Development and Validation of UV‐ Spectrophotometric Method for Determination of Naftopidil in Bulk

Pharmaceutical Chemistry Review Volume 1 2015

http://www.bacpl.org/J/pcr

Development and Validation of UV‐ Spectrophotometric Method for Determination of Naftopidil in Bulk and in Formulation Pritam S Jain*, Kajal D Bobade and Sanjay J Surana Department of Pharmaceutical Chemistry, R. C. Patel Institute of Pharmaceutical Education and Research, Shirpur, Dist‐ Dhule,Maharashtra, (India), 425405 North Maharashtra University, Jalgaon pritash79@yahoo.com

Abstract: A simple, rapid, accurate and economical spectrophotometric method has been developed for estimation of Naftopidil from bulk and pharmaceutical formulation. Materials and methods: The λmax of Naftopidil in methanol and water was found to be 281 nm. The drug followed linearity in the concentration range 10‐45 μg/ml with correlation coefficient value 0.998.

The proposed method was applied to pharmaceutical formulation and % amount of drug estimated 99.58 % was found in good agreement with the label claim. The accuracy of the method was checked by recovery experiment performed at three different levels i.e., 80%, 100% and 120 %. The % recovery was found to be in the range 98.8%– 99.06%. The low values of % R.S.D. were indicative of the accuracy and reproducibility of the method. The precision of the method was studied as an intra‐day, inter‐day variations and repeatability. The % R.S.D. value less than 2 indicated that the method was precise. Ruggedness of the proposed method was studied with the help of two analysts. The above method was a rapid and cost‐effective quality‐control tool for routine analysis of Naftopidilin bulk and in pharmaceutical dosage form.

Key words: Naftopidil; UV‐Spectrophotometric; Quantitative determination

I. Introduction Naftopidil is chemically 1‐[4‐(2‐methoxyphenyl) piperazin‐1‐yl]‐3‐(1‐naphthyloxy) propan‐2‐ol (Fig.1), having molecular formula): C24H28N2O3 with molecular weight 392.5. It is white to off white crystalline powder with melting point 127.70C and soluble in methanol[1]. It acts as an anti‐hypertensive agent.Naftopidil exerts its antihypertensive action via alpha1‐adrenoceptor blockage and Ca2+antagonism in vascular smooth muscle[2].

Naftopidil, a phenylpiperazine derivative, is a novel alpha1‐adrenoceptor antagonist and is a new drug for the bladder outlet obstruction in patients with benign prostatic hyperplasia (BPH). Naftopidil competitively inhibited specific [3H]prazosin binding in prostatic membranes of humans. Naftopidil was selective for the alpha 1d‐ adrenoceptor with approximately 3‐ and 17‐fold higher affinity than for the alpha 1a‐ and alpha 1b‐adrenoceptor subtypes, respectively. In clinical studies, naftopidil has been demonstrated to be effective in the treatment of bladder outlet obstruction in patients with BPH[3]. In addition to the antagonistic action of this agent on the alpha1 adrenergic receptors of prostatic smooth muscle naftopidil may also act on the lumbosacral cord and thus may improve collecting disorders in patients with benign prostatic hyperplasia[4]. In this way, they reduce the pressure on the urethra and so help increase the flow of urine. Newly developed alpha1 adrenoceptor antagonists including naftopidil are free from the ʺprazosin‐likeʺ side effect of orthostatic hypotension and associated symptoms[5]. Naftopidil a novel antihypertensive compound, possesses Agonistic properties in addition to being an alpha1 adrenoceptor antagonist[6]. Various methods are reported for the analysis of individual drug as HPLC[7‐8], and LCMS/MS[9] but no spectrophotometric method is reported estimation of drug in pharmaceutical dosage form. Accordingly, the objective of this study is to develop and validate the spectrophotometric method for

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http://www.bacpl.org/J/pcr Pharmaceutical Chemistry Review Volume 1 2015

the estimation of naftopidil in bulk and pharmaceutical formulation as per ICH guidelines[10].

FIGURE 1 CHEMICAL STRUCTURE OF NAFTOPIDIL

II. Experimental Work 1. Materials and Methods Naftopidil was supplied as a gift sample by Mepro pharmaceutical Ltd. (Gujarat). All chemicals and reagents used were of analytical grade and purchased from Qualigens Fine Chemicals, Mumbai, India.

2. Preparation of standard stock solution Accurately weighed 50 mg of Naftopidil was transferred to 100 ml volumetric flask, dissolved in 100 ml methanol by shaking manually for 10 min and sonicated for 5 min. 10 ml of this was transferred to 200ml volumetric flask and volume was adjusted up to the mark withpH 4 dissolution media to give final concentration 25 μg/ml.

