A Novel Method Development and Validation for Related Substances of Adapalene in Bulk Drug Product by HPLC

 

G. Kumar^a, T B. Patrudu^b*, M.V. Basaveswara Rao^a and Tentu. Nageswara Rao^a

^aDepartment of Chemistry, Krishna University, Machilipatnam, A.P., India.

^bDepartment of Chemistry, GITAM University, Hyderabad Campus, Telangana India.

 

ABSTRACT:

A simple and inexpensive method was developed with high performance liquid chromatography with PDA detection for determination of Adapalene and related impurities ((3-Adamantyl-4-methoxy) phenyl boronic acid, 6-(toluene-4-sulfonyloxy)-naphtalene -2-carboxylic acid methyl ester, 1-adamantyl-2-methoxybenzene, 6-(3-adamantan-1-yl-4-methoxy-phenyl)-naphtalene-2-carboxylic acid methyl ester and 3,3’-di-(1-adamantyl)-4,4’-dimethoxy-1,1’-biphenyl).  The chromatographic separations were achieved on (250×4.6 mm), 5.0 µm make: Phenomenex Luna column employing methanol, 0.1% orthophosphoric acid buffer, and Tetrahydrofuran in the ratio of 55:30:15 as mobile phase with gradient programmed at flow rate 1.0 mL/min was chosen. Five impurities were eluted within 35 minutes. The column temperature was maintained at 25^oC and a detector wavelength of 260 nm was employed.   The method was successfully validated by establishing System Suitability, Specificity, Linearity, Accuracy, limit of detection and Limit of quantification.

 

 

 

INTRODUCTION:

Adapalene is a synthetic naphthoic acid derivative with retinoid activity ^1,2. Adapalene is used to in the treatment of acne vulgaris ^3,4. Acne vulgaris was not cured, but it will control the acne with adapalene. This results also will be clearly seen after the prolong usage^5-8. Oral antibiotics is used to kill the bacteria in the body, but on prolong usage, this antibiotics causes side effects and shows adverse affect on main organs in human body ^{9, 10}.

 

To overcome these situations, topical medicines are very useful in the field of dermatology¹¹. At present Adapalene was available as gel in different compositions and combination with another molecule¹². Treatment with topical retinoids, such as adapalane lead to good results for acne vulgaris with less side effects and high efficacy. The accumulation of adapalene on skin is very low, decreased the risk of side affect and works effectively on targeted areas ¹². In the present study, a novel HPLC method was developed and successfully validated for Adapalene and its impurities. The details are given below. As on date, there were no research articles for method validation of related substances of adapalene.

 

 

Name	IUPAC Name	Structure	Molecular Formula	Molecular Weight
Adapalene	6-(3-Adamantan-1-yl-4-methoxy-phenyl)-naphtalen-2-carboxylic acid		C28H28O3	412.5
Impurity A	(3-Adamantyl-4-methoxy) phenyl boronic acid		C17H23BO3	286.18
Impurity B	6-(toluene-4-sulfonyloxy)-naphtalene -2-carboxylic acid methyl ester		C19H16O5S	356.40
Impurity C	1-adamantyl-2-methoxybenzene		C17H22O	242.35
Impurity D	6-(3-adamantan-1-yl-4-methoxy-phenyl)-naphtalene-2-carboxylic acid methyl ester		C29H30O3	426.56
Impurity E	3,3’-di-(1-adamantyl)-4,4’-dimethoxy-1,1’-biphenyl		C34H42O2	482.71

 

MATERIAL AND METHODS:

Materials:

Standard gift samples of Adapalene and impurities were provided by Dr Benarji Patrudu, Associate Professor, Gitam University, and Hyderabad. All the chemicals and reagents used were of analytical grade.

 

HPLC Chromatographic Parameters:

Chromatographic separation was performed on The HPLC-UV system used, consisted shimadzu high performance liquid chromatography with LC- 20AT pump and SPD-20A interfaced with LC solution software, equipped with a reversed   phase C18 analytical column of 250 mm x 4.6 mm and particle size 5 µm (PhenomenexLuna-C18)  Column oven temperature was maintained at 25°C An HPLC method was developed for Adapalene and related impurities by using photo diode array detector.  Adaplane   and all related impurities were injected into HPLC system by changing the different composition of Methanol (MeOH): 1% v/v H₃PO₄ in water: Tetrahydrofuran (THF). Finally the Adaplane and related impurities are separated in the composition as given below.

