научная статья по теме A SENSITIVE SPECTROFLUORIMETRIC METHOD FOR THE DETERMINATION OF RANITIDINE HYDROCHLORIDE IN PHARMACEUTICAL PREPARATION Физика

Текст научной статьи на тему «A SENSITIVE SPECTROFLUORIMETRIC METHOD FOR THE DETERMINATION OF RANITIDINE HYDROCHLORIDE IN PHARMACEUTICAL PREPARATION»

ОПТИКА И СПЕКТРОСКОПИЯ, 2012, том 113, № 2, с. 147-151

СПЕКТРОСКОПИЯ ^^^^^^^^

КОНДЕНСИРОВАННОГО СОСТОЯНИЯ

УДК 535.37

A SENSITIVE SPECTROFLUORIMETRIC METHOD FOR THE DETERMINATION OF RANITIDINE HYDROCHLORIDE IN PHARMACEUTICAL PREPARATION

© 2012 г. Sevgi Tatar Ulu, Mahmut Bulent Cakar

Department of Analytical Chemistry, Faculty of Pharmacy, Istanbul University, Istanbul 34416, Turkey

E-mail: sevgitatar@yahoo.com Received March 6, 2011; in final form, November 10, 2011

A simple, precise, and sensitive spectrofluorimetric method was developed and validated for the determination of ranitidine in pharmaceutical preparations. The method is based on derivatization of ranitidine with 4-flu-oro-7-nitrobenzofurazan (NBD-F). The method was successfully validated in accordance to ICH guidelines. The validation characteristics included linearity, limit of detection, limit of quantification, accuracy, precision, specificity and robustness. The method is linear over the range of 40—1200 ng/mL. The recoveries were ranged from 98.97 to 99.43%. The proposed method was applied for the determination of ranitidine in commercially available tablets. The results were compared with those obtained by reference method using tand F-tests.

INTRODUCTION

Ranitidine (RAN), ^-(2-[(5-(dimethylaminome-thyl)furan-2-yl)methylthio]ethyl)-^-methyl-2-ni-troethene-1,1-diamine (Fig. 1), is a specific potent H2-receptor antagonist. The drug is widely used in the treatment and prophylaxis of gastrointestinal lesions aggravated by gastric acid secretion [1, 2].

Several methods for the determination of RAN in biological fluids have been developed including re-versed-phase high performance liquid chromatography (HPLC) [3—13], high-performance thin layer chromatography (HPTLC) [14], supercritical fluid chromatography [15], capillary electrophoresis [16] and polarography [17].

Various analytical methods reported for the determination of RAN in pharmaceutical formulations such as voltammetry [18], titrimetry and visible spectrophotometry [19], kinetic spectrophotometry [20], flow injection potentiometry and spectrophotometry

[21], flow injection extraction spectrophotometry

[22], near-infrared reflectance spectroscopy [23], conductimetry [24], HPLC [25, 26], HPTLC [27], and capillary electrophoresis [28].

This paper describes the development and validation of spectrofluorimetric method for the determination of RAN in pharmaceutical preparations. This proposed method is based on the derivatization of RAN with NBD-F. NBD-F an activated halide derivative was first introduced as a fluorogenic reagent for the determination of secondary and primary amines [29].

MATERIALS AND METHODS

RAN was supplied from Sigma (St. Louis, MO, USA) and its pharmaceutical preparations Ranitab film tablets (150 mg) (Deva, Istanbul, TR) was purchased from a local pharmacy. NBD-F was purchased from Fluka (Buchs, Switzerland). All other chemicals and solvents were of analytical grade.

Stock solutions of RAN of 1.0 mg/mL were prepared by dissolving in methanol. The standard solutions of RAN 100 ^g/mL were prepared in the same solvent.

The NBD-F solution was prepared by dissolving 2 mg NBD-F in 10 mL of methanol. Borate buffer so-

Fig. 1. Chemical structures of ranitidine and NBD-F.

147

2*

Fluorescence intensity

500 250 0

(a)

350 490 630

Wavelength, nm

Fluorescence intensity

500 250

490 630

Wavelength, nm

Fig. 2. Emission (a) and excitation (b) spectra of the blank with chloform.

Fluorescence intensity 1000 r

(a)

500

350 490 630

Wavelength, nm

Fluorescence intensity (b)

500

350 490 630

Wavelength, nm

Fig. 3. Excitation (a) and emission (b) spectra on the reaction derivative RAN with NBD derivative (600 ng/mL) after extraction with chloroform.

lution (0.025 M) was prepared by dissolving sodium tetraborate in water and pH was adjusted sodium hydroxide solution to pH 9.

Fluorescence measurements were made on a Shi-madzu RF-1501 spectrofluorimeter (Kyoto, Japan), equipped with xenon lamp and using 1.0 cm quartz cells. The excitation and emission wavelengths were 458 and 521 nm, respectively.

Ten Ranitab® tablets (each containing 150 mg RAN) were weighted and finely powdered in a mortar. The net weight of each tablet was calculated and portions equivalent to 100 mg RAN were transferred into a 100 mL volumetric flask; 50 mL methanol was added, shaken thoroughly to dissolve. Then, it was brought to volume, mixed well and centrifuged the supernatant was used to prepare solutions of 1 mg/mL of RAN using the methanol as the diluents. The working sample solution (100 |g/mL) obtained by dilution of supernatant which was used to set up the concentrations in the range of calibration studies.

