Rakesh Yadav; Rajkumar Yadav; Divya Yadav*

Research Article

Austin J Anal Pharm Chem. 2018; 5(2): 1099.

New Multi Wavelength Method for the Estimation of Tazarotene and Hydroquinone in Gel Formulation

Rakesh Yadav*, Rajkumar Yadav and Divya Yadav

¹Department of Pharmacy, Banasthali University, Banasthali-304022, Rajasthan, India

*Corresponding author: Rakesh Yadav, Department of Pharmacy, Banasthali University, Banasthali-304022, Tonk, Rajasthan, India

Received: April 20, 2018; Accepted: May 25, 2018; Published: June 01, 2018

Abstract

A simple, sensitive, rapid, precise & accurate, UV spectrophotometric method has been developed for simultaneous estimation of Tazarotene & Hydroquinone from their gel formulation. This method is based on multi wavelength spectroscopic method. For the simultaneous estimation of both the drug sampling wavelength 294nm and 351nm were selected. Tazarotene and Hydroquinone showed linearity in concentration range of 1-5 μg/ml and 10-50 μg/ml respectively. Recovery for tazarotene and hydroquinone was obtained in the range of 99.38% to 100.0%. All three methods showed good reproducibility and recovery with % RSD less than 2.0%. Statistical validation of data shows that the proposed methods can be successfully applied for routine analysis of drugs in gel formulation.

Keywords: Tazarotene; Hydroquinone; Simultaneous estimation; Multicomponent

Introduction

Hydroquinone is benzene-1, 4-diol chemically or also known as quinol having the chemical formula C₆H₄(OH)₂ [1]. It is topical agent used for treatment of certain skin conditions. In skin creams it is also used as de-pigmenter agent and antioxidant in the photography industry. Hydroquinone acts by inhibiting the melanin formation. Due to toxicological effects of hydroquinone it can cause dermatitis. In skin toning creams hydroquinone and some of its derivatives are present. So the determination of hydroquinone and its derivative in cosmetics is very important for the protection of human health [2-4].

Tazarotene is member of the acetylenic class of retinoids. Tazarotene is chemically (ethyl 6-[2-(4, 4-dimethyl-3, 4-dihydro- 2H-1-benzothiopyran-6-yl)-ethynyl]-pyridine-3-carboxylate). It is a third-generation topical retinoid. It is available in the form of cream, gel, or foam. Tazarotene is used for treatment of psoriasis, acne and photo damage skin.

Tazarotene is a prodrug which is converted to its active form by rapid de-esterification in humans and animals. Tazarotenic acid binds to all three members of the retinoic acid receptor (RAR) family: RARa, RARβ, and RARγ but shows relative selectivity for RARβ, and RARγ which may modify gene expression. The clinical significance of these findings is unknown [5] (Figure 1).

Figure 1:

    
    
    Figure 1:

Tazarotene plus hydroquinone is used in treatment of photo damaged facial skin. Literature review reveals that the efficacy of tazarotene is improved, when applied in combination with hydroquinone [6]. A very few analytical methods are available for the estimation of drugs like tazarotene and hydroquinone in combination in pharmaceutical dosage formulation [7-13]. The non-availability of analytical methods as on date for the concurrent analysis of multicomponent formulations made it worthwhile to pursue the present research work. The developed method is also been validated as per ICH guidelines [14]. The scope of developing and validating and analytical method is to ensure a suitable method for a particular analyte more specific, accurate and precise. The main objective for that is to improve the condition and parameters, which should be followed in the development and validation.

Materials and Methods

Instruments

A thermospectronic model is Lab India 3000+ (Double beam) spectrophotometer with 1cm matched quartz cells.

Chemicals & reagents

All chemicals are of analytical grade reagent and solutions were prepared in methanol: water (80:20). Tazarotene & hydroquinone gift samples were obtained from Lupin Pharmaceutical Ltd. Pune. Methanol is procured from Merck India Ltd. In-house formulation was prepared for gel formulation.

