Novel Spectrophotometric Method Development for the Estimation of Boceprevir in Bulk and in Pharmaceutical Formulations

 

P. Govardhan Reddy¹, V. Kiran Kumar¹, VVSS. Appala Raju² J.Raghu Ram³,

N. AppalaRraju³*

¹Department of Pharmaceutics, Unity college of Pharmacy, Raigir (V), Bhongir (M), Yadagiri(D), Telanagan State.

²Deparment of Chemistry, Faculty of Pharmacy, MAHSA University, Koula Lumpur, Malaysia.

³Department of Pharmaceutical Chemistry, Sultan Ul-Uloom College Of Pharmacy, Mount Pleasant, 8-2-249, Road No.3, Banjara Hills, Hyderabad, Telangana – 50034, India

 

ABSTRACT:

A New, Simple, Rapid and economical extractive spectrophotometric methods were developed for the determination of Boceprevir (anti-retroviral drug). Boceprevir is a direct acting protease inhibitor for the treatment of hepatitis C. It also has two isomers in which the S isomer is more active than the R-isomer.  The methods were based on the formation of color chromogens with Bromo cresol green, Bromo thymol blue, Bromo phenol blue and Methyl orange indicator. The extractive spectrophotometry was carried out with phthalate buffer and chloroform. The absorbances of the chromogens were measured at 410 nm and 415 nm against the corresponding reagent blank. The proposed methods have been successfully applied to the bulk drug. The method has been statistically evaluated and was found to be precise and accurate.

 

 

 

INTRODUCTION:

Boceprevir (Victrelis™)  is indicated for the treatment of chronic hepatitis C genotype 1 infection, in combination with peginterferon alfa and ribavirin, in adult patients (18 years and older) with compensated liver disease, including cirrhosis, who are previously untreated or who have failed previous interferon and ribavirin therapy ^1-3. Boceprevir has the following chemical name:(1R,5S)-N-[3-Amino-1- (cyclo butyl methyl)- 2,3dioxo propyl] -3-[2(S)-[[[(1,1-dimethyl ethyl) amino] carbonyl] amino]-3,3-dimethyl-1-oxobutyl] -6,6-dimethyl-3azabicyclo [3.1.0] hexan-2(S)-carboxamide^4-6. The molecular formula is C₂₇H₄₅N₅O₅ and its molecular weight is 519.7. Boceprevir is manufactured as an approximately equal mixture of two diastereomers.

 

Boceprevir is a white to off-white amorphous powder^7-9. It is freely soluble in methanol, ethanol and isopropanol and slightly soluble in water. Victrelis ® 200 mg capsules are available as hard gelatin capsules for oral administration. Each capsule contains 200 mg of boceprevir and the following inactive ingredients: sodium lauryl sulfate, microcrystalline cellulose, lactose monohydrate, croscarmellose sodium, pre-gelatinized starch, and magnesium stearate. The red capsule cap consists of gelatin, titanium dioxide, D and C Yellow #10, FD and C Blue #1, and FD and C Red #40. The yellow capsule body contains gelatin, titanium dioxide, D and C Yellow #10, FD and C Red #40, and FD and C Yellow #6. The capsule is printed with red and yellow ink. The red ink contains shellac and red iron oxide, while the yellow ink consists of shellac, titanium dioxide, povidone and D and C Yellow #10 Aluminum Lake. Boceprevir is an inhibitor of the HCV NS3/4A protease that is necessary for the proteolytic cleavage of the HCV encoded polyprotein into mature forms of the NS4A, NS4B, NS5A and NS5B proteins. Boceprevir covalently, yet reversibly, binds to the NS3 protease active site serine (S139) through an (alpha)-ketoamide functional group to inhibit viral replication in HCV-infected host cells¹⁰.