To a set of 12 mL volumetric flasks, increasing volumes from the standard solution of the RAN were quantitatively transferred so as to contain the drug within the concentration range 40—1200 ng/mL. Next, 100 |L of 0.02% NBD-F solution was added, followed by 100 |L of borate buffer (pH 9.0) and mixed well. The reaction was allowed to proceed at 70° C in a water bath for 10 min. The mixture was cooled and acidified

with 100 |L of 0.1N HCl. The content of tube was extracted three times with 2 mL of chloroform. The combined organic phases were adjusted to 10 mL with the chloroform. The fluorescence intensity of the resulting solutions was measured at 521 nm after excitation at 458 nm against reagent blanks treated similarly.

The method was validated by analysis of samples of pharmaceutical formulations with label claims of 150 mg and determination of linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, precision, robustness, and specificity in accordance with ICH guidelines [30].

The calibration curves were constructed by plotting concentration versus intensities, using linear regression analysis. The calibration curves (F = aC + b) were constructed by the plots of the fluorescence intensities (F) of the RAN versus the concentrations (C) of the calibration standards.

The limit of detection was calculated by LOD = = 3.3g/S, where a is the standard deviation of the response of the blank and S is the slope of the calibration curve. The limit of quantification was calculated by LOQ = 10a/S under the ICH guidelines.

Accuracy and precision of the assay were determined by intra-day and inter-day for 3 consecutive days. Three different concentrations of RAN were analyzed in six independent series in the same day (intra-day precision) and 3 consecutive days (inter-day preci-

Fluorescence intensity

^L added of 0.02% NBD-F

Fig. 4. Effect of added 0.02% NBD-F.

sion). The accuracy and precision of the method were expressed by relative mean error (RME %) and relative standard deviation (RSD %).

Recovery studies were carried out by standard addition method. Different amount of standard drugs were added to a known pre-analyzed formulation sample and the total concentration was determined using the proposed method.

Robustness was examined by evaluating the influence of small variations in different experimental conditions such as reaction time (±1 min), reagent volume (±5 ^L), borate buffer pH (±1). These variations did not have significant effect on the measured responses.

Potential interfering substances in the spectrofluo-rimetric determination of RAN were studied by selecting excipients (microcrystalline cellulose, magnesium stearate and titanium dioxide) often used in tablet formulations.

RESULTS AND DISCUSSION

NBD-F has a highly labile floride atom which is displaceable by nucleophilic groups such as thiol, primary and secondary amines [29]. RAN contains several amino groups and can therefore react with NBD-F to yield a strongly fluorescent product. Under the described experimental conditions, the fluorophore (NBD-RAN derivative) affords favorable characteristics of the wavelengths of excitation and emission of 458 and 521 nm, respectively (Figs. 2 and 3).

Optimization of Experimental Parameters

To improve the sensitivity and accuracy of RAN analysis the derivatization conditions volume of NBD-F, pH, temperature and time, diluting solvent and hydrochloride acid were optimized.

Fig. 5. Effect of pH on the intensity of the reaction of RAN with NBD-F.

The influence of the concentration of NBD-F was studied using different volumes (25—250 ^L) of 0.02% solution of the reagent. The maximum fluorescence intensity was obtained with 100 ^L of the reagent (Fig. 4).

As the derivatization reaction ofRAN with NBD-F is a typical nucleophilic reaction, basic reaction conditions were expected to be beneficial. The pH was varied over the pH range of 8—10 using borate buffer where the maximum fluorescence was obtained at pH 9.0 (Fig. 5).

The influence of different heating temperatures and times was studied using a water bath. Effect of heating time at different temperatures 50—80°C for NBD derivatives. The best results were obtained at 70°C within 10 min.

In order to select the most appropriate organic solvent the reaction solutions, different solvents were tested: methanol, acetonitrile, dichloromethane, chloroform, and ethyl acetate. The highest reading was obtained when chloroform was used.

Table 1. Experimental and analytical data

Parameter Value

^ex (nm) 458

^em (nm) 521

Linear range (ng/mL) 40-1200

Correlation coefficient (r) 0.9998

F = aC + b 0.8818C-2.2559

Slope (a) 0.8818

SD of slope (Sa) 3.3 x 10-4

Intercept (b) 2.2559

SD of intersept (Sb) 1..1 x 10-2

LOD (ng/mL) 0.04

LOQ (ng/mL) 0.12

Table 2. Intra-day precision and accuracy of RAN (n = 5)

Concentration, ng/mL Relative standard deviations RSD, % Relative mean error RME, %

Added Found ± SD

Intra-day 40 40.6 ± 1 x 10-1 0.25 + 1.50

200 200.3 ± 4.3 x 10-1 0.21 + 0.15

1200 1200.4 ± 4.0 x 10-1 0.03 + 0.03

Inter-day 40 40.7 ± 1.2 x 10-1 0.29 + 1.75

200 200.5 ± 4.5 x 10-1 0.22 + 0.25

1200 1200.7 ± 6.3 x 10-1 0.05 +0.06

The fluorescence of the hydrolysis product of NBD-F, n

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