Procedure

Formula for preparation of carbopol gel:

Tazarotene: 0.1 % w/w

Hydroquinone: 4% w/w

Carbopol 940: 2% w/w

Triethanolamine: q.s.

Methyl hydroxy benzoate: 0.15 % w/w

Propyl hydroxy benzoate: 0.05 % w/w

Distilled water: 95.8 % w/w

Carbopol 940 was sprinkled slowly to 5ml of water as medium and the medium was continuously stirred to get a uniform dispersion of carbopol. The other ingredients that are methyl hydroxy benzoate and propyl hydroxyl benzoate were pre dissolved in separate portion of water (5ml) and added to carbopol dispersion. Final volume was adjusted with water and pH brought to neutral by using the triethanolamine.

Determination of solubility of Drug:

Preparation of standard stock solutions:

10mg of each tazarotene and hydroquinone was weighed accurately and transferred into two different 10ml volumetric flask respectively, and the volume was adjusted up to the mark with the methanol (80%), to give a stock solution of 1000ppm.

Determination of λmax of Drugs: Standard solution (10μg/ ml) of pure tazarotene and hydroquinone were scanned on UV spectrophotometer, which showed maximum absorbance at 351nm and 294nm for Tazarotene and Hydroquinone respectively. The UV spectra are shown in (Figure 2 & 3).

Figure 2: Selection of λ_max of tazarotene.

    
    
    Figure 2: Selection of λ_max of tazarotene.

Figure 3: Selection of λ_max of hydroquinone.

    
    
    Figure 3: Selection of λmax of hydroquinone.

Preparation of standard stock solution for test of linearity: From stock solutions of tazarotene 1 ml was taken and diluted up to 10ml. From this solution 0.1, 0.2, 0.3, 0.4, 0.5 ml solutions were transferred to five different 10ml volumetric flasks respectively and make volume up to 10ml with methanol (80%), gives standard drug solution of 1, 2, 3, 4, 5 μg/ml concentration and From stock solutions of hydroquinone 1ml was taken and diluted up to 10 ml. From this solution 1.0, 2.0, 3.0, 4.0, 5.0 ml solutions were transferred to five different 10ml volumetric flasks respectively and make the volume up to 10ml with methanol (80%), gives standard drug solution of 10, 20, 30, 40, 50 μg/ml concentration. The absorbance of resulting solutions for these drugs was measured at 294.0nm, and 351.0nm respectively.

Multi-component method: In this method six mixed standards of tazarotene and hydroquinone in the ratio of 0.1:4 having concentrations in μg/ml 1:10, 2:20, 3:30, 4:40, and 5:50 were prepared in methanol: water (80:20) solution by diluting appropriate volumes of the standard stock solutions and scanned in the region of 400nm to 200nm. Sampling wavelengths (294.0nm and 351.0nm) were selected on the trial and error basis. The concentration of individual drug was feed to the multi-component mode of the instrument. The instrument collects and compiles the spectral data from mixed standards and concentration of each component were obtained by spectral data of sample solution with reference to that of six mixed standards (Figure 4).

Figure 4: Overlay spectra of mixed standard of tazarotene and hydroquinone.

    
    
    Figure 4: Overlay spectra of mixed standard of tazarotene and hydroquinone.

Analysis of the gel formulations: A quantity of Gel equivalent to 0.1mg of tazarotene was transferred to separate 10ml volumetric flask and dissolved in 5ml of diluent with frequent shaking for 15 minutes and final volume was made up with diluent. The sample solution was then filtered through Whatman filter paper No.41 and first few ml were rejected. From the above solution 1.0ml of solution was taken and diluted to 10ml with diluent to get a solution containing 1μg/ml of tazarotene, and 40μg/ml of hydroquinone respectively. Absorbance of the sample was recorded at 294.0 and 351.0nm respectively and analysis procedure was repeated six times with gel formulation. Concentration of drugs in the sample was determined using absorbance of sample.

