Analytica Chimica Acta 467 (2002) 9196

Simultaneous determination of methylparaben, propylparaben, sodium diclofenac and its degradation product in a topical emulgel by reversed-phase liquid chromatography
R. Hjkov a , P. Solich a, , M. Posp ilov a , J. cha b
a

Department of Analytical Chemistry, Faculty of Pharmacy, Charles University, Heyrovskho 1203, 500 05 Hradec Krlov, Czech Republic b Bochemie Group, Herbacos-Bofarma Ltd., trossova 239, 530 02 Pardubice, Czech Republic Received 19 November 2001; accepted 13 February 2002

Abstract A novel reversed-phase liquid chromatographic method with UV spectrophotometric detection was developed and validated for the determination of compounds in topical emulgel. The method describes determination of active component sodium diclofenac, its degradation product 1-(2,6-dichlorophenyl)-indolin-2-one (occurring in formulation after long-term stability tests) and two preservatives presented in the emulgel, methylparaben and propylparaben, using urbiprofen as an internal standard. The chromatographic separation was performed on a SUPELCO Discovery C18 column; the mobile phase for separation of all the compounds was methanol/phosphate buffer, pH 2.5 (65:35, v/v). The analysis time was <17 min. The method was found to be applicable for routine analysis (stability tests) of active compound sodium diclofenac, preservatives and degradation product in a pharmaceutical product, topical diclofenac emulgel 1%. 2002 Elsevier Science B.V. All rights reserved.
Keywords: Liquid chromatography; Methylparaben; Propylparaben; Sodium diclofenac

1. Introduction Diclofenac (DCF, Fig. 1), as the sodium salt, is a benzeneacetic acid derivative, designated chemically as 2-[2,6-dichlorophenyl)amino]benzeneacetic acid, monosodium salt. Diclofenac is a non-steroidal anti-inammatory drug (NSAID). In pharmacological studies, DCF has shown anti-inammatory, analgesic and antipyretic activity. As with other NSAIDs, its mode of action is not known; its ability to inhibit prostaglandin synthesis, however, may be involved in its anti-inammatory activity, as well as contribute to
Corresponding author. Tel.: +42-49-506-7294; fax: +42-49-521-0718. E-mail address: solich@faf.cuni.cz (P. Solich).

its efcacy in relieving pain related to inammation. With regard to its analgesic effect, diclofenac is not a narcotic [1,2]. Although the chemical stability of the compound is relatively high, several potential impurities are described in different pharmacopoeias. The main impurity, 1-(2,6-dichlorophenyl)indolin-2-one (DPI, Fig. 1), may occur in pharmaceutical formulations after long-term storage. Methylparaben (MP) and propylparaben (PP) (Fig. 1) are effective antibacterial and anti-fungal agents, which are commonly used as preservatives in foods, beverages, cosmetics and pharmaceuticals [3]. Methylparaben and propylparaben are used together since they have a synergistic effect [4]. Recently there have been a number of reports dealing with various analytical techniques for the

0003-2670/02/$ see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 0 2 ) 0 0 1 3 1 - 9

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in a topical emulgel, viz. active component sodium diclofenac, two preservatives methylparaben and propylparaben and the degradation product of sodium diclofenac, 1-(2,6-dichlorophenyl)indolin-2-one, using an internal standard. Thereafter, this method was successfully applied for the separation, quantication and a stability study of all the compounds in the pharmaceutical formulation diclofenac emulgel 1%. 2. Experimental 2.1. Reagents The in-house secondary standards used during this study, standards of sodium diclofenac and 1-(2,6dichlorophenyl)indolin-2-one, were obtained from Amoli Organics Ltd. (Mumbai, India); methylparaben and propylparaben were from Galena a.s. (Opava, Czech Republic), internal standard urbiprofen was from SigmaAldrich (Prague). All compounds were assayed against the EP reference standards. Standard solutions were prepared in methanol. The nal concentrations of the sample or reference standards were ca. 250 g ml1 sodium diclofenac, 25 g ml1 methylparaben, 12.5 g ml1 propylparaben, 5 g ml1 1-(2,6-dichlorophenyl)indolin-2-one and 10 g ml1 internal standard urbiprofen. Methanol (Chromasolv, for LC) was obtained from SigmaAldrich, orthophosphoric acid, 85% p.a. and sodium dihydrogenphosphate p.a. were from Merck (Darmstadt, Germany). All other chemicals were reagent grade from Merck. Diclofenac emulgel 1% was supplied from Herbacos-Bofarma Ltd. (Bochemie Group, Pardubice, Czech Republic). The deionised water was puried by a Milli-Q system (Millipore Corp., Bedford, MA) and meets the European Pharmacopoeia specications. 2.2. Chromatographic system The LC system, consisting of a binary pump LCP 4100 (Ecom, Prague), Waters autosampler 717 plus, variable wavelength UV detector Waters 486 (Waters, Milford, MA) and a PC for data processing, was controlled by chromatographic software CSW v.1.7 for Windows (Data Apex s.r.o., Prague). Analyses were performed on a 5 m SUPELCO Discovery C18 125 mm 4 mm i.d. column (SigmaAldrich) with