Pump A: Methanol;

Pump B: 1% v/v H₃PO₄ in water and

Pump C: Tetrahydrofuran (THF)

 

Time	% of A	% of B	% of  C	Flow rate (mL)
0	55	30	15	1.00
1.5	55	30	15	1.00
20	75	10	15	1.00
35	75	10	15	1.00
45	75	10	15	1.00
46	55	30	15	1.00
60	55	30	15	1.00

 

The absorption maxima for Adapalene, Impurity –A, Impurity-B,  Impurity –C, Impurity-D and Impurity –E were found to be at 260 nm. the compound was scanned form 200 – 400 nm. Column temperature was set up at 25°C and injection volume as set to 20µL. By follow this analytical method conditions, Adapalene and related impurities were separated. Hence, it was concluded that HPLC method was suitable for method validation.

 

Method Validation:

System Suitability:

Preparation of test solution:

In a 50 ml volumetric flask dissolve about 150.0 mg of the substance to be examined, accurately weighed, with dissolution phase and dilute to volume with dissolution phase.

 

Preparation of Reference Solution (a):

In a 50 ml volumetric flask dissolve about 15 mg of reference standard accurately weighed with dissolution phase and dilute to volume with dissolution phase. In a 100 ml volumetric flask transfer 1 ml of this solution, dissolve and dilute to volume with dissolution phase (0.1% with respect to test solution).

 

Preparation of Reference Solution (b) for System Suitability:

Dissolve 15 mg of Impurity D in a 50 ml volumetric flask and dilute to volume with dissolution phase. Take 0.5 ml of the latter solution and dilute in a 50 ml flask containing 150 mg of Adapalene.

 

System Suitability Test:

Resolution:

The resolution between the peaks of Adapalene and Imp D not be less than 5.0 in the chromatogram obtained with reference solution (b).

Procedure:

Program the method. Inject the reference solution b and check the resolution between the peak of Adapalene and Impurity D. Inject six times 10µl of reference solution (a) and record the chromatograms. Inject the test solution once. Calculate reference standard mean area of Adapalene with all six injections.

 

The test is invalid if RSD of reference standard areas is more than 3 %.

Specificity and Selectivity:

In a separate 50 mL volumetric flasks, dissolve 15 mg of the Adapalane reference standard, impurity-A, impurity-B, impurity-C, impurity-D and impurity-E and dissolve with dissolution phase(each solution conc. - 300 µg/ml) and labelled as standard stock solutions.  1.0 mL of each solution was transferred into separate 100 ml volumetric flask transfer, dissolve and dilute to volume with dissolution phase (each solution conc. - 3 µg/ml) and the resulting solutions were injected in HPLC system and peak purity was determined for adaplane and related impurities. In a 100 ml volumetric flask pipette 1 ml of the standard stock solutions of each impurity and of Adapalene, previously prepared, dissolve and dilute to volume with dissolution phase. Inject the obtained solution 6 times for its selectivity.

 

Linearity:

Standard stock solution of Adapalane and impurities having concentrations of 300 µg/mL was taken for linearity test. 0.2, 0.5, 1.0, 1.5 and 2.0 mL of standard stock solution of Adapalane and impurities was transferred into separate 100 mL volumetric flask and diluted up to the mark with dissolution phase to get the final concentration of 0.6, 1.5, 3.0, 4.5 and 6.0 µg/mL respectively. The linearity was in the range of 20 – 200 % for A.I and impurities. The resulting solutions were injected into HPLC in three replications at 260 nm. Correlation coefficient was calculated for A.I and impurities by plotting the graph between concentrations versus peak Area.

 

Precision:

The Precision was determined in agreement with ICH guidelines, injecting six different solutions containing both impurities and Adapalene at the test concentration (six solutions with different weights are prepared and injected). In a separate 50 mL volumetric flasks, dissolve 15 mg of the Adapalane , impurity-A, impurity-B, impurity-C, impurity-D and impurity-E and dissolve with dissolution phase (each impurity conc. - 300 µg/ml) and labelled as standard stock solutions.  1.0 mL of solution was transferred into separate 100 ml volumetric flask transfer, dissolve and dilute to volume with dissolution phase (each impurity conc. - 3 µg/ml).