Validation of developed method

Linearity: Linearity of analytical procedure is its ability (within a given range) to obtain test, which are directly proportional to absorbance of analyte in the sample. The calibration plot was constructed after analysis of five different (from 1 to 5 μg/ml for tazarotene and 10 to 50 μg/ml for hydroquinone) concentrations and absorbance for each concentration were recorded three times, and mean absorbance was calculated (Table 1 & 2).

Table 1: Optical characteristics and linearity data.




  
    Parameter 
    Tazarotene (TAZA) 
    Hydroquinone (HYDRO) 
  
  
    lmax (nm) 
    351
    294 
  
  
    Beer’s Law limit (μg/ ml) 
    1-5
    10-50 
  
  
    Correlation coefficient 
    0.9998
    0.9973 
  
  
    Regression Equation 
    1.22x-0.001
    0.0053x+0.006 
  
  
    Intercept 
    0.001
    0.006 
  
  
    Slope 
    1.22
    0.0053 
  
  
    LOD (μg/ ml) 
    0.20
    0.60 
  
  
    LOQ (μg/ ml) 
    0.35 
    1.20



Table 1: Optical characteristics and linearity data.

Table 2: Analysis data of marketed formulations.




  
    Conc. (μg/ml) 
    Conc. found (μg/ml) 
    % Found 
  
  
    TAZA 
    HYDRO
    TAZA
    HYDRO
    TAZA
    HYDRO 
  
  
    2
    20
    2.05
    19.90
    102.50
    99.50
  
  
    2
    20
    2.00
    19.95
    100.00
    99.75
  
  
    2
    20
    2.01
    19.98
    100.50
    99.90
  
  
    2
    20
    1.98
    20.00
    99.00
    100.00



Table 2: Analysis data of marketed formulations.

Accuracy: Recovery studies were performed to validate the accuracy of developed method. To pre-analysed sample solution, a definite concentration of standard drug (80%, 100%, and 120%) was added and then its recovery was analyzed and result was shown in Table 3 and statistical validation of recovery studies shown in Table 4.

Table 3: Recovery studies for accuracy of formulation.




  
    Level of Recovery (%) 
    80 
    100 
    120 
  
  
    TAZA 
    HYDRO 
    TAZA 
    HYDRO 
    TAZA 
    HYDRO 
  
  
    Amount Present (mg) 
    2 
    10 
    2 
    10 
    2 
    10 
  
  
    2 
    10 
    2 
    10 
    2 
    10 
  
  
    2 
    10 
    2 
    10 
    2 
    10 
  
  
    Amount of Std. Added (mg) 
    1.6 
    8 
    2 
    10 
    2.4 
    12 
  
  
    1.6 
    8 
    2 
    10 
    2.4 
    12 
  
  
    1.6 
    8 
    2 
    10 
    2.4 
    12 
  
  
    Amount Recovered (mg) 
    1.58 
    8.01 
    2 
    10.01 
    2.39 
    11.98 
  
  
    1.59 
    7.99 
    1.99 
    10 
    2.4 
    12 
  
  
    1.6 
    8 
    2.01 
    9.99 
    2.39 
    11.99 
  
  
    % Recovery 
    98.75 
    100.13 
    100 
    100.1 
    99.58 
    99.83 
  
  
    99.38 
    99.88 
    99.5 
    100 
    100 
    100 
  
  
    100 
    100 
    100.5 
    99.9 
    99.58 
    99.92



Table 3: Recovery studies for accuracy of formulation.

Table 4: Results of analysis data of gel formulation.




  
    Level of Recovery (%) 
    Drug 
    % Recovery 
    Standard Deviation* 
    % RSD 
  
  
    80 
    Tazarotene 
    99.38 
    0.625 
    0.628931 
  
  
    Hydroquinone 
    100 
    0.125 
    0.125 
  
  
    100 
    Tazarotene 
    100 
    0.5 
    0.5 
  
  
    Hydroquinone 
    100 
    0.1 
    0.1 
  
  
    120 
    Tazarotene 
    99.72 
    0.240563 
    0.241233 
  
  
    Hydroquinone 
    99.92 
    0.083333 
    0.083403



Table 4: Results of analysis data of gel formulation.

