Saudi Pharmaceutical Journal (2012) 20, 249253

King Saud University

Saudi Pharmaceutical Journal

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ORIGINAL ARTICLE

Simultaneous determination of retinoic acid and hydroquinone in skin ointment using spectrophotometric technique (ratio dierence method)
Eman S. Elzanfaly, Ahmed S. Saad *, Abd-Elaziz B. Abd-Elaleem
Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr-El Aini Street, 11562 Cairo, Egypt Received 31 January 2012; accepted 25 March 2012 Available online 31 March 2012

KEYWORDS Spectrophotometry; Binary mixtures; Retinoic acid; Hydroquinone and ratio difference method; DP method

Abstract An innovative spectrophotometric method was developed for simultaneous determination of compounds with interfering spectra in binary mixtures without previous separation, showing signicant advantages over the conventional methods regarding minimal data manipulation and applicability. The proposed method was applied for the determination of retinoic acid and hydroquinone in laboratory-prepared mixtures with mean percentage recoveries 100.13 0.31 and 99.99 0.04, respectively, and in their pharmaceutical formulation with mean percentage recoveries 100.13 0.86 and 100.07 0.58, respectively. The method was validated according to USP guidelines and can be applied for routine quality control testing.
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1. Introduction Many methods have been introduced for the analysis of binary mixtures among which the spectrophotometric based methods were the most simple, fast and applicable in almost all laboratories. Several manipulations were performed on the raw overlapping spectral data to enable mixture resolution for example,
* Corresponding author. Tel.: +20 1146106003. E-mail address: ahmedss_pharm@yahoo.com (A.S. Saad). 1319-0164 2012 King Saud University. Production and hosting by Elsevier B.V. All rights reserved. Peer review under responsibility of King Saud University. http://dx.doi.org/10.1016/j.jsps.2012.03.004

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using different order derivatives (Altino s and Tekeli, 2001; Bonazzi et al., 1997; Challa et al., 2010; Erk, 2002; Gallego and Arroyo, 2001; Nanda et al., 2008), derivatives of the ratio spectrum (Darwish et al., 2011; Hassib et al., 2011; Salinas et al., 1990), ratio subtraction technique (El-Bardicy et al., 2008), and dual wavelength method (Bindaiya et al., 2010). The aim of the present work was to develop a new simple, rapid, selective method for the simultaneous determination of components having overlapping spectra in binary mixtures, having the advantages of minimal data processing and a wider range of applications over the previously mentioned methods. To prove the ability of the newly described method in resolving the overlapping spectral data and simultaneous determination of each component, it was applied for the analysis of a mixture of retinoic acid (RA) and hydroquinone (HQ) formulated together in the form of skin ointment widely used for the treatment of hyperpigmentation problems accompanying several skin disorders (Gilman et al., 2001).

250 1.1. Theory For any two drugs X and Y with overlapping spectra, upon dividing the spectrum of X by a divisor of a certain concentration of Y, a ratio spectrum will result, and a linear relationship between the difference in amplitudes at any two wavelengths and the corresponding concentration of X will result. While the ratio spectrum of Y will be a straight line of constant amplitude parallel to the x-axis and the difference in amplitudes of Y at any two wavelengths will be zero, as shown in Figs. 1 and 2. Mathematically it can be explained as follows: In the ratio spectrum of a lab mixture of X and Y divided by a divisor Y0 P1 P1X K P2 P2X K 1 2 2.3. Pharmaceutical formulation 2. Experimental 2.1. Apparatus

E.S. Elzanfaly et al.

