INTRODUCTION:

Pravastatin sodium ((3R,5R)-3,5-dihydroxy-7-[(1S,2S,6S, 8S,8aR)-6-hydroxy-2-methyl-8-[[(2S)-2-methyl butanoyl] oxy]-1,2,6,7,8,8a-hexahydronaphthalen-1-yl] heptanoate) (Fig. 1),a competitive inhibitor of HMG Co A reductase, is mainly used in the treatment of atherosclerosis and hypercholestremia¹. Various methods have been reported for estimation of pravastatin sodium in biological fluids and biological samples, which include use of LC with ultraviolet (UV) detection^2–5. LC/MS^6–8 methods are also reported for estimation of pravastatin sodium; however mass-selective detection is cost intensive and often not available for routine analysis. Also capillary electrophoresis⁹, spectrophotometric¹⁰ and electrochemical volta metric method¹¹ are reported. Only a single HPLC method is reported for estimation of pravastatin sodium in tablet dosage form¹² while another describes estimation of pravastatin in production media¹³. The reported methods^12-13used acetonitrile and buffer for quantification of pravastatin sodium.

Present study involved in development of RP-HPLC (reverse phase high performance liquid chromatography) method using simple mobile phase containing methanol which is cheaper than acetonitrile and no buffer for quantitative estimation of pravastatin sodium in tablet dosage forms. Since there was no buffer used in proposed method less chance of HPLC and column damage and require less time in column washing.

MATERIAL AND METHODS:

Material:

Pravastatin sodium was a gifted by Astron Research Ltd., Ahmedabad, Gujarat, India. Methanol of HPLC grade and o-phosphoric acid of analytical grade were purchased from S.D. fine chemicals Ltd., Mumbai, India. Tripled distilled water was made by glass distillation assembly and used throughout experiment. Commercially available pravastatin sodium tablets claimed to contain 20 mg of the pravastatin sodium were purchased from the local market.

Instrument and Chromatographic Condition:

Quantitative HPLC was performed on an isocratic high pressure liquid chromatograph (Shimadzu^® HPLC system, Japan) with a LC-10 AT pump, an ultraviolet detector SPD 10 A, a Luna C18 100 A column (250 X 4.6 mm i.d., 5 µ particle size) (phenomenex, Germany) and a manual Rheodyne 7725(i) injector with a 20 µl loop. The HPLC system was equipped with the software “LC solution®” (Shimadzu).

Fig 1: Chemical structure of Pravastatin sodium

Table 1 : Summary of validation parameter

Parameters	Values
Linearity range	14 - 26 µg/ml
Correlation Co-efficient	0.9997
Precision (% CV) Intra day Inter day	0.51 - 0.80 % 0.40 - 1.04 %
% Mean Recovery	98.9 - 100.2 %
Limit Of Detection	33.3 ng/ml
Limit Of Quantification	100.9 ng/ml

The contents of the mobile phase were methanol and aqueous solution of o-phosphoric acid (0.6% v/v) in the ratio of 70:30 (v/v). Before mixing the organic solvent with aqueous solution of o-phosphoric acid (0.6% v/v), aqueous solution of o-phosphoric acid (0.6% v/v) was filtered through a 0.45 µm Millipore filter. The mobile phase was degassed by ultrasonication for 20 min. The mobile phase was pumped from the solvent reservoir to the column at a flow rate of 1.0 ml/min. The run time was set at 12 min and the column temperature was maintained at 25 ± 2° C. The volume of injection loop was 20 µl. The diluent consisted of methanol: water in the ratio 50:50 (v/v). The column was equilibrated for at least 30 min with the mobile phase flowing through the system, prior to injection of the solutions. The eluent was monitored at 239 nm and data were acquired, stored and analyzed with the software LC solution®.

Preparation of Standard Stock Solution:

Standard stock solution of the drug was prepared freshly by dissolving each time 50 mg of pravastatin sodium in a 100 ml volumetric flask containing 70 ml of the diluent, sonicated for about 20 min and then made up to volume with the diluent.

Preparation of sample solution:

Twenty tablets were weighed to obtain the average tablet weight, which were then powdered. Sample of the powdered tablets, claimed to contain 50 mg of the active ingredient, were mixed with 70 ml diluent in a 100 ml volumetric flask. This mixture was sonicated for 20 minute to ensure complete solubility of the drug. The volume was made up to mark with the diluent. This mixture was shaken well and then filtered through a 0.20 µm Millipore filter. The filtrate (1.0 ml) was transferred to a volumetric flask (25 ml) and made up to sufficient volume with the diluent to give an expected concentration of 20.0 µg/ml.

Method Validation:

The develop method was validated as per US FDA guideline¹⁴ and ICH guideline¹⁵ for linearity, range, accuracy, precision, specificity, limit of detection, limit of quantification and robustness parameter.

Specificity:

To perform the specificity blank sample was injected in chromatograph and chromatogram was recorded.

Table 2 : Results of recovery study

Amount of Pravastatin sodium added to tablet solution (mg/ml)	Amount of Pravastatin found (mg/ml) Mean ± S.D. (n =3)	% Recovery Mean ± S.D. (n =3)
16.0	15.8 ± 0.10	98.9 ± 0.63
18.0	17.8 ± 0.10	99.2 ± 0.53
20.0	20.0 ± 0.07	100.2 ± 0.35

inearity:

The linearity of the method was determined at the seven concentration levels ranging from 14.0-26.0 μg/ml. working standard solutions of pravastatin sodium were prepared by suitable dilution of the standard stock solution with the diluent to obtain concentration of pravastatin sodium in the range of 14.0 to 26.0 µg/ml. Each of these drug solutions (20 µl) were injected into the injector and the peak areas were recorded. The calibration graph was constructed by plotting amount injected (µg/ml) versus mean peak area (m AU).

