 5 N NaOH was added and these
mixtures were heated for up to 8 h at 70 0 in dark, in order to exclude the possible degradative
effect of light. Forced degradation in acidic media was performed by keeping the drug in contact
with 1N HCl for upto 30 h at ambient temperature as well as heating for up to 8 h at 70 0 in dark.
Degradation with hydrogen peroxide was performed by taking 10 ml of stock solution of MEP and
DDEA in two different flasks and adding 10 ml of 30% (w/v) hydrogen peroxide in each of the
flasks. These mixtures were kept for upto 4 days in the dark. To study neutral degradation, 10 ml of
stock solution of MEP and DDEA taken in two different flasks, then 10 ml of HPLC grade water
was added in each flask, these mixtures were heated for 6 h at 700 in the dark. For dry heat
degradation, solid drugs were kept in Petri dish in oven at 1000 for 12 h. Thereafter, 10 mg each of
MEP and DDEA were weighed and transferred to two separate 10 ml volumetric flasks and diluted
up to the mark with methanol. The photostability was also studied by exposing above stock
solutions (1000 µg/ml) of both the drugs to direct sunlight in summer days for 5 h on a wooden
plank. For UV degradation study, the stock solutions of both drugs (1000 µg/ml) were exposed to
UV radiation of a wavelength of 256 nm and of 1.4 flux intensity for 12 h in UV chamber.
For HPLC analysis, all the degraded sample solutions were diluted with mobile phase to
obtain final concentration of 30μg/ml of MEP and DDEA. Similarly mixture of both drugs in a
concentration of 30 μg/ml of MEP and DDEA each was prepared prior to analysis by HPLC.
Besides, solutions containing 30 μg/ml of each drug separately were also prepared without being
performing the degradation of both the drugs. Then 20 μl solution of above solutions were injected
into HPLC system and analyzed under the chromatographic condition described earlier.
RESULTS AND DISCUSSION
The mobile phase consisting of methanol: water (70:30, v/v) pH 3.0 adjusted with o-
phosphoric acid, at 1ml/min flow rate was optimised which gave two sharp, well-resolved peaks
with minimum tailing factor for MEP and DDEA (fig. 3). The retention times for MEP and DDEA
were 3.9 min and 14.5 min, respectively. UV overlain spectra of both MEP and DDEA showed that
both drugs absorbed appreciably at 221 nm, so this wavelength was selected as the detection
wavelength. The calibration curve for MEP and DDEA was found to be linear over the range of 50-
300 μg/ml and 10-60 μg/ml, respectively. The data of regression analysis of the calibration curves
is shown in Table 1. The proposed method was successfully applied to the determination of MEP
and DDEA in their combined gel dosage form. The results for the combination were comparable
with the corresponding labelled amounts. The developed method was also found to be specific,
since it was able to separate other excipients present in gel from the two drugs (fig. 4).
The LOD for MEP and DDEA were found to be 0.20 μg/ml and 0.25 μg/ml, respectively,
while LOQ were 0.50 μg/ml and 0.45 μg/ml, respectively. The results for validation and system
suitability test parameters are summarized in Table 2. Results for robustness evaluation for both the
drugs are presented in Table 3. Insignificant differences in peak areas and less variability in
The degradation study indicated that MEP was susceptible to neutral hydrolysis while it was
stable to acid, base, H2O2, direct sunlight, UV radiation and dry heat under experimental conditions.
In neutral hydrolysis the drug degrades as observed by the decreased area in the peak of the drug
when compared with peak area of the same concentration of the nondegraded drug, without giving
any additional degradation peaks (fig. 5).
DDEA was found to be susceptible to acid, H2O2, direct sunlight and UV radiation with
maximum degradation under acidic and photolytic conditions; however it shows stability towards
alkaline and neutral hydrolysis as well as dry heat degradation. DDEA gets degraded into one or
two degradation products in the stress conditions of acid hydrolysis as well as photolytic exposure,
while both the drugs showed no degradation at 0 h in all the degradation conditions. The
chromatogram of the acid degraded sample of DDEA showed one additional peak at tR 7.9 (fig. 6)
and chromatogram of photo induced degraded sample showed two additional peaks at tR 6.3 and
10.8 min, respectively (fig. 7) In oxidative and UV degradation, the drug degrades as shown by the
decreased areas in the peaks when compared with peak areas of the same concentration of the
nondegraded drug, without giving any additional degradation peaks. Per cent degradation was
calculated by comparing the areas of the degraded peaks in each degradation condition with the
corresponding areas of the peaks of both the drugs under non degradation condition. Summary of
degradation studies of both the drugs is given in Table 3.
