INTRODUCTION:

Ranitidine hydrochloride is a histamine H₂-receptor antagonist that inhibits stomach acid production. It is commonly used in treatment of peptic ulcer disease (PUD) and gastroesophageal reflux disease (GERD). Ranitidine is also used alongside fexofenadine and other antihistamines for the treatment of skin conditions such as hives. Ranitidine HCl is marketed under the brand name Zinetac.

Chemically it is dimethyl {5-[2-(1- methylamino-2-nitrovinylamino)ethylthiomethyl]furfuryl}- amine hydrochloride ^[1-3]. The molecular formula is C₁₃H₂₂N₄O₃S• HCl, representing a molecular weight of 350.87. It has the following structural formula:

Ranitidine hydrochloride is a white to pale yellow, crystalline substance that is freely soluble in water; soluble in methanol and in ethanol (95%); sparingly soluble in ethanol; very slightly soluble in chloroform and in dichloromethane^1-3.

Each tablet, for oral administration, contains 168 mg or 336 mg of ranitidine hydrochloride equivalent to 150 mg or 300 mg of ranitidine, respectively.

Literature survey revealed that various analytical methods such as UV spectroscopy^4-7, HPLC⁸ and HPTLC^9-10 methods are used for estimation of Ranitidine hydrochloride in single as well as in combination with various drugs. No nanodrop spectrophotometric method has been reported for estimation of Ranitidine hydrochloride in single component formulation. Hence, an attempt has been made to develop new nanodrop spectrophotometry method for its estimation either in bulk or pharmaceutical tablet dosage form with good accuracy, simplicity, precision and economy.

The Nanodrop ND-1000 is a full-spectrum spectrophotometer (UV and visible spectrum, 220-750 nm) for measuring the absorbance, the sample droplet is held in place by surface tension when it is slightly compressed between the pedestal and the sample arm; this generates the defined pathway of 1 mm. The spectrum measurement is then performed with two optical fibers installed in the pedestal (emitting light of a Xenon lamp) and the sample arm (spectrometer with linear CCD array). Quantification is performed based on the spectrum measurement at the defined pathway of 1 mm. Unlike traditional spectrophotometers, the nanodrop does not require cuvettes or capillaries. Instead, the sample is pipette directly onto the measurement pedestal.

EXPERIMENTAL:

Instrumentation: Spectral and absorbance measurements were made on Nanodrop spectrophotometer. Denver TB-215D balance was used for weighing the samples. Commercially available tablets of Ranitidine hydrochloride were procured from the local market and estimated.

Chemicals and reagents: -

1. Water (Milli Q grade): Millipore water filter

2. Ranitidine hydrochloride (98%): Wockhardt Pharma, Mumbai

Apparatus / Instruments: -

Name	Model	Manufacturer / Supplier
Nanodrop spectrophotometer	ND-1000	Thermo-scientific
Micropipettes	DH-43394	Thermo-scientific
Millipore water purification unit	BM5SN3112A	Millipore (India) Pvt. Ltd.
Tube and micro-pipette tips	-	Eppendorf

OPTIMIZATION:

Scanning and determination of maximum wavelength (l_max): In order to ascertain the wavelength of maximum absorption (λ _max) of the drug, different solutions of the drug (40 ppm and 60 ppm) in distill water were scanned using nanodrop spectrophotometer within the wavelength region of 220 – 700 nm against distill water as blank. The resulting spectra are shown in fig. A, B, C and the absorption curve showed characteristic absorption maxima at 316 nm for Ranitidine hydrochloride.

Fig (A) Nanodrop spectrum of blank

Fig (B) Spectrum of Ranitidine HCl std. 40 ppm

Fig (C) Spectrum of Ranitidine HCl std. 60 ppm

METHODS:

Preparation of standard stock solutions: Standard stock solution was prepared by dissolving 25 mg of Ranitidine hydrochloride in distill water and volume made up to 25 ml with distill water to get concentration of 1mg/ml (1000 ppm) solution.