SHIMADZU dual beam (Kyoto/Japan) UVvisible spectrophotometer model UV-1601 PC connected to IBM compatible and an hp1020 laserjet printer. The bundle software, UV.PC personal spectroscopy software version 3.7 (SHIMADZU) was used to process absorption and ratio spectra, the spectral band width was 2 nm and scanning speed was 2800 nm/min. 2.2. Reference samples RA and HQ reference standards were kindly supplied by CID Pharmaceutical Co. (Cairo, Egypt).

where P1 and P2 are the amplitudes of the mixture spectrum at k1 and k2, respectively. P1X and P2X are the amplitudes of X at k1 and k2, respectively. K is the constant resulting from Y/Y0 DPk1 k2 P1 P2 P1X K P2X K P1X P2X 3

Lucocid-R 3% ointment, labeled to contain 0.025 g% RA and 3 g% HQ, batch no. 291, manufactured by CID Pharmaceutical Co., Egypt. 2.4. Materials and reagents Methanol (Adwic). 2.5. Standard solutions 2.5.1. Stock standard solutions 2.5.1.1. Stock standard solution of RA (1 mg/ml). An accurately weighed 25 mg of RA was dissolved in 5 ml methanol in 25 ml volumetric ask and completed to the volume with the same solvent. 2.5.1.2. Stock standard solution of HQ (3 mg/ml). An accurately weighed 75 mg of HQ was dissolved in 5 ml methanol in 25 ml volumetric ask and completed to the volume with the same solvent. 2.5.2. Working standard solutions 2.5.2.1. Working standard solution of RA (20.0 lg/ml). RA working solution was prepared by transferring 2 ml of the previously prepared standard RA stock solution into two 100 ml volumetric asks and completing to volume with methanol to get a nal concentration of 20.0 lg/ml.

A calibration curve relating the difference in amplitudes in the ratio spectrum at k1 and k2 (DPk1 k2 ) using a certain concentration of Y as a divisor to the corresponding concentration of X will be used for the determination of X in the unknown samples of the binary mixture. Similarly component Y can be obtained by using certain concentration of X as a divisor.

Figure 1 Division spectra of HQ 330 lg/ml using 10 lg/ml of RA as a divisor and methanol as a blank.

Figure 2

Division spectra of RA 110 lg/ml and using 10 lg/ml of RA as a divisor and methanol as a blank.

Simultaneous determination of retinoic acid and hydroquinone in skin ointment 2.5.2.2. Working standard solution of HQ (60.0 lg/ml). HQ working solution was prepared by transferring 2 ml of the previously prepared standard HQ stock solution into two 100 ml volumetric asks and completing to volume with methanol to get a nal concentration of 60.0 lg/ml. 2.6. Procedures 2.6.1. Construction of calibration curves (linearity) Aliquots equivalent to 10100 lg of RA working standard solution (20.0 lg/ml) were transferred into two series of 10 ml volumetric asks and completed to mark with methanol. The zero order (D0) absorption spectra of each solution were recorded against methanol as a blank. Three calibration curves were constructed relating the absorbance at 339.0 nm to the corresponding RA concentration, and the regression equation was computed. Aliquots equivalent to 30300 lg of HQ working standard solution (60.0 lg/ml) were transferred into a series of 10-ml volumetric asks and completed to mark with methanol. The zero order (D0) absorption spectra of each solution were recorded against methanol as a blank. The spectra of HQ were divided by the spectrum of (10 lg/ml) RA. Two calibration curves were constructed relating the difference in the amplitudes of the resulting ratio spectrum at 289.2 and 257.8 nm (DP289.2257.8 nm) and the amplitude difference at 225.8 and 289.2 nm (DP225.8289.2 nm) to the corresponding HQ concentration, and the regression equations were computed. 2.6.2. Analysis of laboratory-prepared mixtures Laboratory-prepared mixtures containing different ratios of RA and HQ were prepared. By applying the procedure under linearity, the D0 absorbances at 339.0 nm were recorded for RA and the DP289.2257.8 nm and DP225.8289.2 nm were recorded for HQ. The concentration of each drug in each mixture was calculated from its corresponding regression equation. Validity of the method was assessed by spiking the pharmaceutical formulation by known amounts of standard drug powders (standard addition technique). The recovery of the added standards was then calculated after applying the proposed method. 2.6.3. Application of the proposed method for the simultaneous determination of RA and HQ in Lucocid-R skin ointment 0.1 g ointment was accurately weighed into a 100-ml beaker and sonicated in 15 ml methanol for 10 min, ltered into 50ml volumetric ask. The residue was washed three times each using 10 ml methanol and completed to the mark with the same solvent. Transfer accurately 2 ml of the extracted solution into a 10-ml measuring ask. Two milliliters of RA working solution (20 lg/ml) equivalent to 40 lg RA was added and completed to the mark with methanol. The general procedure under linearity was followed. 3. Results and discussion Several approaches have been developed to remove the overlapping constant in the ratio spectrum, either using certain order derivative (Darwish et al., 2011; Hassib et al., 2011; Salinas et al., 1990) or through a sophisticated subtraction followed by a multiplication procedure (El-Bardicy et al., 2008), the latter