Limit of detection (LOD) and Limit of Quantification (LOQ):

The LOD and LOQ values were calculated by using calibration curve. For the said purpose calibration curve of pravastatin sodium was prepared in the lower concentration range 100-300 ng/ml. The LOD and LOQ are calculated by using formula given in the guideline^14,15.

Accuracy:

To examine the accuracy of the developed method, recovery studies were carried out by standard addition method at three different concentration levels in triplicate by spiking standard pravastatin sodium solution in previously analyzed tablet solution.

Fig. 2: A chromatogram of tablet dosage form of Pravastatin sodium. Observed retention time for excipients at about 2 min and pravastatin sodium 6.73 min.

Precision:

Intra-day and inter-day precision were determined by analyzing samples of pravastatin sodium at three different concentration level in triplicate within range. Intra-day and inter-day precision were performed by injecting 14.0 µg/ml, 20.0 µg/ml and 26.0 µg/ml of pravastatin sodium solution in triplicate and calculate percentage relative standard deviation (% RSD). Repeatability was performed by injecting 20.0 µg/ml of pravastatin sodium solution six times in chromatograph and calculate % RSD.

Robustness:

Robustness of the method was studied by changing the composition of organic phase composition (65:35, and 75:25) and the flow rate (0.8ml/min and 1.2 ml/min).

RESULTS AND DISCUSSION:

The goal of the present study was to develop a rapid, precise, accurate and cost effective HPLC method for the analysis of pravastatin sodium in its pharmaceutical tablet formulation, using the reverse phase (RP) C₁₈ column with UV detection and validate develop method as per US FDA guideline and ICH guideline. The validation parameters were summarized in Table 1. In order to obtain suitable peak shape, retention time and separation of pravastatin sodium peak from excipients peak methanol and aqueous solution of o-phosphoric acid mobile phases were used. Satisfactory peak shape (tailing factor 1.2 and theoretical plate > 5200) was obtained using the mobile phase methanol: aqueous solution of water (0.6 % v/v) in the ratio of 70: 30 (v/v) with a flow rate 1 ml/min. As pravastatin sodium showed maximum absorption at 239 nm, the detector was set at 239 nm. Chromatogram of tablets formulation of Pravastatin sodium was shown in Fig. 2. It showed pravastatin sodium peak around 6.73±0.3 min which is separated from the excipients peak which was found around 2 min. It indicate that the develop method was successful to separate the drug and excipients peak. The method was found to be specific. There was no peak found in blank sample chromatogram at pravastatin sodium peak retention time.

Table 3: Results of intra-day and inter-day precision study

Conc. of Pravastatin sodium (µg/ml)	Mean found concentration of Pravastatin sodium (n =3)		% RSD
Conc. of Pravastatin sodium (µg/ml)	Intra day	Inter day	Intra day	Inter day
14.0	14.0	13.8	0.73	1.04
20.0	19.8	20.0	0.51	0.40
26.0	26.1	25.7	0.80	0.72

Table 4: Estimation of pravastatin sodium in tablet dosage form by the proposed HPLC method.

Brand of

the Pravastatin sodium tablet

Labeled amount

Mg/tablet

Observed amount

Mg/tablet Mean ± S.D. (n = 3)

% Assay

Mean ± S.D. ( n= 3)

20.2 ± 0.06

100.9 ± 0.64

20.3 ± 0.12

101.7 ± 0.58

The method was linear in the concentration range from 14.0 - 26.0 µg/ml of pravastatin sodium. The calibration curve was constructed by plotting concentration (X) versus the mean peak area of pravastatin sodium standard (Y). The correlation coefficient was found to be 0.9997. The regression equation was found as Y = 59401X - 8909. Where, Y is the peak area of pravastatin sodium and X, the concentration of measured solution in µg/ml. The results showed excellent correlation between the peak area and the concentration of pravastatin sodium in the range tested. The calculated LOD and LOQ values were found to be 33.3 ng/ml and 100.9 ng/ml, respectively.

The mean absolute recovery determined by spiking known amounts of pravastatin sodium solution 16, 18 and 20 µg/ml to the previously analyzed tablet solution in triplicate. The results were shown in Table 2. The average percent recoveries obtained as 98.9 - 100.2 % indicate good accuracy of the method.

This method was validated for intra-day and inter-day precision. % RSD for intra-day and inter-day precision were found to be 0.51 - 0.80 % and 0.40-1.04 % respectively, indicating good precision. Repeatability was found to be 0.75%. The results of precision were shown in Table 3.

The proposed method was found to be robust because change in flow rate and mobile phase composition did not change system suitability criteria such as symmetry of peak and number of theoretical plate. In order to demonstrate the stability of both standard and sample solutions during analysis, both the solutions were analyzed over a period of 12 h at room temperature. The results showed that for the solutions, retention time and peak area of pravastatin sodium almost unchanged and no significant degradation was observed within the indicated period.

The proposed method was successfully applied to the analysis of two marketed tablet formulation of pravastatin sodium (20 mg/tablet) and the results obtained are given in Table 4. The average drug content was found to be 100.9 % and 101.7 % respectively.

CONCLUSION:

The results of the study showed that the proposed HPLC method is simple, rapid, precise, accurate, specific and sensitive for determination of pravastatin sodium, without any interference from the excipients. Hence, this method is suitable for routine analysis of pravastatin sodium in its tablets dosage form.

ACKNOWLEDGEMENTS:

The authors are thankful to M/s Astron Research Ltd., Ahmedabad, Gujarat, India for providing gift samples of pravastatin sodium.

REFERENCES:

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Received on 10.11.2009 Modified on 30.12.2009

Asian J. Research Chem. 3(1): Jan.-Mar. 2010; Page 197-200