INTRODUCTION:

Pitavastatin calcium¹ (PTC), monocalciumbis {(3R, 5S,6E)-7-[2-cyclopropyl-4-(4-flurophenyl)-3-quinolyl]-3-5-dihydroxy-6-heptenoate}, is a lipid-lowering agent² , used in hyperlipidemia. The analytical methods reported in literature includes HPLC³ and polarographic methods⁴for CPH and LC/MS method⁵ for PTC, however, no spectrophotometric method has so far been reported for these drugs. Hence, it was thought worthwhile to develop advanced spectrophotometric method for the same. This paper describes difference spectrophotometric methods for the estimation of CPH and PTC separately in bulk and their tablet formulations. All the chemicals were of analytical reagent grade and solutions were prepared with purified water of IP⁶ grade. The solutions of 0.1N HCl and 0.1N NaOH were prepared in water as per IP^7..Several analytical techniques have been used for the determination of Pitavastatin, which rely upon sophisticated and expensive instrumentation. The low cost and the case of operation make the spectrophotometric techniques highly desirable for the determination of Pitavastatin in pharmaceuticals. Simple and facile method for the determination of Pitavastatin in pharmaceuticals has been developed. The proposed method is simple, accurate and easy to apply for routine use.

EXPERIMENTAL:

Apparatus: A secomam Anthelic NUA 002 UV-vis spectrophotometer with 1 cm quartz cell was used for the absorbance measurements.

Reagents: All solutions were prepared with doubly distilled water. Chemicals used were of analytical reagent grade.

Procedure:

Pitavastatin: A 1000 µg ml^-1 standard Pitavastatin drug (Fig 1) solution was first prepared by dissolving 0.1 g in methanol and diluting to the mark in 100 ml calibrated flask. The stock solution was diluted approximately to get the working concentration.

Hydrochloric acid (5M), potassium iodide (2%), sodium acetate buffer (2M) were sued. A 0.05% solution of Toluidine blue was prepared by dissolving 0.05 g of TB in distilled water and making up to 100ml.

Figure1. Pitavastatin

Spectrophotometry:

Determination of Pitavastatin using as TB reagent: Different aliquots (19.0 – 132.0 µg ml^-1) of Pitavastatin is transferred into a series of 10 ml calibrated flasks by means of micro burette. Then, 1 ml each of 2% KI and 5M HCl were added to each flask and the mixture was gently shaken until the appearance of yellow color, indicating the liberation of iodine. To this system, 0.5 ml of 0.05% TB is added followed by 2ml of 2M sodium acetate solution and the reaction and the reaction mixture was shaken for 5 min. The contents were diluted to the mark with distilled water and mixed well. The absorbance of the colored solution was measured at 630 nm against the corresponding reagent blank and the absorbance corresponding to the bleached color, which in turn corresponds to the drug concentration, was obtained by subtracting the absorbance of the blank solution by that of the test solution and measured at 630 nm.

RESULTS AND DISCUSSION:

The method involves the reaction of H₂O₂, with Pitavastatin in acidic medium, and its reaction with potassium iodide to liberate iodine. This liberated iodine bleaches the colored TB to colorless leucoform, which was measured at 630 nm and are represented in scheme 1. The decrease or increases in absorbance are directly proportional to the analyte concentration.

Optimization of reagents: The oxidation of iodide to iodine was effective in the pH range of 1.3 -1 .9 which could be maintained by adding 1ml of 5 ml HCl in a final volume of 10 ml. The liberation of iodine from potassium iodide in an acidic medium was quantitative. The appearance of yellow color indicates the liberation of iodine. It was found that 1 ml of each 2% potassium iodide and 5 M HCl were sufficient for the liberation of iodine from iodide by Pitavastatin. This liberated iodine is sufficient to bleach the blue colored TB to the colorless leucoform. Constant and maximum absorbance values were obtained in the pH range of 4.0 – 5.0. This could be achieved by adding 2ml of 2M acetate buffer solution to a total volume of 10 ml. Maximum absorbance was obtained instantaneously and required no heating under the reaction condition. Under the optimum reaction conditions, the color system was stable for a period of over 4hours

Analytical data: A linear relation was found between absorbance at λ_max and concentration ranges given in (Table 1). Regression analysis of the Beer’s law data using the method of least squares was made to evaluate the slope (b), intercept (a) and correlation coefficients (R), for each system of Pitavastatin, which are also presented in Table 1.

Sensitivity parameters such a molar absorptivity, the Sandell sensitivity, detection and quantification were calculated according to ICH guidelines. The accuracy of the method was established by analyzing the pure drug at the three levels (within working limits) and the precision was ascertained by calculating the relative standard deviation of five replicate determinations on the same solution containing the drug at three levels (Table. 2)

Table1. Analytical parameters

Parameters

Pitavastatin

λ max (nm)

The Beer’s law limits (μgml^-1)

Molar absorptivity (1 mol^-1 cm^-1)

The Sandell Sensitivity (μgml^-1)

Limit of detection* (μgml^-1)

Limit of quantification* (μgml^-1)

Regression equation**

Slope (b)

Intercept(a)

Correlation coefficient (R)

630

17.0-109.0

0.232× 104

0.159

0.5314

1.8642

y = a + bx

0.0075

0.0048

0.9892

*Calculated according to ICH-Guidelines; ** y is the absorbance and x is he concentration in _μgml^-1

Table 2. Evaluation of accuracy and precision

Pitavastatin Using Toluidine Blue reagent

Amount taken (μgml^-1)	Amount found* (μgml^-1)	RE (%)	SD (μgml^-1)	RSD (%)
36.0	36.02	0.051	0.038	0.105
55.0	55.04	0.068	0.019	0.027
75.0	75.03	0.036	0.024	0.029
92.0	92.02	0.019	0.028	0.032

· Mean Value of four determinations; RE – relative error; SD – standard deviation; RSD – relative standard deviation

Interference study: In the pharmaceutical analysis, it is important to test the selectivity towards the recipients and fillers added to the pharmaceutical preparations. Several species which can occur in the real samples together with drug were investigated. The level of interference was considered acceptable. Commonly encountered excipients such as starch, glucose, lactose did not interfere in the determination.

Method validation: Accuracy and precision: The accuracy of the proposed method was established by analyzing the pure drug at four levels (within the working limits) and precision was ascertained by calculating the relative standard deviation (RSD) of five replicate determinations.

The relative error and relative standard deviation values which are less than 1% indicate the high accuracy and precision of the methods (Table 2). For a better picture of reproducibility on a day to day basis, a series of experiments were performed in which standard drug solution at four different levels was determined each day for five days with all solutions being prepared afresh each day. The day to day relative standard deviation values represent the best appraisal of the method in routine use.

Applications: the proposed method can be used for the determination of Pitavastatin in bulk and pharmaceuticals.

CONCLUSIONS:

Simple and rapid methods for the determination of Pitavastatin using Toluidine blue reagent have been developed. The method is simple and easy to perform compared too many other method and do not entail any stringent experimental variables which affect the reliability of results. The methods can thus be used for the determination of Pitavastatin in pure and dosage forms.

ACKNOWLEDGEMENTS:

The authors are thankful to M/s. Alembic Ltd. Vadodara, India, for providing the gift sample of Pitavastatin calcium, respectively.

REFERENCES:

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Received on 21.02.2010 Modified on 12.03.2010

Asian J. Research Chem. 3(3): July- Sept. 2010; Page 643-645