 

The literature survey showed the different studies which were carried out on Boceprevir include - Intracellular and plasma pharmacokinetics of Boceprevir, High performance liquid chromatography using UV detection for the simultaneous quantification of Boceprevir and 4 protease inhibitors in human plasma and LC-MS/MS determination of Boceprevir in rat plasma.^11-12 Extractive spectrophotometry is the method of analysis for Boceprevir which involves the formation of colored ion-association complexes. Boceprevir being a base forms an ion-association complex with acidic dyes Bromo Cresol Green (BCG) and Bromo Thymol Blue (BTB), which is extractable into chloroform from an aqueous phase. The protonated nitrogen of drug molecule in acid medium is expected to attract the oppositely charged part (negative charge) of the dye and behave as a single unit being held together by electrostatic forces of attraction.

 

MATERIALS AND METHODS:

Instruments used:

A shimadzu UV-1700 Pharmaspec with 1 cm matched quartz cell were used for the spectral and absorbance measurements. A Digisun digital pH meter was used for pH measurements.

 

Preparation of standard drug solution for Boceprevir:

A standard stock solution containing 1 mg/ml was prepared by dissolving 10 mg of Boceprevir in 10 m1 of acetone (1000 μg/m1). 1 ml of above solution is taken and dissolved in 10 ml of acetone to obtain 100 μg/m1 and it was further diluted i.e., 1 ml of the second stock solution was taken and diluted to 10 ml, to produce a concentration of 10 μg/ml. From the former stock solution 10 – 100 μg/ml concentrations and from the later 1-10 μg/ml concentrations were made respectively.

 

Preparation of reagents:

All the chemicals and reagent used were of analytical grade and the solutions were prepared in double distilled water. The various methods proposed are described in the Table I.

Table I: List of chemicals and reagents used in the proposed methods

S.NO	METHODS	REAGENTS AND CHEMICALS	PREPARATIONS
1.	METHOD 1 (BCPV 1)	POTASSIUM HYDROGEN PHTHALATE BUFFER (pH 2.4)	Accurately weighed quantity of 4.08 gm of Potassium hydrogen phthalate was dissolved in 100 ml of distilled water(0.2M) To 25 ml of above solution, 21.1 ml of 0.2 M HCl was added and diluted to 100 ml with distilled water to obtain a buffer solution of pH 2.4.
		BROMO CRESOL GREEN (0.1% W/V) (BCG)	Dissolve 100 mg of BCG in 10 ml of distilled water.
		CHLOROFORM	AR grade
2.	METHOD 2 (BCPV 2)	BROMO THYMOL BLUE (0.1% W/V) (BTB)	Dissolve 100 mg of BTB in 10 ml of distilled water.

 

Table II: Optical and regression characteristics and precision of proposed methods for Boceprevir

S.NO.	PARAMETERS	BCPV 1	BCPV 2
1.	λmax (nm)	415	415
2.	Beer’s law limits (μg/ml)	1 - 10	1 - 10
3.	Molar absorptivity (1 mole-1 cm-1)	2.77*10-6	0.171*10-6
4.	Sandell’s sensitivity (μg.cm-2/0.001 AU)	0.003	0.055
5.	Regression equation Slope (m) Intercept (c)	0.131 0.4724	0.0081 0.1883
6.	Correlation coefficient (r)	0.994	0.9899
7.	Precision (% RSD)	0.2754	0.1856

 

Parameter Fixation:

In developing these methods, a systematic study of the effects of various parameter in the method concerned were undertaken by varying one parameter at a time and controlling all other parameters to get maximum color development, minimum color in blank solution, reproducibility and reasonable period of stability of final colored species formed.

 

Fig 1: Structure of Boceprevir

 

The optimum conditions in these methods were fixed based on the study of the effects of various parameters such as type and concentration of buffer, concentration of dye, choice of organic solvent, effect of pH on color development, ratio of aqueous to organic phase, shaking time and intensity as well as stability of the colored species in organic phase after final dilution and by measuring the absorbance at 415 nm.

