Estimation of Rizatriptan in bulk and pharmaceutical formulation.

Jane Jacob*, Devin M. Dadhaniya, Patel Vikas, Jani Vishal M, Amit Bangad

Department of Pharmaceutical Chemistry, NGSM Institute of Pharmaceutical Sciences, Paneer,

Deralakatte-575018

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

ABSTRACT:

Four simple, sensitive, accurate, precise, and economical methods were developed for the quantitative estimation of Rizatriptan in bulk drug and its pharmaceutical formulation. Method-1 was a HPLC method and the chromatographic separation was carried out on column of Zorbex-eclips XDB, C18, 150 x 4.6mm, 5µ. using a mobile phase composition of acetonitrile,methanol and phosphate buffer of pH 2.5 in ratio 45:30:25 v/v pumped at flow rate of 1 ml/min. The eluent was monitored at 278 nm. The method was linear over the range of 50-150 µg/ml. The method was statistically validated for precision, accuracy, robustness and recovery. Method-2 and Method-3 are colorimetric methods based on condensation reaction involving the formation of violet colored complex between Rizatriptan and vanillin in presence of conc. Sulphuric acid which showed a linearity range 3-18 µg/ml at 558nm and formation of red colored complex between Rizatriptan and chromogenic agent 4-aminophenazone in presence of potassium ferricyanide which obeyed Beer’s law in the concentration range of 20-100 µg/ml and showed absorption maxima of 531nm. Method-4 was a simple UV method developed and validated for the estimation of Rizatriptan in methanol. The linearity range for Rizatriptan was obtained as 10-50 µg/ml and its wavelength of detection was 278nm. The results of analysis for all the four methods have been validated statistically according to ICH guidelines.

KEY WORDS: Rizatriptan, HPLC, colorimetric methods.

INTRODUCTION:

Rizatriptan¹ chemically is N,N-dimethyl-2-[5-(1H-1,2,4-triazol-1-ylmethyl)-1H-indol-3-yl] Ethanamine. Three distinct pharmacological actions have been implicated in the antimigraine effect of the triptans: (1) stimulation of presynaptic 5-HT_1D receptors, which serves to inhibit both dural vasodilation and inflammation; (2) direct inhibition of trigeminal nuclei cell excitability via 5-HT_1B/1D receptor agonism in the brainstem and (3) vasoconstriction of meningeal, dural, cerebral or pial vessels as a result of vascular 5-HT_1B receptor agonism. Rizatriptan is marketed with brand name of RITZA, available in strength of 5 and 10 mg and the pure sample of Rizatriptan was obtained as a gift from Aurobindo Pharmaceutical, India. Rizatriptan is official in Indian pharmacopoeia, British and European Pharmacopoeia. Literature survey revealed analytical methods reported for the determination of Rizatriptan in pharmaceutical formulation. The methods so far reported include HPLC^2-5, derivative spectroscopy^6-8. The main aim of this work was to develop simple, accurate, reproducible and economical methods for the routine analysis of Rizatriptan in dosage forms.

MATERIALS AND METHODS:

All the chemicals and solvents used for spectrophotometric procedures were of analytical grade and solvents used to carry out HPLC studies were of HPLC grade obtained from Merck. Spectrophotometric studies were carried out on Jasco V -530 UV Visible Spectrophotometer using matched quartz cuvettes of thickness 1 cm. The HPLC system employed in the method development and validation was Integrated system of model HP Hawlettpackard Agilent 1100 technology. The chromatographic separation was achieved on Zorbex-eclipsXDB, C₁₈, 150 x 4.6mm, 5µ column and G1311A Quac pump serial S#DE91607737, Hydraulic system low pressure pump and UV/VIS detector.