3. Preparation of dissolution media Accurately weighed 5.25 g citric acid monohydrate and was added in 1 litre water.Accurately weighed 8.90 g disodium hydrogen phosphate dihydrate in 1 litre water. SolutionA was added to solutionB to adjust pH 4.

4. Selection of wavelength for analysis of Naftopidil Appropriate volume4 ml of standard stock solution of Naftopidil was transferred into 10 ml volumetric flask, diluted to mark with dissolution media to give concentration of 10μg/ml. The resulting solution was scanned in UV range (200 nm – 400 nm). In zero order, spectrum Naftopidil showed absorbance maximum at 281 nm (Figure 2).

FIGURE 2 UV SPECTRUM OF NAFTOPIDIL AT 281 NM

III. Validation of the Method The method was validated in terms of linearity, accuracy, precision, and ruggedness.

1. Linearity study Different aliquots of Naftopidil in range 0.4‐1.4 ml were transferred into series of 20 ml volumetric flasks and the volume was made up to the mark with dissolution media to get concentrations 10, 15, 20, 25, 30 and 35g/ml, respectively. The solutions were scanned on spectrophotometer in the UV range 200 ‐ 400 nm. . The calibration plot was constructed as concentration vs. amplitude (Figure 3).

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Pharmaceutical Chemistry Review Volume 1 2015

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FIGURE 3 CALIBRATION CURVE OF NAFTOPIDIL AT 281 NM

2. Accuracy To the preanalyzed sample solutions, a known amount of standard stock solution was added at different levels i.e.

80%, 100% and 120 %. The solutions were reanalyzed by proposed method.

3. Precision Precision of the method was studied as intra‐day and inter‐day variations. Intra‐day precision was determined by analyzing the 10, 20 and 30 μg/ml of Naftopidil solutions for three times in the same day. Inter‐day precision was determined by analyzing the 10, 20 and 30 μg/ml of Naftopidil solutions daily for three days over the period of week.

4. Sensitivity The sensitivity of measurements of Naftopidil by the use of the proposed method was estimated in terms of the Limit of Quanfication (LOQ) and Limit of Detection (LOD). The LOQ and LOD were calculated using equation LOD = 3.3 x N/B and LOQ = 10 x N/B, where, ‘N’ is standard deviation of the peak areas of the drugs (n = 3), taken as a measure of noise, and ‘B’ is the slope of the corresponding calibration curve.

5. Repeatability Repeatability was determined by analyzing 25 μg/ml concentration of Naftopidil solution for six times.

6. Ruggedness Ruggedness of the proposed method was determined for 25 μg/ml concentration of Naftopidil by analysis of aliquots from homogenous slot by two analysts using same operational and environmental conditions.

IV. Determination of Naftopidil in Bulk Accurately weighed 50 mg of Naftopidil was transferred into 100 ml volumetric flask containing 50 ml methanol and volume was made up to the mark using same. Appropriate volume 4 ml of this solution was transferred to 200 ml volumetric flask and volume was adjusted to mark using dissolution media. The resulting solution was scanned on spectrophotometer in the UV range 200 ‐ 400 nm and absorbance was measured at 281 nm. The concentrations of the drug were calculated from linear regression equations.

V. Application of Proposed Method for Pharmaceutical Formulation For analysis of commercial formulation 50mg of Naftopidil orally dispersible tablet was taken in 100 ml volumetric flask and the volume was made up to the mark with methanol to give 500μg/ml concentration. From this, 10 ml was taken and transferred to 200 ml volumetric flask and volume was made up to the mark with dissolution media to give 25 μg/ml concentration. It was scanned on spectrophotometerin the UV range 200 ‐ 400 nm. The spectrum

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http://www.bacpl.org/J/pcr Pharmaceutical Chemistry Review Volume 1 2015

was recorded at 281 nm. The concentrations of the drug were calculated from linear regression equation.

VI. Results and Discussion 1. Method Validation The proposed method was validated as per ICH guidelines. The solutions of the drugs were prepared as per the earlier adopted procedure given in the experiment.

1). Linearity studies The linear regression data for the calibration curves showed good linear relationship over the concentration range 10 – 35 μg/ml for Naftopidil. Linear regression equation was found to be Y = 0.0215 X + 0.0002 (r2 = 0.999). The result is expressed in Table 1.