 

Intermediate Precision:

Intermediate precision was assessed by six injections of test solutions prepared on different days, using fresh mobile phase, as in Precision.

 

Accuracy:

The Accuracy was determined as prescribed by ICH guidelines. Known quantities of impurities are going to be added to Adapalene at 50 –100 –150% of the nominal limit of  0.10 % for each impurity. The results obtained have to meet the proposed limits.  The scheme to carry out applied to every impurity. The experiment is performed in triplicate at each level.

 

LOD and LOQ

LOD and LOQ were assessed in accordance with ICH guidelines ¹³. The method chosen is based on Signal-to-noise ratio, using the following formulas:

 

LOD=	3 x s		LOQ=	10 x s
	S			S

 

RESULTS AND DISCUSSIONS:

System Suitability:

The resolution between the peaks of adapalane and Impurity 2 are more than 1.5 and % RSD of reference standard areas is less than 3 % on each day of analysis. Hence the system suitability passes the acceptance criteria. The suitability of method was confirmed by verifying the USP’s parameters like retention times (RT), theoretical plates (N) and tailing factors (T).The results were presented in Table. 1.

 

Table. 1 System suitability parameter results

Product	Approximate RT	N	T
Impurity - A	10.0	20154	1.02
Impurity - B	4.9	17845	0.98
Impurity - C	16.1	16984	0.78
Impurity - D	23.0	21899	1.12
Impurity - E	31.0	15742	1.29
Adapalane	19.5	13891	1.37

 

Specificity and Selectivity:

The specificity of the method was determined by injecting the individual solutions of A.I (3 µg/mL) and impurities (3 µg/mL) and peak purity was checked. The peak purity was more than 99 % for Adapalene and impurities confirm the specificity of the method.

 

Linearity:

The linearity regression curve for Adapalene   and their impurities were drawn between concentrations and peak areas. The correlation coefficient is above 0.99 at wavelength of 260 nm for adapalene and their impurities. The results are mentioned in Table 2. A calibration curves were showed in Figure 1 and Representative chromatograms were presented in Figure 2 and figure 3. 

 

Table 2. Linearity Data of adapalene and its impurities

Range	Concentration in µg/mL	Area in mAU-sec of Adapalene	Area in mAU-sec Impurity-A	Area in mAU-sec Impurity-B	Area in mAU-sec Impurity-C	Area in mAU-sec Impurity-D	Area in mAU-sec Impurity-E
20%	0.6	35141	5347	6929	6868	19503	24954
50%	1.5	98959	13358	18680	15259	52925	53247
100%	3	189278	26810	39668	33392	113120	111789
150%	4.5	290217	40256	55576	47639	159865	171657
200%	6	378942	53265	75254	64635	213465	224455
Slope		63595.48	8892.27	12556.99	10728.41	35800.11	37562.96
Intercept		89.49	63.32	43.59	85.98	79.27	52.61
correlation coefficient		0.9997	1.0000	0.9991	0.9994	0.9992	0.9992

 

 

Figure 1. Linear regression curve of adapalene its impurities

 

Figure. 2. Representative chromatogram of diluent as mobile phase

 

 

Figure. 3. Representative chromatogram of linearity 4.5 µg/mL

 

Precision:

The precision test is carried out with six homogenous solution of Adapalene test item and the content of Adapalene and their impurities were calculated. The results are mentioned in Table 3 to Table 8.

 

Table. 3. Intermediate Precision Impurity A

Injection	Weight in mg	Area in mAU.Sec	Day
1	11.92	38638	Day 1
2	11.60	37885		Average	38273
3	11.92	37851		STDV	1069
4	12.24	36879		RSD	2.79%
5	12.40	37475
6	12.40	36375
7	15.6	37659	Day 2
8	15.12	39546
9	14.64	39732
10	14.48	39259
11	14.64	39064
12	15.04	38806

 

Table. 4. Intermediate Precision Impurity B

Injection	Weight in mg	Area in mAU.Sec	Day
1	15.31	34597	Day 1
2	15.06	33971		Average	35122
3	15.11	36152		STDV	644
4	14.50	34258		RSD	1.83%
5	14.91	35630
6	15.3	35507
7	15.62	35100	Day 2
8	14.75	34712
9	15.23	35299
10	15.27	35747
11	15.03	35502
12	15.31	34994