251

Figure 3 Zero order absorption spectra of 7 lg/ml RA (- - - -) and (30 lg/ml) HQ (____) using methanol as a blank.

was capable of determining only the component with the less extended spectrum in the mixture. A simple innovative method was developed capable of determining RA and HQ in the binary mixture with minimal data processing, and high selectivity regardless which component has more extended spectrum. The zero order spectra of the two components revealed spectral overlap at the spectrum of HQ while RA can be directly determined at its wavelength of maximum absorption (kmax) 339.0 nm with no interference of HQ, while HQ cannot be measured in the presence of RA, as shown in Fig. 3. Calibration curves for RA were constructed relating the absorbances at kmax 339.0 nm to the corresponding concentrations in the range of 110 lg/ml. The regression equations were computed and found to be A 0:1122C 0:0065; r 0:9997; for RA at k 339:0 nm

where A is the absorbance, C is the concentration of the drug in lg/ml and r is the correlation coefcient. For HQ, different divisor concentrations of RA were tried. The divisor concentration 10 lg/ml was found to be optimal regarding minimal noise. A linear correlation was obtained between DP289.2257.8 nm and DP225.8289.2 nm against the corresponding concentration of HQ. The regression equations were found to be DP289:2 and 257:8 nm 0:0576C 0:0125; DP225:8 and 289:2 nm 0:1365C 0:0203; r 0:9999 r 0:9998 4 5

The proposed ratio difference procedure was successfully applied for the determination of HQ in laboratory-prepared mixtures containing different ratios of RA and HQ, as shown in Table 1. The suggested method was found to be applicable and valid for the analysis of RA and HQ in Lucocid-R skin ointment with mean percentage recoveries 100.13 0.86 and 100.07 0.58, respectively, without interference of the excipients. The validity of the proposed procedure was assessed by applying the standard addition technique, as shown in Table 2. The validation parameters according to USP guidelines (The United States Pharmacopoeia and National Formulary, 2005) of accuracy, repeatability and intermediate precision are presented in Table 3. The data showed that the results obtained by the new method are accurate, precise, robust and specic over the specied range. Ruggedness was evaluated by applying the proposed method to the analysis of different laboratory-prepared mixtures

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Table 1 Determination of RA and HQ in laboratory-prepared mixtures by the proposed method.

E.S. Elzanfaly et al.

Ratio RA:HQ Concentration (lg/ml) RA 1:4 1:3 1:2 2:3 3:4 1:1 2:1 Mean SD RSD% 3 4 6 8 9 6 12 HQ 12 12 12 12 12 6 6

RA recovery % at 339.0 nm D0 HQ recovery % DP289.2257.8 nm HQ recovery % DP225.8289.2 nm 100.11 99.94 100.21 100.78 99.89 100.02 99.95 100.13 0.31 0.31 99.92 99.98 100.06 100.34 99.97 99.99 100.03 100.04 0.14 0.14 99.95 100.02 100.02 99.97 100.06 99.96 99.98 99.99 0.04 0.04

Table 2 Determination of RA and HQ in Lucocid-R ointment by the proposed method and application of standard addition technique.
Producta Method Standard addition Taken (lg/ml) Added (lg/ml) Found (lg/ml) RA 100.13 0.86 D0 4.10 3.00 4.00 5.00 100.11 0.75 0.75 3.02 3.97 5.02 Recovery (%) 100.67 99.25 100.4
b

Mean SD RSD%

Found DP289.2257.8 nm Found DP225.8289.2 nm Recovery % (lg/ml) (lg/ml) DP289.2257.8 nm HQ 100.07 0.58 at 12.00 DP225.8289.2 nm 9.00 12.00 15.00 99.93 0.18 0.18 9.01 11.97 14.99 100.20 0.06 0.06 9.02 12.03 15.02 100.11 99.75 99.93

Recovery % DP225.8289.2 nm 100.22 100.25 100.13

Mean SD RSD%
a b

Lucocid-R ointment 0.025 g% RA and 3 g% HQ B. no. 291. Average of three determinations.