 

Fig. 2: Absorption spectrum of Boceprevir standard with Bromocresol green (0.1 % w/v) 2 μg/ml

 

Fig. 3: Absorption spectrum of Boceprevir standard with Bromo Thymol blue (0.1 % w/v) 2 μg/ml

 

Fig. 4: Beer’s law plot of Boceprevir with Bromo Cresol green (0.1% w/v)

 

 

Fig. 5: Beer’s law plot of Boceprevir with Bromo Thymol blue (0.1% w/v)

 

Recommended procedure:

The following procedure was recommended for the assay of BCPV in bulk samples by using different dyes like BCG (BCPV 1) and BPB (BCPV 2). Aliquots of standard drug solution of BCPV were taken and from that 1 – 10 μg/ml concentrations (1, 2, 4, 6, 8 and 10 μg/ml) were made with acetone for BCPV 1 and BCPV 2.  A properly cleaned and dried separating funnel (125 ml) was taken; to it 1 ml of drug concentration (1 μg/ml) was added. Then to it 1 ml each of phthalate buffer and dye (BCG/BPB/) was added. It was mixed properly and after sometime 5 ml of chloroform was added. The reactions mixture was shaken gently for 5 minutes. Reaction mixture was allowed to stand, so as to separate the aqueous and chloroform layer. Colored chloroform layer was separated out and absorbance was measured at 415 nm against the blank.

 

Similarly the method was repeated by taking different concentrations to obtain the absorbance. Calibration curve was plotted from absorbance values obtained.

 

RESULTS AND DISCUSSIONS:

Spectral characteristics:

In order to ascertain the optimum wavelength absorption (λ_max) of the colored species formed in each method, specified amounts of Boceprevir at concentration (10 μg/m1 for methods BCPV 1, BCPV 2) were taken and the colors were developed (bluish colors for BCPV 1 and BCPV 2) separately by the above mentioned procedure. The absorption spectra were scanned on the spectrophotometer in a wavelength of 400 - 500 nm against a corresponding reagent blank. The reagent blank absorption spectrum of each method was also recorded against appropriate solvents. The results were graphically presented in Figures: 2, 3, 4 and 5. The absorption curves of colored species formed show characteristic absorption maxima whereas the blank in each method has low or no absorption in this region.

 

Optical Characteristics:

In order to test whether the colored species formed in the proposed methods adhere to Beer's law, the absorbance at appropriate wavelength of a set of solutions containing varying amount of BCPV and specified amount of reagent (as given in the recommended procedures for each method) were recorded against the corresponding reagent blanks. The Beer's plots of these systems were recorded graphically (Fig. 4 and 5). Beer's law limits, molar absorptivity and Sandell’s sensitivity for BCPV in each of the mentioned reagents (BCPV 1 and BCPV-2) were calculated and recorded in Table II. Least square regression analysis was carried out for getting the slope, intercept and correlation co-efficient values.

 

Precision:

The precision of the proposed methods was ascertained from the absorbance values obtained by actual determination of six replicates of a fixed amount of Boceprevir in final solution (10 μg/m1 for all the methods. The percent relative standard deviation and percent range of error (0.05 and 0.01 confidence limits) were calculated for the proposed methods and presented in Table II.

 

Molar absorptivity:

When C is in moles per liter, the constant is called as the molar absorptivity, and the symbol being ‘Є’. Beer’s law limits and λmax values are expressed as μg/ml and 1 mole‾ ¹ cm‾ ¹ respectively (represented in Table II).

 

Sandell’s sensitivity:

Sandell’s sensitivity refers to the number of “mg” of drug determined converted to the colored product, which in a column solution of cross section 1cm² shows an absorbance of 0.001 (expressed as μg cm‾ ²). Its satisfactory results were recorded in Table II.

 

DISCUSSION:

There are very few methods reported for the estimation of Boceprevir (anti-viral drug). It can be seen from the results presented, that all the methods has good sensitivity. The extractive spectrophotometric method was found to be simple, precise, economic, and less time consuming.  The colored chromogen developed in each method helps us to distinguish the reaction occurred visually and analytically. The method has also been statistically evaluated and the results obtained are accurate, precise and sensitive.

 

ACKNOWLEDGMENT:

The authors are thankful to Hetero Pharmaceuticals Ltd for providing the pure drugs as gift samples and Sultan-Ul-Uloom College of Pharmacy for providing necessary equipment’s and instruments used in the work.

 

CONFLICT OF INTEREST:

There is no conflict of interest.

 

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Accepted on 01.10.2017      © RJPT All right reserved

Research J. Pharm. and Tech 2017; 10(12): 4313-4316.