 

Table. 5. Intermediate Precision Impurity C

Injection	Weight in mg	Area in mAU.Sec	Day
1	15.71	32795	Day 1
2	14.83	32880		Average	31753
3	14.81	32624		STDV	903
4	14.95	32274		RSD	2.84%
5	15.63	32147
6	14.82	32629
7	14.63	31131	Day 2
8	15.25	30290
9	14.66	31431
10	15.13	30953
11	15.74	30745
12	14.93	31140

 

Table. 6. Intermediate Precision Impurity D

Injection	Weight in mg	Area in mAU.Sec	Day
1	15.92	107386	Day 1
2	15.66	106148		Average	108550
3	15.63	109107		STDV	2136
4	16.31	105493		RSD	1.97%
5	15.9	111567
6	22.54	111015
7	15.16	108044	Day 2
8	15.31	105186
9	15.24	109088
10	14.82	110341
11	15.37	110299
12	15.26	108925

 

Table. 7. Intermediate Precision Impurity E

Injection	Weight in mg	Area in mAU.Sec	Day
1	15.82	101289	Day 1
2	15.72	100830		Average	101794
3	15.84	101547		STDV	962
4	15.94	102525		RSD	0.95%
5	15.78	101319
6	15.84	101958
7	15.82	101827	Day 2
8	15.46	103017
9	15.52	103593
10	15.27	100008
11	15.35	101465
12	15.44	102156

 

Accuracy:

Preparation of Test solutions:

Prepare a solution containing all the impurities at a concentration of 300 µg/ml each (15 mg/50 ml; solution from the linearity test can be used). Transfer respectively 0.5 ml, 1 ml and 1.5 ml of this solution to three different 100 ml volumetric flasks, containing 300 mg of Adapalene each one. The dilutions have to be carried out for each solution of the linearity test (total: 3x3 test solutions). The representative chromatogram showed in Figure 4 and results were presented in Table 9.

 

 

Table. 8.  Intermediate Precision Adapalene

Injection	Weight in mg	Area in mAU.Sec	Day
1	15.3	123358	Day 1
2	15.07	122587		Average	125289
3	14.96	131140		STDV	3193
4	14.98	125115		RSD	2.55%
5	15.52	127939
6	15.43	129948
7	15.41	120169	Day 2
8	15.42	124927
9	15.55	122046
10	14.92	125677
11	15.23	126037
12	15.81	124531

Table 9. Recovery results of adapalene impurities.

%	Impurity -A	Impurity -B	Impurity -C	Impurity -D	Impurity -E
50	98.3	97.4	95.4	96.3	95.4
50	99.1	97.9	97.2	96.8	94.8
50	98.8	96.7	98.5	97.1	96.3
100	99.2	97.4	98.7	98.8	97.4
100	98.9	98.8	98.1	99.7	97.8
100	99.7	97.8	99.2	99.6	98.2
150	99.2	99.2	99.7	99.8	98.7
150	99.8	99.7	98.7	99.9	98.5
150	99.5	96.3	99.7	99.6	99.2

 

Figure. 4. Representative chromatogram of 100 % petrification level of impurities

 

LOD and LOQ:

The LOD and LOQ are established successfully for each impurity in Adapalene based on Signal-to-noise ratio method. The results were presented in Table 10.

 

 

Table 10. Limit of quantification and Limit of detection results of adapalene impurities

Impurity	Average S/N		% Conc
Imp A	26.28	LOD	0.01
		LOQ	0.03
Imp B	57.48	LOD	0.005
		LOQ	0.02
Imp C	29.07	LOD	0.01
		LOQ	0.03
Imp D	85.65	LOD	0.004
		LOQ	0.01
Imp E	110.38	LOD	0.003
		LOQ	0.01

 

CALCULATIONS:

The Adapalene impurities are determined by comparison of peaks areas with the following formula:

 

Percentage Adapalene impurity =	At x C x D x PS	x 100
	Ar x W sample x Fc

 

Where:

At: peak area of Impurity obtained by test solution

Ar: peak area of Adapalene obtained by reference solution (a)

C: Adapalene concentration in reference solution (a) (mg/ml)

D: sample dilution (ml)

W sample: sample weight in test solution (mg)

PS: Purity of reference standard

Fc: Response Factor of Impurity

 

% Recovery	=	Recovered Concentration	×	100
		Fortified Concentration

 

CONCLUSIONS:

The method developed for quantitative determination of adapalene and its impurities is rapid, precise, accurate and selective. The method was completely validated showing satisfactory data for all method - validated parameters tested. The mobile phase composition of methanol (MeOH), 1% H3PO4 in water and Tetrahydrofuran showed good separation and resolution. Satisfactory validation parameters such as linearity, recovery, precision LOD and LOQ were established by following ICH guidelines. Therefore, the proposed analytical procedure could be useful for regular monitoring, pharma manufacturing labs and research scholars

.