Method-1 Stock solution of Rizatriptan (1 mg/ml) was prepared by dissolving 100mg of Rizatriptan in 100 ml of diluent( Aectonitrile and methanol in the ratio of 50:50). Working solution was prepared by further diluting stock solution suitably with diluent to get a conc. of 100µg/ml. The HPLC analysis was performed on reversed-phase high-performance liquid chromatographic system with isocratic elution mode. Aliquot samples of Rizatriptan were pipetted into different 10 ml calibrated flask so as to obtain linearity range of 50-150 µg/ml. the mobile phase selected was a combination of phosphate buffer of pH 2.5, acetonitrile, methanol and triethylamine in the ratio 25:45:30:5 v/v. pH is adjusted to 2.5 with ortho phosphoric acid. The mobile phase was filtered through a 0.45 μm miilipore membrane filter.The solutions were injected using a 20 μl fixed loop system under optimized chromatographic conditions and chromatograms were recorded at 278 nm at a flow rate of 1ml/min. Limit of Detection is 4 μg/ml and limit of Quantification is 50 μg/ml.

CHROMATOGRAPHIC CONDITION:

Mobile Phase : MeOH: ACN: 5mM potassium

dihydrogen Phosphate (45:30:25v/v/v)

Analytical column: Zorbex-eclips XDB, C₁₈, 150 x

4.6mm, 5µ column and G1311A Quac pump serial S#DE91607737, Hydraulic system low pressure pump

Column Temperature	:	35˚C
λmax	:	278nm.
Flow rate	:	1ml / min.
Injection Volume	:	20 µL
Run Time	:	10 minutes
Retention Time	:	3.373 min
Range	:	0.0100 AUFS
Operation Pressure	:	60-65 bar

The developed method was extended to Pharmaceutical dosage form. Rizatriptan is marketed as Ritza tablet of strength 5 and 10 mg which was procured from the local market and marked as F-1 and F-2 respectively. Twenty tablets were weighed accurately and powder weight equivalent to 100 mg was transferred to a100 ml volumetric flask and made up to the mark with the diluent and further dilutions were made to get a concentration of 100 μg/ml. The same procedure carried out for pure Rizatriptan was done for tablet dosage form.

Forced degradation studies

Forced degradation was carried out under acid and alkali hydrolysis, oxidation by peroxide and thermal studies. Accelerated studies in acidic(0.1N HCl) and basic (0.1N NaOH) media at 70˚C for 1 hr as well as oxidation in 1% H₂O₂ and thermal stability studies were carried out. Comparison of peak parameters such as peak height and area and the retention time of pure and formulation of Rizatriptan showed no changes in oxidation and thermal degradation studies.

Method-2 Stock solution of Rizatriptan was prepared in methanol to give a concentration of 1mg/ml, further dilutions were with methanol to get aworking concentration of 100 μg/ml. From the working standard solution aliquots of drug solution were placed in 5 different calibrated 10ml volumetric flasks and absorbance was measured against corresponding reagent blank at 278nm. The linearity range for Rizatriptan was observed in the range 10-50 µg/ml and the solution was found to be stable for more than 10 hours.

Method-3 Pure sample of Rizatriptan was accurately weighed (100 mg) into a 100 ml volumetric flask dissolved and made upto the mark with methanol. Further dilutions were done to get a working concentration of 100 µg/ml. Fresh aliquots of Rizatriptan ranging from 0.3 to 1.8 ml ( 100 µg/ml ) were transferred into a series of 10ml volumetric flask to provide final concentration of 3-18 µg/ml. To each volumetric flask, 2 ml of 5% of vanillin and 1 ml of concentrated sulphuric acid were added. The solution in each flask was made up to the mark with methanol and the absorbance of violet colored chromogen was measured at 558nm against reagent blank within 90 mins.

