Development and Validation of Analytical Methods for Pravastatin

 

Jane Jacob*, Jani Vishal M.

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

Deralakatte-575018

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

 

Three simple, sensitive, accurate, precise, reliable and economical methods have been developed for the quantitative estimation of Pravastatin in bulk and in its pharmaceutical formulation. Method-1 was   an HPLC method and the chromatographic separation was carried out on column of Hypersil ODS,3µm,10cm×4.6mm. The mobile phase comprised of acetonitrile, methanol and 0.08M ortho phosphoric acid in the ratio of 23:20:57 v/v/v. The pH of the mobile phase was adjusted to 2.8 by TEA (Tri Ethyl Amine), pumped at flow rate of 1 ml/min and eluent was monitored at 234 nm. Methods 2 & 3 were colorimetric estimations based on condensation reaction between Pravastatin and vanillin in presence of concentrated Sulphuric acid resulting in the formation of blue colored chromogen which showed a linearity range 2-12 µg/ml at  λmax of 610nm and a oxidative coupling reaction Pravastatin and chromogenic agent MBTH (3-Methyl -2-  benzothiazolinonehydrazone hydrochloride) in presence of cerric ammonium sulphate which obeyed Beer’s law in th concentration range of 2-10 µg/ml at absorption maxima of 616nm respectively. The three methods were validated statistically following ICH guidelines.

 

 

INTRODUCTION:

Pravastatin is chemically (+)-(3R,5R)-3,5-Dihydroxy-7-[(1S,2S,6S,8S,8aR)-6-hydroxy-2-methyl-8-[(S)2-methylbutyryloxy]-1,2,6,7,8,8a-hexahydro-1- naphthyl]-heptanoic acid, and it is uses as antihyperlipidemic agent. Statins act by competitively inhibiting HMG-CoA reductase, the first committed enzyme of the HMG-CoA reductase pathway. Because statins are similar to HMG-CoA on a molecular level they take the place of HMG-CoA in the enzyme and reduce the rate by which it is able to produce mevalonate, the next molecule in the cascade that eventually produces cholesterol, as well as a number of other compounds. This ultimately reduces cholesterol via several mechanisms. Pravastatin is marketed under the brand name of PRAVATOR, available in strengths of 10 & 20 mg. Pravastatin is official in Indian Pharmacopoeia¹. Literature survey reveals stability indicating quantification of Pravastatin in human plasma^2,3, determination of  simvastatin, pravastatin sodium and rosuvastatin calcium in tablet dosage forms by HPTLC⁴, LC/MS method⁵, capillary electrophoretic⁶ and a simple uv spectrophotometric⁷ were reported . the developed methods can be employed for routine analysis in laboratories as well as in industries.

 

MATERIALS AND METHODS:

All chemicals and solvents used were of HPLC/ AR grade. Acetonitrile, Potassium dihydrogen phosphate, O-phosphoric acid (Merck, India). All spectral measurements were done Jasco V530 series using 1cm matched cuvettes. The HPLC system employed in the method development and validation was Integrated system of model HP Hewlett Packard Agilent 1200 technology. The chromatographic separation was achieved on Hypersil ODS, 3µm, 10cm×4.6mm  column and Hydraulic system low pressure pump equipped with a PDA detector.

 

Method-1: 

Stock solution of Pravastatin (1 mg/ml) was prepared by dissolving 100mg of Pravastatin in 100 ml of diluent comprising of methanol and water in the ratio 50:50. Working solution was prepared by further diluting stock solution suitably with diluent to get a conc. of 100µg/ml. The suitable mobile phase and composition chosen for chromatographic run was acetonitrle, methanol and 0.08M Ortho phosphoric acid in the ratio 23:20:57 v/v/v.  The pH of the mobile phase was adjusted to 2.8 by TEA (Tri Ethyl Amine). The injection volume was 25 μl and the flow was fixed at 1 ml/min with the PDA detector set at wavelength of 234 nm. The standard solution of different concentration ranging from 2-12 μg/ml were prepared and injected into this fixed chromatographic condition and chromatograms were obtained.

CHROMATOGRAPHIC CONDITION:

Mobile Phase: ACN: MeOH: 0.08M Orthophosphoric acid (23:20:57 v/v/v)

Analytical column:   Hypersil ODS, 3μm,10cm×4.6mm.

Column Temperature: 30° C

λmax:                       234nm.

Flow rate:                 1 ml / min.

Run Time:                30 minutes

Retention Time:       9.39 min.

Operating pressure:   230-240 Kgf

Injection volume:      25μl

 

The developed procedure was extended to formulation by weighing twenty tablets and powdered using a mortar and pestle, powder weight equivalent to 50 mg of pravastatin was transferred to 50ml of volumetric flask and dissolve it in methanol: water (50:50), and filter through  a Millipore filter of size 22 μ. Whatman filter paper, so as to get a conc. of 50μg/ml and same procedure as for standard solution is followed. 

 

The accuracy of the method was studied by the recovery study. To the preanlaysed sample solution (50 μg/ml of pravastatin) a known quantity of pravastatin was added at the 50% level and analysed by the proposed RP-HPLC method. Sensitivity of the method was estimated in term of limit of detection (LOD) and limit of quantitation (LOQ).

Various system suitability parameters were calculated. It was observed that all the values were within the limits. The statistical evaluation of the method revealed its good linearity, reproducibility and its validation of different parameters and led us to the conclusion that it could be used for the rapid and reliable determination of pravastatin in tablet formulation.