ACKNOWLEDGEMENT:

The authors are thankful to the Dr. Benerjee patrudu, Gitam University , Hyderabad for providing the gift sample of Adapalene and providing necessary facilities to carry out the research work with keen interest and help.

 

REFERENCES:

1.     Martins LA, Meneghini LZ, Junqueira CA, Ceni DC, Bergold A. A simple HPLC-DAD method for determination of adapalene in topical gel formulation. J Chromatogr Sci. 49(10); 2011 :796-800.

2.     Tolba MM and El-Gamal RM. Determination of adapalene in gel formulation by conventional and derivative synchronous fluorimetric approaches, application to stability studies and in vitro diffusion test. Chem Cent J 2016; 10: 33.

3.     Yi-Cheng Chen, Pi-Ju Tsai, Yaw-Bin Huang, and Pao-Chu Wu. Optimization and Validation of High-Performance Chromatographic Condition for Simultaneous determination of Adapalene and Benzoyl Peroxide by Response Surface Methodology. PLoS One. 10(3); 2015: e0120171.

4.     Prakash Modi  B and Nehal Shah J.  Novel Stability-Indicating RP-HPLC Method for the Simultaneous Estimation of Clindamycin Phosphate and Adapalene along with Preservatives in Topical Gel Formulations. Sci Pharm. 82(4); 2014: 799–813.

5.     Laura Martins A, Leonardo Meneghini Z, César Junqueira A, Danieli Ceni C and Ana Bergold M. A Simple HPLC-DAD Method for Determination of Adapalene in Topical Gel Formulation. Journalof Chromatographic Science. 49; 2011: 796-800.

6.     Mailvelan R, Selvamani P, Rameshkumar T, Raviraj T. HPLC Method Development and Validation for the Estimation of Adapalene in Pharmaceutical Formulation. Asian Journal of Pharmaceutical Analysis and Medicinal Chemistry. 1(3); 2013:166 - 171.

7.     Rohit Khatri H, Rashmin Patel B and Mrunali Patel R. A new RP-HPLC method for estimation of clindamycin and Adapalene in gel formulation: development and validation consideration. The Thai Journal of Pharmaceutical Sciences. 38 (1); 2014: 1-56.

8.     Deo SS, Inam F and Karmarkar NP. Analytical Method Development for Determination of Performance of Adapalene in Adapalene 0.1% Gel Formulation Using Manual Diffusion Cell. Chem Sci Trans. 2(1): 2013:251-257.

9.     Mudasir M, Tabassum N, Ali J, Khan NA & Jan R. Qualitative and Quantitative Estimation by HPLC Method in Transdermal Formulations: A Technical Note.  RJPBCS. 2(1); 2011:231-241.

10.   Khatri Rohit H, Patel Rashmin B, Patel Mrunali R. A new RP-HPLC method for estimation of clindamycin and Adapalene in gel formulation: development and validation consideration. Thai Journal of Pharmaceutical Sciences. 38 (1); 2014: 44-48.

11.   Prakash Modi B, Nehal Shah J. Novel Stability-Indicating RP-HPLC Method for the Simultaneous Estimation of Clindamycin Phosphate and Adapalene along with Preservatives in Topical Gel Formulations. Sci. Pharm. 82; 2014: 799–813.

12.   Barrios, Jerusa Goi. Validation of analytical method by HPLC for determination of Adapalene in suspensions of nanocapsules. Quím. 34(8); 2011:1464-1467.

13.   International Conference on Harmonisation, Validation of Analytical Procedures. ICH Q2B.  1996.

 

 

Accepted on 06.11.2016        © RJPT All right reserved

Research J. Pharm. and Tech 2016; 9(12):2234-2240.