In the proposed study, stability-indicating HPLC method was developed for the
simultaneous determination of MEP and DDEA and validated as per ICH guidelines. Statistical
analysis proved that method was accurate, precise, and repeatable. The developed method was
found to be simple, sensitive and selective for analysis of MEP and DDEA in combination without
any interference from the excipients. The method was successfully used for determination of drugs
in a pharmaceutical formulation. Assay results for combined dosage form using proposed method
showed 99.06±1.40 % of MEP and 98.95±0.66 % of DDEA. The results indicated the suitability of
the method to study stability of MEP and DDEA under various forced degradation conditions viz.
acid, base, dry heat, neutral, photolytic and UV degradation. It can be concluded that the method
separates the drugs from their degradation products; it may be employed for analysis of stability
samples of MEP and DDEA. However characterization of degradation products was not carried out.
The authors thank Nulife Pharmaceuticals, Pune, for providing Mephenesin and Diclofenac
diethylamine as gift samples for this work. They also thank Prof. M. N. Navale, Founder President
and Dr. (Mrs). S. M. Navale, Secretary, Sinhgad Technical Education Society, Vadgaon (Bk),
Pune, for providing required facilities to carry out this research work.
1. Indian Pharmacopoiea, Controller of Publication, Govt. of India, Ministry of Health and Family
2. British Pharmacopoiea, HMSO Publication: London; 2007, Vol. 1, 664.
3. Guinebault P, Colafranceschi C, Bianchetti G. Determination of mephenesin in plasma by high-
performance liquid chromatography with fluorimetric detection. J Chromatogr 1990;507:221-
4. Patravale V, Deshpande S, Krishnan K. Estimation of Mephenesin and Ibuprofen in
combination. Indian Drugs 1990;27(3):580-82.
5. Sheen J, Her G. Analysis of neutral drugs in human plasma by fluoride attachment in liquid
chromatography/negative ion electrospray tandem mass spectrometry. Rapid Comm Mass
6. Cai S, Hanold A, Syage A. Comparison of atmospheric pressure photoionization and
atmospheric pressure chemical ionization for normal-phase LC/MS chiral analysis of
pharmaceuticals. Anal. Chem 2007;79(6):2491-98.
7. Chmielewska A, Konieczna L, Plenis A. Determination of diclofenac in plasma by high-
performance liquid chromatography with electrochemical detection. Biomed Chromatogr
8. Mukherjee B, Mahapatra S, Das S. HPLC detection of plasma concentrations of diclofenac in
human volunteers administered with povidone-ethylcellulose based experimental transdermal
matrix-type patches. Meth Find Exp Clin Pharmacol 2006;28(5):301-06.
9. Arcelloni C, Lanzi R, Pedercini S. High-performance liquid chromatographic determination of
diclofenac in human plasma after solid-phase extraction. J Chromatogr B Biomed Sci Appl
10. Zecca L, Ferrario P, Costi P. Determination of diclofenac and its metabolites in plasma and
cerebrospinal fluid by high-performance liquid chromatography with electrochemical detection.
11. Robles B, Pérez-Urizar J, Flores-Murrieta J. Determination of diclofenac in micro-whole blood
samples by high-performance liquid chromatography with electrochemical detection
Application in a pharmacokinetic study. Arzneim-Forsch/Drug Res. 1997;47(9):1040-43.
12. Jiao H, Xu F, Zhang Z. Simultaneous determination of ZLR-8 and its active metabolite
diclofenac in dog plasma by high-performance liquid chromatography. Biomed Chromatogr
13. Blagbrough S, Daykin M, Doherty M. High-performance liquid chromatographic determination
of naproxen, ibuprofen and diclofenac in plasma and synovial fluid in man. J Chromatogr
14. Miller B. High-performance liquid chromatographic determination of diclofenac in human
plasma using automated column switching. J Chromatogr 1993;616(2):283-90.
15. Hanses A, Spahn-Langguth H, Mutschler E. A new rapid and sensitive high-performance liquid
chromatographic assay for diclofenac in human plasma. Arch Pharm (Weinheim)
16. Avgerinos A, Karidas T, Malamataris S. Extractionless high-performance liquid
chromatographic method for the determination of diclofenac in human plasma and urine. J
17. Lee S, Jeong K, Choi J. Simultaneous determination of aceclofenac and diclofenac in human
plasma by narrowbore HPLC using column-switching. J Pharm Biomed Anal 2000;23(5):775-
18. ICH, Q1A, Stability Testing of New Drug Substances and Products, in: Proceedings of the
International Conference on Harmonisation, Geneva, October, 1993.