Precision:

(A) Repeatability

Standard dilutions were prepared and three replicates of each dilution were analyzed in same day for repeatability and results were subjected to statistical analysis. Standard dilutions were prepared and three replicates of each dilution were analyzed in different days and by different analysts. Statistical analysis was carried out (Table 5).

Table 5: Results of analysis data of gel formulation.




  
    Drug 
    %    Label claim 
    Amount    found* 
    Label    claim (%) 
    S.D.    * 
    %    RSD 
  
  
    Tazarotene 
    0.1 
    0.099 
    99.66 
    0.052 
    0.058 
  
  
    Hydroquinone 
    4 
    3.99 
    98.6 
    0.252 
    0.125 
  
  
    *Denotes    average of three determinations.



Table 5: Results of analysis data of gel formulation.

(B) Intermediate Precision

(a) Day to Day

(b) Analyst to Analyst

The intermediate precision expresses with in laboratories variation: different days, different analysts, different equipment etc. The standard dilution was prepared and three replicate of each dilution were analyzed by different analysts for all the developed methods. The statistical analysis method was carried out and the data is presented in Table 6.

Download PDF

Citation: Yadav R, Yadav R and Yadav D. New Multi Wavelength Method for the Estimation of Tazarotene and Hydroquinone in Gel Formulation. Austin J Anal Pharm Chem. 2018; 5(2): 1099.

Instruction for Authors

Submit Your Article

Parameter	*Tazarotene* (TAZA)	*Hydroquinone* (HYDRO)
lmax (nm)	351	294
Beer’s Law limit (μg/ ml)	1-5	10-50
Correlation coefficient	0.9998	0.9973
Regression Equation	1.22x-0.001	0.0053x+0.006
Intercept	0.001	0.006
Slope	1.22	0.0053
LOD (μg/ ml)	0.20	0.60
LOQ (μg/ ml)	0.35	1.20

Conc. (μg/ml)		Conc. found (μg/ml)		% Found
TAZA	HYDRO	TAZA	HYDRO	TAZA	HYDRO
2	20	2.05	19.90	102.50	99.50
2	20	2.00	19.95	100.00	99.75
2	20	2.01	19.98	100.50	99.90
2	20	1.98	20.00	99.00	100.00

Level of Recovery (%)	Drug	% Recovery	Standard Deviation*	% RSD
80	*Tazarotene*	99.38	0.625	0.628931
80	*Hydroquinone*	100	0.125	0.125
100	*Tazarotene*	100	0.5	0.5
100	*Hydroquinone*	100	0.1	0.1
120	*Tazarotene*	99.72	0.240563	0.241233
120	*Hydroquinone*	99.92	0.083333	0.083403

Intra-day Precision			Inter-day Precision
	% Label claim			% Label claim
	*Tazarotene*	*Hydroquinone*		*Tazarotene*	Hydroquinone
After 1hr	99.52	99.15	First day	98.00	98.00
After2hr	99.10	99.10	Second day	97.10	98.10
After3hr	98.78	99.05	Third day	97.05	97.50
After4hr	98.70	98.56
After5hr	98.60	98.00
After6hr	98.50	98.00
Mean	98.87	98.64	Mean	97.38	97.87
S.D.*	0.38	0.54	S.D.*	0.53	0.32
% RSD	0.38	0.55	% RSD	0.55	0.33
*Denotes average of three determinations.

New Multi Wavelength Method for the Estimation of Tazarotene and Hydroquinone in Gel Formulation

Abstract

Introduction

Materials and Methods

Instruments

Chemicals & reagents

Procedure

Validation of developed method

Results and Discussion

Result of Precision

Conclusion

Acknowledgements

References