Table 3
Parameter

Assay validation sheet of the proposed ratio difference method for the determination of HQ.
Ratio dierence DP289.2257.8 nm DP225.8289.2 nm 99.81 1.50 99.99 0.04 100.02 0.70 99.97 0.56 0.28 0.85 0.1365 0.0203 0.9998 330

Accuracy (mean SD) Specicity Precision Repeatabilitya Intermediate precisionb Linearity LOD (lg/ml) LOQ (lg/ml) Slope Intercept Correlation coecient (r) Range (lg/ml)

99.89 0.58 100.04 0.14 99.99 0.55 99.97 0.63 0.24 0.73 0.0576 0.0125 0.9999 330

a The intraday (n = 3), average of three concentrations (4, 5, 6 lg/ml) for RA and (12, 15, 18 lg/ml) for HQ repeated three times within the day. b The interday (n = 3), average of three concentrations (4, 5, 6 lg/ml) for RA and (12, 15, 18 lg/ml) for HQ repeated three times in three successive days.

Simultaneous determination of retinoic acid and hydroquinone in skin ointment using the same conditions but different instrument (SHIMADZU dual beam (Kyoto/Japan) UVvisible spectrophotometer model UV-1650 PC). Results obtained were found to be reproducible. 3.1. Comparing the ratio difference method to three conventional spectrophotometric methods 3.1.1. Advantages over ratio subtraction method A sophisticated four step procedure including (1) division, (2) subtraction of the constant at a certain wavelength, (3) multiplication and (4) calibration curve construction is adopted in the ratio subtraction method, while in the described ratio difference method a simple two step procedure of (1) division and (2) calibration curve construction is applied. The choice of the wavelength at which the constant is subtracted represents a limitation for the method applicability, where the overlapped component must have proper linearity and the measured component should have zero contribution at that wavelength, whereas the ratio difference method can be applied for two wavelengths in the ratio spectrum, even in the overlapped part of the two spectra. Ratio subtraction method fails to determine the extended component, i.e. can determine only the component with the less extended spectrum in the binary mixture, while the ratio difference method succeeded in the determination of both components. 3.1.2. Advantages over derivative of the ratio spectrum method Although the ratio derivative was able to determine both components of the binary mixture, but the procedure comprises three steps, where a division followed by certain order derivative with a specied Dk and nally calibration curve construction, c.f. ratio difference method which was able to determine both components of the binary mixture using the simple two step procedure described above. 3.1.3. Advantages over the dual wavelength method Although the measurements are taken in the absorption spectrum without division (single step procedure) to determine both components in binary mixture, but the linearity of the measured component always comes in the second place after nding the wavelength couple at which the overlapped component has exactly the same absorptivity (Bindaiya et al., 2010), a limitation that superimposes itself over the linearity of the measured component, (i.e. the overlapped component has a higher priority over the measured component in wavelengths choice) and may sometimes have conicts with it, whereas linearity of the measured component always comes in the rst place in the ratio difference method, since the overlapped compound contributes equally at all wavelengths throughout the whole ratio spectrum. The main condition for the application of the dual wavelength method is the presence of two wavelengths in the spectrum of the overlapped component having exactly the same

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absorptivity; a condition that is mainly met at peak shoulders, where Dk/DA is said to be maximum, therefore selectivity and robustness toward the measured component are extremely affected, while in the ratio difference method measurements can be done at any two wavelengths throughout the whole ratio spectrum, where no contribution of the overlapped component in the amplitude difference at any wavelength couples. 4. Conclusion The present work described a simple innovative method for simultaneous determination of compounds having overlapped spectral data in binary mixtures. The method was successfully applied for simultaneous determination of RA and HQ in pure powder form and in pharmaceutical formulation. The method was validated and showed that it can be used for the regular quality control testing. References
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