A New Analytical method Validation and Quantification of Entacapone and its Related Substance in bulk Drug Product by HPLC

 

G. Kumar¹, T.B. Patrudu²*, Tentu Nageswara Rao¹, M.V. Basaveswara Rao¹

¹Department of Chemistry, Krishna University, Machilipatnam, A.P., India.

²Department of Chemistry, GITAM University, Hyderabad Campus, Telangana India.

*Corresponding Author E-mail: tnraochemistry@gmail.com

ABSTRACT:

A simple and inexpensive method was developed with high performance liquid chromatography with PDA detection for determination of entacapone and its related impurities are (N, N diethyl cyano acetamide), (3,4 Dihydroxy 5-Nitro benzaldehyde) and (2-Z)-2-Cyano-3-(3,4-Dihydroxy-5-nitrophenyl)N,N-diethyl-2-propenamide. The chromatographic separations were achieved on (250×4.6 mm), 5.0 µm make: Phenomenex Luna column employing Acetonitrile: HPLC Water in the ratio of 50:50 (v/v) as mobile phase with isocratic at flow rate 0.9 mL/min was chosen. Three impurities were eluted within 15 minutes. The column temperature was maintained at 30^oC and a detector wavelength of 210 nm was employed. The method was successfully validated by establishing System Suitability, Specificity, Linearity, Accuracy, limit of detection and Limit of quantification.

 

 

 

INTRODUCTION:

Entacapone API is nitrocatechol derivative and it is used for treatment of Parkinson’s disease^1,2. Maximum recommended daily dose for adults is 2000 mg per day if necessary. Parkinson’s disease is a neurodegenerative, slow progressive disorder, resting tremor, rigidity and postural reflex impairment with associated characteristic eosinophilic cytoplasmatic inclusions. Entacapone should be used in combination with levodopa³. It is film coated tablet containing 200 mg Entacapone API in one tablet ^4,5.

 

It is orally taken medicine. Entacapone is rapidly absorbed in the gastro-intestinal tract and undergoes extensive first pass metabolism. entacapone is converted to its (cis)-isomer i.e. Z- entacapone, the main metabolite in plasma followed by direct glucuronidation to inactive glucuronide conjugates ^6,7. 

 

High performance liquid chromatography (HPLC) is a technique used for analysis of drug substance, drug product and determination and quantification of known as well as unknown impurities at lower level, food and drug administration (FDA) also trust on the purity method of analysis by using HPLC, because of high accuracy and reproducibility of results⁸. By using this technique we can separate drug related process impurities, degradation impurities as well as reactants⁹. In the present study, a novel HPLC method was developed and successfully validated for entacapone and its impurities. The details are given below. As on date, there were no research articles for method validation of related substances of entacapone.

Name	IUPAC Name	Structure	Origin
entacapone	(2E)-2-cyano-3-(3, 4-dihydroxy-5-nitrophenyl)-N, N- diethyl-2-propenamide		-
Impurity -I	N, N-Diethyl cyano acetamide		Key raw material
Impurity - II	3,4-Dihydroxy-5-nitrobenzaldehyde		Key raw material
Z-isomer	(2Z)-2-cyano-3-(3,4-dihydroxy-5-nitro phenyl)N,N-diethyl-2-propenamide		Process impurity

 

MATERIAL AND METHODS:

MATERIALS:

Standard gift samples of  entacapone 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 30°C and flow rate 0.9 mL/min An HPLC method was developed for entacapone and related impurities by using photo diode array detector. Entacapone and all related impurities were injected into HPLC system by changing the different composition of Acetonitrile: HPLC Water in the ratio of 50:50 (v/v). The absorption maxima for  entacapone, Impurity –I, Impurity-II, Z-isomer was found to be at 215 nm. the compound was scanned form 200 – 400 nm. Column temperature was set up at 30°C and injection volume as set to 10µL. By follow this analytical method conditions,  entacapone and related impurities were separated. Hence, it was concluded that HPLC method was suitable for method validation.

 

Method Validation:

Specificity:

Entacapone impurity –I (N, N diethyl cyano acetamide, Entacapone impurity-II (3,4-Dihydroxy-5-nitrobenzaldehyde,Z-Isomer:(2-Z)-2-Cyano-3-(3,4-Dihydroxy-5-nitrophenyl) N,N-diethyl-2-propenamide and Entapone2E)-2-Cyano-3-(3,4-Dihydroxy-5-nitrophenyl) N,N-diethyl-2-propenamide were injected in to the HPLC at the target concentration (1.5 ppm) individually Then injected a sample of Entacapone spiked with known impurities (0.15 %) to demonstrate the separation of each impurity in the sample matrix.

Preparation of Working Reference Standard Stock Solution:

Weighed 15.10 mg of Entacapone impurity –I, 15.1 mg of Entacapone impurity –II, 15.17 mg of Z-Isomer and 10.30 mg of Entacapone into a 100 ml volumetric flask, dissolved and diluted to volume with the diluent.

 

Preparation of Working Reference Standard Solution:

From the above working reference stock solution, 1.0ml taken into a 100 ml volumetric flask and diluted to volume with the diluent.

 

Preparation of Entacapone impurity-I, II and Z-Isomer standard solutions (Conc.1.5 µg/ml):

Weighed 15.10 mg Entacapone impurity-I, II and  Z-Isomer reference standards in 100 ml volumetric flask, and diluted up to the mark with diluent. From the above working reference stock solution, 1 ml taken into a 100 ml volumetric flask and diluted to volume with the diluent. The reference standard solutions were injected into HPLC.