Fig. 1. Major components and degradation product of diclofenac emulgel 1%: (a) sodium diclofenac; (b) 1-(2,6-dichlorophenyl)indolin-2-one; (c) methylparaben; (d) propylparaben.

determination of DCF, such as spectrouorimetry [5,6], capillary electrophoresis [710], thin-layer chromatography [11,12], ow injection analysis [1315], gas chromatographymass spectrometry (GCMS) [16,17], supercritical uid chromatography [18,19] and liquid chromatography (LC) [1,2024], as well as LCMS [25]. Only one LC method for simultaneous determination of DCF and its degradation product DPI was found in the literature [26], but was not applied in the presence of preservatives methylparaben and propylparaben. LC analysis of MP and PP is frequently described [3,4,27], but to our surprise there is no LC method described for the simultaneous determination of all four components, DCF, DPI, MP and PP, in pharmaceutical preparations. The current United States Pharmacopoeia: USP 24 [28] or European Pharmacopoeia, third edition, specify an LC method for the determination of DCF and its degradation product DPI, but not simultaneously in the presence of MP and PP. Moreover, the pharmacopoeia methods do not use an internal standard. The purpose of this study was to develop a new LC method for the determination of the four compounds

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a pre-column SupelGuard 20 mm 4 mm i.d., 5 m (SigmaAldrich). The optimal mobile phase for separation of DCF, MP, PP and DPI was a mixture of methanol and phosphate buffer, pH 2.5 (65:35, v/v). Phosphate buffer was a mixture of orthophosphoric acid, 85% (1 g l1 ) and sodium dihydrogenphosphate (1.6 g l1 ), 50:50, v/v adjusted to an apparent pH of 2.5. Mobile phase was degassed before application by means of helium. The nally selected optimised conditions were as follows: injection volume 20 l, the mobile phase was isocratically pumped at 0.7 ml min1 at ambient temperature, and the detection wavelength was 245 nm. Fig. 2 shows a chromatogram of standard solutions of sodium diclofenac, preservatives, degradation product 1-(2,6-dichlorophenyl)indolin-2-one and urbiprofen as internal standard. 2.3. Sample preparation An accurately weighed portion (ca. 0.5 g) of the pharmaceutical emulgel was transferred into a 50 ml centrifuge tube and supplemented with 20.00 ml of internal standard (10 g ml1 urbiprofen in methanol). The mixture was placed into the ultrasonic bath for

10 min and then centrifuged at 3000 rpm for 15 min. A volume of 20 l of supernatant as analysed by LC. Identication of peaks in the emulgel samples was based on comparison of retention times of compounds in standard solutions. Peak identity was conrmed by UVVIS spectra.

3. Results and discussion 3.1. Method development and optimisation The main criteria for developing a successful LC determination of sodium diclofenac, preservatives and degradation product in topical emulgel were as follows: the method should be stability indicating, free of interference from excipients, robust and straightforward enough for routine use in a quality control laboratory. The European Pharmacopoeia, third edition, species a method for LC determination of sodium diclofenac and its degradation product with 254 nm detection, a 4.6 mm 25 cm column and a mobile phase containing methanol and potassium dihydrogenphosphate buffer, pH 2.5 (2:1) with a ow rate ca. 1.0 ml min1 . Using these conditions, the preservatives present in the topical cream (MP and PP) were not adequately separated. Therefore it was necessary to change several variables. From the UV spectra of all the analysed compounds, the optimal detection wavelength, which is a compromise bearing in mind that the concentration of degradation product DPI will be very low in comparison with that of the main active compound sodium diclofenac, was chosen to be 245 nm. The choice of appropriate internal standard was made from several compounds (urbiprofen, ibuprofen and naproxen), all having similar characteristics to the compounds determined. The retention time of naproxen was similar to that for DPI so this compound was unsuitable. Both ibuprofen and urbiprofen (Fig. 3) were sufciently separated from other compounds if interest; the second compound was nally chosen because of its better sensitivity. For good separation of DCF, DPI, MP and PP, the mobile phase was changed a little give a ratio of methanol to phosphate buffer, pH 2.5 of 65:35, v/v. Using a 5 m packing of the SUPELCO Discovery C18 column and decreasing the ow rate to 0.7 ml min1 ,

Fig. 2. Chromatogram of separation of standard solutions of methylparaben (25 g ml1 ), propylparaben (12.5 g ml1 ), 1-(2,6-dichlorophenyl)indolin-2-one (5 g ml1 ), internal standard urbiprofen (10 g ml1 ) and sodium diclofenac (250 g ml1 ).