19. ICH, Q2A, Hamonised Tripartite Guideline, Test On Validation of Analytical Procedures,
IFPMA, in: Proceedings of the International Conference on Harmonization, Geneva, March,
20. ICH, Q2B, Hamonised Tripartite Guideline, Validation of Analytical Procedure: Methodology,
IFPMA, in: Proceedings of the International Conference on Harmonization, Geneva, March
21. ICH Guidance on Analytical Method Validation, in: Proceedings of the International
Convention on Quality for the Pharmaceutical Industry, Toronto, Canada, and September, 2002.
22. United States Pharmacopoeia/National Formulary, 24th ed. Rockville, MD: Pharmacopeial
TABLE 1: LINEAR REGRESSION DATA FOR CALIBRATION CURVES
TABLE 2: SUMMARY OF VALIDATION AND SST PARAMETERSParameter (Units)
TABLE 3: SUMMARY OF DEGRADATION STUDIES FOR MEP AND DDEA
Degradation % Degradation tR (min) of degradation products Condition MEP DDEA MEP DDEA ----- ---- ---- . ---- . . ---- Fig. 1: structure of mephenesin (MEP) Fig. 2: Structure of Diclofenac diethylamine (DDEA) Fig. 3: Chromatogram of mixture of MEP and DDEA Fig. 4: Chromatogram of market formulation of MEP and DDAE Fig. 5: Chromatogram of mixture of MEP and DDEA degraded with neutral hydrolysis
Fig. 6: Chromatogram of mixture of MEP and DDEA degraded under acidic conditions
Fig. 7: Chromatogram of mixture of MEP and DDEA exposed to direct sunlight
Table and figure titles and legends: TABLE 1: LINEAR REGRESSION DATA FOR CALIBRATION CURVES MEP is mephenesin, DDEA is diclofenac diethyl amine, SE is the standard error of the mean, SD is standard deviation for n = 3 observations. TABLE 2: SUMMARY OF VALIDATION AND SST PARAMETERS SST stands for system suitability test. TABLE 3: ROBUSTNESS EVALUATION OF METHOD FOR MEP AND DEEA Concentrations level used for robustness evaluation was 50 µg/ml, aThree factors were slightly changed at three levels (-1, 0, 1) and bretention time. TABLE 4: SUMMARY OF DEGRADATION STUDIES FOR MEP AND DDEA MEP is mephenesin, DDEA is diclofenac diethylamine, tR stands for retention time, ND represents no degradation observed. ** Represents that no rise of additional degradation peak was observed. Fig. 1: structure of mephenesin (MEP) Fig. 2: Structure of Diclofenac diethylamine (DDEA) Fig. 3: Chromatogram of mixture of MEP and DDEA Mephenesin (MEP, peak 1) with tR of 3.9 min and diclofenac diethylamine (DDEA, peak 2) with tR of 14.5 min. Fig. 4: Chromatogram of market formulation of MEP and DDAE Mephenesin (MEP, peak 1) with tR of 3.9 min and diclofenac diethylamine (DDEA, peak 2) with tR of 14.5 min resolved form other excipients (peaks 3, 4 and 5) with tR of 3.5, 4.8 and 7.4 min, respectively. Fig. 5: Chromatogram of mixture of MEP and DDEA degraded with neutral hydrolysis Mephenesin (MEP, peak 1) with tR of 3.9 min shows decrease in peak area, but on additional degradation product is observed, diclofenac diethylamine (DDEA, peak 2) with tR of 14.5 min Fig. 6: Chromatogram of mixture of MEP and DDEA degraded under acidic conditions Mephenesin (MEP, peak 1) with tR of 3.9 min and diclofenac diethylamine (DDEA, peak 2) with tR of 14.5 min and degradation product of DDEA (peak 3) with a tR of 7.9 min Fig. 7: Chromatogram of mixture of MEP and DDEA exposed to direct sunlight Mephenesin (MEP, peak 1) with tR of 3.9 min and diclofenac diethylamine (DDEA, peak 2) with tR of 14.5 min and degradation products of DDEA (peaks 3 and 4) with tR 6.3 min and 10.8 min, respectively
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