 

Linearity:

Standard stock solution of entacapone and impurities having concentrations of 100 µg/mL was taken for linearity test. 0.2, 0.5, 1.0, 1.5 and 2.0 mL of standard stock solution of entacapone and impurities was transferred into separate 100 mL volumetric flask and diluted up to the mark with acetonitrile as diluent to get the final concentration of 0.2, 0.5, 1.0, 1.5 and 2.0 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 210 nm. Correlation coefficient was calculated for A.I and impurities by plotting the graph between concentrations versus peak Area.

 

Intermediate Precision:

The Precision was determined in agreement with ICH guidelines, injecting six different solutions containing both impurities and  entacapone at the test concentration (six solutions with different weights are prepared and injected). In a separate 100 mL volumetric flasks, dissolve 10 mg of the  entacapone, impurity-I, impurity-II and Z-isomer and dissolve with acetonitrile as diluent (each impurity conc. - 100 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. - 1 µg/ml). 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 entacapone 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 guidelines¹⁰. 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:

Specificity:

The resolution between Z-Isomer and entacapone is  more than 2.0 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
Entacapone	11.4	31247	0.89
Impurity - I	4.3	24714	0.74
Impurity - II	6.1	20478	1.04
Z-isomer	10.9	19801	0.92

 

Linearity:

The linearity regression curve for entacapone  and their impurities were drawn between concentrations and peak areas. The correlation coefficient is above 0.999 at wavelength of 210 nm for entacapone 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 entacapone and its impurities

Range	Concentration in µg/mL	Area in mAU-sec of entacapone	Area in mAU-sec of Impurity-I	Area in mAU-sec of Impurity-II	Area in mAU-sec of  Z-isomer
20%	0.2	21274	16254	13895	18754
50%	0.5	51843	40004	34111	47109
100%	1	104074	81987	69217	96208
150%	1.5	156231	124685	103548	140987
200%	2	208785	161514	137595	189524
Slope		104254.93	81554.19	68885.02	94632.51
Intercept		16.27	72.44	32.7805	98.59
correlation coefficient		1.0000	0.9997	0.9998	0.9999

 

 

 

Figure 1. Linear regression curve of entacapone its impurities

 

Figure. 2. Representative chromatogram of diluent as acetonitrile

 

 

Figure. 3. Representative chromatogram of linearity 1.0 µg/mL

 

Precision

The precision test is carried out with six homogenous solution of entacapone test item and the content of  entacapone and their impurities were calculated. The results are mentioned in Table 3 to Table 6.

 

Table. 3. Intermediate Precision entacapone

 

Table. 4. Intermediate Precision Impurity -I

Injection	Weight in mg	Area in mAU.Sec	Day
1	10.41	82254	Day 1
2	10.35	82047		Average	81780.42
3	10.29	81787		STDV	548.65
4	10.37	82111		RSD	0.67
5	10.44	82874
6	10.28	81625
7	10.19	81475	Day 2
8	10.35	81958
9	10.38	81978
10	10.27	81305
11	10.22	80956
12	10.25	80995

 

Table. 5. Intermediate Precision Impurity - II

Injection	Weight in mg	Area in mAU.Sec	Day
1	10.30	72541	Day 1
2	10.22	71984		Average	72215.83
3	10.28	72458		STDV	870.88
4	10.17	72169		RSD	1.21
5	10.36	73004
6	10.26	71547
7	10.09	70895	Day 2
8	10.05	70421
9	10.29	72524
10	10.33	72965
11	10.31	72854
12	10.39	73228

Table. 6. Intermediate Precision Z-isomer

 

Accuracy:

Preparation of Test Solutions:

Prepare a solution containing all the impurities at a concentration of 100 µg/ml each (10 mg/100 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 100 mg of entacapone 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 7 and results were presented in Table 7.

 

Table 7. Recovery results of  entacapone impurities.

%	Impurity -I recovery in %	Impurity -II recovery in %	Z - Isomer recovery in %
50	95.23	94.29	96.78
50	96.14	95.06	96.29
50	95.87	95.43	96.74
100	97.23	96.74	98.25
100	97.58	97.12	98.21
100	97.96	96.89	98.77
150	98.74	98.06	98.94
150	98.09	97.96	99.18
150	98.28	98.56	99.27

 

Figure. 9. Representative chromatogram of 100 % petrification level of impurities

 

LOD and LOQ:

The LOD and LOQ are established successfully for each impurity in entacapone based on Signal-to-noise ratio method. The results were presented in Table 8.

 

Table 8. Limit of quantification and Limit of detection results of  entacapone impurities

Impurity	Average S/N		Concentration in µg/mL
Impurity -I	11.45	LOD	0.02
		LOQ	0.05
Impurity -II	14.72	LOD	0.01
		LOQ	0.03
Z - Isomer	12.38	LOD	0.003
		LOQ	0.01

 

CALCULATIONS

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

 

Percentage  entacapone 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  entacapone obtained by reference solution (a)

C:  entacapone 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 entacapone 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 acetonitrile and HPLC water 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  entacapone and providing necessary facilities to carry out the research work with keen interest and help.

 

REFERENCES:

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Received on 25.09.2016       Accepted on 27.10.2016     

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