TABLE 1 LINEARITY STUDY OF NAFTOPIDIL

Sr. no.

Concentration μg/ml

1 2 3 4 5 6

10 15 20 25 30 35

Absorbance* Mean ± S.D. (n=6) 0.217± 0.0010 0.322± 0.0011 0.419± 0.0018 0.535± 0.0027 0.642± 0.0037 0.747± 0.0046

% R.S.D. 0.47 0.36 0.44 0.51 0.58 0.61

* Average of five estimations

2). Accuracy The solutions were reanalyzed by proposed method; results of recovery studies are reported in Table 2 which showed that the % amount found was between 98.8% to 99.06% with %R.S.D. >2.

TABLE 2 RECOVERY STUDIES

Pre‐analyzed sample solution (μg/ml)

Amount of drug added (μg/ml) (n=3)

Amount recovered* (μg/ml) (n=3)

20 25 30

19.76 24.54 29.72

25

% Recovery

% R.S.D.

98.8 98.16 99.06

1.02 1.84 0.15

*Average of three estimates

3). Precision The precision of the developed method was expressed in terms of % relative standard deviation (% RSD). These results showed reproducibility of the assay. The % R.S.D. values were found to be less than 2, which indicate that this method precise for the determination of both the drugs in formulation (Table 3).

TABLE 3 PRECISION STUDIES

Component

Concentration (μg/ml)

Naftopidil

10 20 30

Intra‐day precision* (n=3) Conc. Found % R.S.D. 9.81 19.92 29.79

0.10 0.15 0.05

Inter‐day Precision* (n=3) Conc. Found % R.S.D. 9.90 19.92 29.79

0.10 0.12 0.06

*Average of three estimates

4). Sensitivity The linearity equation was found to be Y = 0.0215 X + 0.0002. The LOQ and LOD for naftopidil were found to be 0.368 μg and 1.1179μg, respectively.

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Pharmaceutical Chemistry Review Volume 1 2015

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5). Repeatability Repeatability was determined by analyzing 25 μg/ml concentration of Naftopidil solution for six times and the % amount found was between 98% to 102% with % R.S.D. less than 2 (Table 4).

TABLE 4 REPEATABILITY STUDIES

Component

Amount taken (μg/ml) (n=6)

Amount found* (%)

%R.S.D.

Naftopidil

25

98.04 ± 0.003

1.84

*Average of six estimations

6). Ruggedness Peak area was measured for same concentration solutions, six times. The results were in the acceptable range for both the drugs. The results were given in Table 5. The result showed that the % R.S.D. was less than 2%.

TABLE 5 RUGGEDNESS STUDIES

Component

Amount taken (μg/ml) (n=3)

Naftopidil

25

Amount Found (%) * Analyst I ±S.D. Analyst II ±S.D. 98.36±0.005 98.16± 0.0051

*Average of six estimations

VII. Determination of Naftopidil in Bulk The concentrations of the drug were calculated from linear regression equations. The % amount found was between 98.0% to 102.0% (Table 6).

TABLE 6 ANALYSIS OF NAFTOPIDIL IN BULK

Concentration (μg/ml)

25

Mean ± S.D. % R.S.D.

Amount found (μg) 24.77 24.81 24.90 24.72 24.63 24.95 24.79± 0.0025 0.47

Amount found (%) 99.08 99.24 99.60 98.88 99.52 99.8 99.18± 0.468 0.472

VIII. Application of Proposed Method for Pharmaceutical Formulation TABLE 7 ANALYSIS OF FORMULATION

Brand name: IOBRIM Conc. (μg/ml)

25

Batch No: RW9111 Amount found (μg)

Amount found (%)

24.50

98

24.59

98.36

24.83

99.32

24.78

99.12

24.91

99.64

24.98

99.92

Mean ± S.D.

24.765 ± 0.003

99.22 ± 0.662

% R.S.D.

0.724

0.667

The spectrum was derivatised into first order derivative and amplitude of the trough was recorded at 281 nm. The concentrations of the drug were calculated from linear regression equation. The % amount found was between 98.0% to 102.0% (Table 7).

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IX. Conclusion This UV spectrophotometric technique is quite simple, accurate, precise, reproducible and sensitive. The validation procedure confirms that this is an appropriate method for their quantification in the plant material and formulation. It is also used in routine quality control of the raw materials as well as formulations containing this entire compound.

ACKNOWLEDGEMENT

The authors are thankful to the Principal and management, R.C. Patel Institute of Pharmaceutical Education and Research, Shirpur (M.S.), India for providing the required facilities to carry out this research work.

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