Preparation of working standard solutions and construction of standard graph: The prepared stock solution was further diluted with distill water to get working standard solutions of 5, 10, 20, 40, 60, 80, and 100 ppm of Ranitidine hydrochloride to construct Beer’s law plot for pure drug, the absorbance was measured at λ _max 316 nm, against distill water as blank. The results are shown in table (1). The standard graph was plotted by taking concentration of drug on X-axis and absorbance on Y-axis and is shown in fig (D). The drug has obeyed Beer’s law in the concentration range of 5-100 ppm. The linearity curve data is shown in table (2).

Table 1: Linearity table of Ranitidine HCl in working standard

Concentration (ppm)	Absorbance
5	0.019
10	0.037
20	0.069
40	0.142
60	0.210
80	0.281
100	0.351

Table (2) Linearity curve data

Beer’s Law limit (ppm)	5-100
Correlation coefficient (R²)	0.9999
Regression equation (y*) y= 0.0035x+0.0011
Slope (m)	0.0035
Y-Intercept (c)	0.0011

* y=mx+c where ‘x’ is the concentration of Ranitidine HCl in ppm and y is the absorbance.

Fig (D) Linearity curve of Ranitidine hydrochloride

Preparation of sample stock solutions and working sample solutions: Ten tablets were accurately weighed and average was calculated. The tablets were then crushed to obtain fine powder. An accurately weighed quantity of tablet powder equivalent to about 25 mg of Ranitidine hydrochloride was transferred to 25 ml volumetric flask, add 10 ml of distill water and shaken for 10 min. The volume was made up to the mark with distill water and required dilutions were made from sample stock solution. The recovery study from formulation is shown in table (3).

Table 3: Recovery from the formulation

Formulation

Labeled amount

(mg)

Nanodrop spectrophotometry method*

Mean ± s. d

(amount mg recovered)

%Labeled amount

% RSD

Zinetac^R

(tablets)

168

165.71±2.39

98.64±1.42

1.44

* Each value is average of three determinations ± standard deviation.

Fig (E) Accuracy study curve

VALIDATION:

Accuracy: To determine the accuracy of the proposed method, recovery studies were carried out by adding different amounts (80%, 100%, and 120%) of bulk samples of Ranitidine hydrochloride within the linearity range were taken and added to the preanalyzed formulation of concentration 10 ppm. From that percentage recovery, values were calculated. The results are shown in table (4).

The response obtained for the various concentrations is plotted and observed to be linear (correlation coefficient – 0.9954 for Ranitidine hydrochloride). The graphical representation of accuracy studies is depicted in fig (E). The accuracy study data is shown in table (5)

Table 4: Accuracy Readings

Sample ID	Concentration (ppm)		%Recovery of Pure drug	Statistical Analysis
Sample ID	Pure drug	Formulation	%Recovery of Pure drug	Statistical Analysis
S₁ : 80 %	8	10	109.37	Mean=105.21 SD=6.15 %RSD=5.85
S₂ : 80 %	8	10	96.86
S₃ : 80 %	8	10	109.37
S₄: 80 %	8	10	100
S₅ : 80%	8	10	103.13
S₆ : 80%	8	10	112.5
S₇ : 100 %	10	10	105	Mean=107.92 SD=2.46 %RSD=2.28
S₈ : 100 %	10	10	110
S₉ : 100 %	10	10	107.5
S₁₀ : 100 %	10	10	110
S₁₁: 100 %	10	10	105
S₁₂ : 100 %	10	10	110
S₁₃ : 120 %	12	10	100	Mean=105.56 SD=4.5 %RSD=4.26
S₁₄ : 120 %	12	10	102.08
S₁₅ : 120 %	12	10	106.25
S₁₆ : 120 %	12	10	104.17
S₁₇ : 120 %	12	10	108.33
S₁₈ : 120 %	12	10	112.5

% Recovery= amount recovered / amount introduced X 100

Table 5: Accuracy studies

S. No.	Concentration (%)	Absorbance (mean)
1	80	0.0657
2	100	0.0752
3	120	0.0827
4	Correlation coefficient (R²)	0.9954
5	Slope (m)	0.0004
6	Y-intercept (c)	0.032

Precision: The precision of the proposed method was ascertained by actual determination of six replicates of fixed concentration of the drug within the Beer’s range and finding out the absorbance by the proposed method. From this absorbance, mean, standard deviation and % RSD was calculated. The system precision and method precision readings are shown in table 6 (a) and 6 (b) respectively.