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3.3. Analysis of degradation products In the pharmaceutical formulation containing sodium diclofenac, a small amount of degradation product DPI can be found after long-term storage. For determination of the amount of the degradation product, it is necessary to realise that it is present in a very low concentration in comparison to DCF (about 501000 times lower). This is also why methods for determination of active compounds and methods for determination of degradation products generally are different. In this case, we nally achieved sufcient separation of all compounds and therefore we could include the whole procedure in one step. Under the optimised conditions, the selectivity of the determination is adequate and the retention time for DPI is sufciently different from that of the other compounds present in the sample. The limit of detection (LOD) for a 20 l injection of DPI standard (signal to noise = 3) was 8 ng ml1 , and the limit of quantication (signal:noise = 10) was 27 ng ml1 . 3.4. Determination in a pharmaceutical product The chromatogram in Fig. 4 was obtained using the LC method with a sample of topical diclofenac emulgel 1% after a long-term stability test (stored for 6 months in the original packaging at 25 2 C and relative humidity 60 5%). All compounds present in the sample, sodium diclofenac, both preservatives, degradation product and internal standard, are clearly

Fig. 3. Internal standard urbiprofen = 2-uoro--methyl-4-biphenylacetic acid.

we also improved the separation of both preservatives. Finally, the analysis time for standards of all compounds was ca. 17 min (Fig. 2). 3.2. Analytical parameters and validation The optimised method was validated by a standard procedure to evaluate if adequate accuracy, precision, selectivity and linearity had been achieved. Accuracy was determined using spiked placebo solutions, three preparations each, two injections of each preparation. Relative standard deviation (R.S.D.) values were calculated for repeated standard injections (system precision) as well as repeated injections of multiple sample preparations (method precision). Linearity was determined in the 20150% range at six different concentrations. Short-term stability of standards was evaluated by comparison of response factors of fresh and stored standards. Visual inspection of chromatograms of standards and placebo solutions was conducted to ensure selectivity. The method validation results obtained under the nal conditions are shown in Table 1. The method meets all common requirements for accuracy, precision and linearity.

Table 1 Method validation results Validation step System precision Method precision Accuracy Linearity (n = 6)d Sample stabilitye
a b

Parameter R.S.D. (%)b R.S.D. (%)c Spike recovery (%)c Recovery R.S.D. (%)c Correlation coefcient Percent change in response factors

Sodium diclofenac 0.25 1.28 98.5 0.47 0.999916 0.19

Methylparaben 0.24 0.74 98.5 0.28 0.99988 0.25

Propylparaben 0.18 0.72 99.0 0.69 0.99986 0.65

DPIa 0.62 1.19 99.1 0.43 0.99919 0.21

Criteria X<2 X<2 X = 100 3 X<2 X > 0.9990 X<2

Degradation product: 1-(2,6-dichlorophenyl)indolin-2-one. Six injections. c Three preparations each, two injections of each preparation. d At 20, 50, 80, 100, 120, 150% levels. e Two-day stability data for all compounds.

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Fig. 4. Chromatogram of all compounds in a topical emulgel after a 6-months stability test.

separated. The average recoveries of DCF, MP and PP in the emulgel were 103.0 0.6, 96.5 0.8 and 104.1 0.9% of the labelled amount, respectively. The amount of degradation product DPI was found to be very low, 0.064% of the amount of the active compound DCF. The results of accelerated stability tests (formulation stored for 6 months in the original packaging at 40 2 C and relative humidity 60 5%) were: remaining DCF, MP and PP were 100.3 0.5, 96.0 0.9 and 103.8 0.8% of the labelled amount, respectively. The proportion of the degradation product DPI was found to be 0.529% of the amount of DCF.

accurate and the limits of detection for the degradation product were sufciently low. The method can be used for routine analysis (batch analysis and stability tests) of compounds in pharmaceutical products containing the active compound sodium diclofenac, preservatives MP + PP and the degradation product of the active compound. This method was successfully applied for the identication, quantitative analysis and stability tests of all major compounds in the topical emulgel diclofenac emulgel 1%.

Acknowledgements The authors gratefully acknowledge the nancial support of the Grant Agency of the Ministry of Education of the Czech Republic: MSM 111600001. References
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4. Conclusions The LC method with UV spectrophotometric detection on a SUPELCO Discovery C18 column was developed successfully for the determination of sodium diclofenac, methylparaben, propylparaben and 1-(2,6-dichlorophenyl)indolin-2-one in a topical emulgel using urbiprofen as an internal standard. The total analysis time was <17 min. The method has been validated; the results obtained were precise and

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