Method-4 Stock solution of Rizatriptan was prepared by weighing accurately 100mg of pure drug in to a 100ml volumetric flask, dissolve and made up to the mark with methanol to give a conc. of 1mg/ml. The working standard solution of Rizatriptan was prepared by further diluting the stock solution suitably with methanol to get a concentration of 100µg/ml. Rizatriptan ranging from 0.2 to 1 ml (1ml/100 µg/ml) from the working solution were transferred into 5 different 10ml volumetric flask so as to get a concentration of 20-100 µg/ml. To each volumetric flask, 1.5 ml of 2.5% 4-aminophenazone followed by 1.5 ml of 3% of potassium ferricyanide and 1 ml of 1% of sodium carbonate was added and the solution in each flask was made up to the mark with 0.1 N H₂SO₄kept aside for 10 min for complete development of reaction and the absorbance of red colored chromogen was measured at 531nm against reagent blank within 80 mins.

Pharmaceutical dosage form: Rizatriptan is marketed as Ritza tablet of strength 5 and 10 mg was procured from the local market and marked as formulation 1(F-1) and (F-2). Six linearities were taken for regression co-efficient and eight linearities were taken for standard deviation separately. Results are shown in table No:1 and 2.

RESULTS AND DISCUSSION:

The developed methods were validated for specificity, linearity, accuracy, precision, limit of detection, limit of quantification and solution stability. Detection limit for Rizatriptan was 4μg/ml and quantification limit was 50 µg/ml by HPLC method. Percentage recovery showed that the proposed methods are free of interference of the excipients used in the formulation and all results obtained are reproducible with coefficient of variance less than 1%. The linear regression of absorbance on concentration with a correlation coefficient (r) of almost 1 indicates a good linearity between absorbance and concentration .The value of percentage relative standard deviation less than 1% and low percentage range of error confirm the high degree of precision and accuracy of the proposed method. The percentage recovery value, which is close to100%, indicates the reproducibility of the method and the absence of the excipients present in the formulation. In method II Rizatriptan was estimated based on a probable condensation reaction between vanillin in the presence of conc.

Table No:1 Assay and % Recovery

Dosage forms	Labelled Amount	Amount found by proposed method(mg)				% Recovery of proposed method
Dosage forms	Labelled Amount	M1	M 2	M 3	M 4	M1	M 2	M 3	M 4
F-1	10 mg	9.59	8.05	8.75	8.80	99.74	97.79	97.65	95.86
F-2	5 mg	-	4.05	4.38	4.73	-	98.90	98.89	97.45

Table No:2 Optical characteristic and precision of the developed methods

Parameter	Method-1	Method-2	Method-3	Method-4
λmax (nm)	278	278	558	531
Beer’s law limit	50-150	10-50	3-18	20-100
Molar absorptivityl/mol (lit/mol^-1cm^-1)	8.44×10⁶	6.2718×10⁴	1.6559×10⁴	3.3858×10⁴
Sandell’s sensitivity (µg/ml/0.001abs units)			0.2347	0.1148
Regression equation( Y*) Slope (b) Intercept (a)	2.148 0.056	0.016 0.008	0.0430 0.007	0.008 0.007
Correlation coefficient (r)	0.9990	0.9996	0.9998	0.9990
% RSD	0.3135	0.6560	0.3226	0.3444
Confidence limits with 0.05 level 0.01 level	0.2621 0.3878	0.5489 0.8115	0.2705 0.4003	0.2879 0.4260

Sulphuric acid resulting in violet colored chromogen which was stable for 90 min and showed an absorption maxima at 558 nm and obeyed beer’s law in the concentration range 3-18 μg/ml. In method III Rizatriptan reacts with 4-Amino Phenazone in the presence of potassium ferricyanide under alkaline conditions to give a reddish colored chromogen which was stable for more than 60 min. probable reactions are shown in Fig 1and2 respectively.

FIG 1

FIG 2

ACKNOWLEDGEMENT:

The authors are grateful to Nitte University, Mangalore for the support given in carrying out the work.

REFERENCES:

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Received on 26.06.2012 Modified on 09.07.2012

Asian J. Research Chem. 5(9): September, 2012; Page 1104-1107