Table 6 (a): System precision readings

Concentration (ppm)	Absorbance	Statistical analysis
40	0.138	Mean = 0.1403 SD = 0.00273 %RSD = 1.95
40	0.138
40	0.144
40	0.138
40	0.141
40	0.143

Table 6 (b): Method precision readings

Concentration (ppm)	Absorbance	Statistical analysis
40	0.139	Mean = 0.1377 SD = 0.00175 %RSD = 1.27
40	0.135
40	0.137
40	0.140
40	0.138
40	0.137

RESULTS AND DISCUSSION:

From the optical characteristics of the proposed method, it was found that Ranitidine hydrochloride obeys linearity within the concentration range of 5-100 ppm. From the results shown in accuracy table (4), it was found that the percentage recovery values of pure drug from the preanalyzed solution of formulation were in between 96.86 – 112.5, which indicates that the proposed method is accurate and also reveals that the commonly used excipients and additives in the pharmaceutical formulations were not interfering in the proposed method. From the results shown in table 6 (a) and 6 (b) it was found that the % RSD is less than 2, which indicates that the system as well as method has good reproducibility.

CONCLUSION:

The proposed method was simple, sensitive and reliable with good accuracy and precision. The proposed method is specific while estimating the commercial formulations without interference of excipients and other additives. Hence, this method can be used for the routine determination of Ranitidine hydrochloride in bulk and pharmaceutical tablet dosage form.

ACKNOWLEDGEMENTS:

The authors thank Wockhardt Pharma, Mumbai for providing the gift sample of Ranitidine hydrochloride. Authors are also thankful to Dr. P.K. Seth, CEO, Biotech Park, Lucknow for providing necessary facilities for the work.

REFERENCES:

1. Indian Pharmacopoeia, 1996.

2. British Pharmacopoeia, vol.1 & 2, The British Pharmacopoeia Commission, London, 2009, Page no- 5168-5173.

3. United State Pharmacopoeia 30- NF 25, 2007, page 1752.

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5. S. Pillai and I. Singhvi. Spectrophotometric simultaneous estimation of Ranitidine hydrochloride and Ondansetron hydrochloride from tablet formulation. Indian J. Pharm. Sci., 2007, 69 (4): 601-604.

6. Tasnuva Haque, Md Mesbah Uddin Talukder, Susmita Laila, Kanij Fatema, Abdul Kalam Lutful Kabir. Simultaneous Estimation of Naproxen and Ranitidine HCl by Using UV Spectrophotometer. S. J. Pharm. Sci. 1(1&2): 18-24.

7. Raut K. N and Sabnis S. D. A new spectrophotometric method for estimation of ranitidine hydrochloride. Indian Journal of Pharmaceutical Sciences.1987 Mar-Apr; 49(2): 65-66.

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9. B. Simonovska, M. Pro ek, I. Vovk and A. Jelen mitek. High-performance thin-layer chromatographic separation of ranitidine hydrochloride and two related compounds. Journal of Chromatography B: Biomedical Sciences and Applications
Volume 715, Issue 2, 18 September 1998, Pages 425-430

10. Shah Shailesh A., Rathod Ishwarsinh S, Savle Shrinivas S., Patel Bipin D. Development of a sensitive high-performance thin-layer chromatography method for estimation of ranitidine in urine and its application for bioequivalence decision for ranitidine tablet formulations. Journal of chromatography. B, Biomedical sciences and applications, 2002, vol. 767, n^o1, pp. 83-91 (11 ref.).

Received on 22.03.2010 Modified on 20.04.2010

Asian J. Research Chem. 3(3): July- Sept. 2010; Page 716-719