 

RESULTS AND DISCUSSION:

Method–1, Pravastatin was assayed by RP-HPLC. The parameters were optimized such as selection of wavelength for detection, LOD & LOQ, ratio of mobile phase and flow rate. System suitability studies were carried out which included column efficiency, resolution capacity factor and peak asymmetry factor. A wavelength of 234nm was selected for the estimation of Pravastatin in methanol:water. A mixture of Acetonitrile, methanol and 0.08M ortho phosphoric acid  in the ratio of 23:20:57, v/v/v was selected as mobile phase and pH was adjusted to 2.8 by triethyl amine. The LOD and the LOQ of the developed method was determined by injecting progressively low concentration of the standard pravastatin into HPLC under optimized chromatographic conditions. A flow rate of 1min/ml seemed to be ideal for the selected drug. The peak were well resolved in the column of Hypersil ODS,3μm, 10cm×4.6mm. Using the chromatography condition mentioned above, the chromatograms of standard solution of Pravastatin were recorded. A computer controlled data was used to plot the peak area ratio of standard versus conc in μg/ml. The Pravastatin showed linearity in the range of 2-12 μg/ml. The retention time of Pravastatin was found to be 9.39min.  reliability and suitability of the method could be seen from the recovery value. Further, there was no interference due to excipients. The proposed RP-HPLC method is rapid, accurate and simple. The optical characteristics such as Beer’s law limit, molar absorptivity and other parameters for the proposed method are summarized in Table III. 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 (Table I II & III). The HPLC method was validated in accordance with ICH guidelines.^[8]

 

Specificity and Degradation studies:

Specificity of the stability indicating method was established by separation of the principal peak with degradants during forced degradation. The stress conditions utilized were acid hydrolysis, alkaline hydrolysis, oxidation by peroxide and thermal degradation. Overall these studies were aimed to degrade 10%-30% of the drug. Overall summary of degradation studies are presented under [Table 1].   

 

Method-2: Stock solution of Pravastatin was prepared by weighing accurately 100mg of pure drug in to a 100ml volumetric flask and dissolve with 95% Ethanol (1mg/ml). The working standard solution of Pravastatin was prepared by further diluting the stock solution suitably with 95% Ethanol to get a concentration of 100µg/ml. Fresh aliquots of Pravastatin ranging from 0.2 to 1.2 ml ( 1ml-100 µg/ml ) were transferred into a series of 10ml volumetric flask to obtain concentration range  of 2-12 µg/ml. To each volumetric flask, 2.5 ml of 5% of vanillin and 1.0 ml of concentrated sulphuric acid were added. The solution in each flask was made upto the mark with distilled water. The absorbance of blue colored chromogen was measured at 610nm against reagent blank within 80 mins. The developed procedure was extended to formulation. Six such linearities were taken for regression co-efficient and eight linearities were taken for standard deviation separately.

 

Method-3: Stock solution of Pravastatin was prepared by weighing accurately 100mg of pure drug in to a 100ml volumetric flask and dissolve with 95% ethanol to give a conc. of 1mg/ml. The working standard solution of Pravastatin was prepared by further diluting the stock solution suitably with 95% ethanol to get a concentration of 100µg / ml. Fresh aliquots of Pravastatin ranging from 0.2 to 1 ml (1ml-100µg/ml) were transferred into a series of 10ml volumetric flask to provide final concentration of 2-10µg/ml. To each volumetric flask, 2.0ml of 0.2% of MBTH followed by 1.0 ml of 2% cerric ammonium sulphate was added. The solution in each flask was made upto the mark with distilled water. The absorbance of greenish blue colored chromogen was measured at 616 nm against reagent blank within 90 mins. The same procedure was extended to formulation.

 

Table I: HPLC Parameters

Sl no.	Parameter	Capacity factor	No. of theoretical plate	Asymmetry
1	Formulation	937.666	9596	0.97
2	LOD	900.607	7580	0.90
3	LOQ	975.666	9880	0.99
4	Acid degradation	Std              Test 940.333     932.333	Std             Test 9176           5106	Std         Test 0.94         0.96
5	Base degradation	938.333      939.667	9467           9488	1.00         0.97
6	Oxidative degradation	938.333      937.666	9580           9519	1.01         0.99
7	Thermal degradation	938.333      937.667	9589           9587	0.98         0.99

 

Table II: Precision data

Drug	Concentration (μg/ml)	Peak area	% RSD
Pravastatin	6	877316 877393 873408 879756 878074 877194	0.3063

 

RESULTS AND DISCUSSION:

The developed colorimetric methods were validated for specificity, linearity, accuracy and precision as per ICH guidelines and are recorded in table no: III. 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. In Method-2 probable reaction may be a condensation reaction between Pravastatin and Vanillin in the presence of concentrated sulphuric acid, resulting in the formation of blue coloured chromogen which showed an absorbance maxima  of 610 nm and was stable more than 2 hours. The method obeyed Beer-Lambert law in the concentration range of 2-12 μg/ml. In method-3, Pravastatin was determined by a probable oxidative coupling reaction between pravastatin and MBTH(-3- Methyl-2- benzothiazolinone hydrazone hydrochloride) in the presence of cerric ammonium sulphate, resulting in the formation of greenish blue coloured chromogen  at 616 nm which was stable for more than two hours. MBTH loses two electrons and one proton on oxidation forming the electrophilic intermediate which is the active coupling species. The intermediate undergoes electrophilic substitution to form the coloured species. The method obeys Beer-Lambert law in the concentration range of 2-10 μg/ml.  The probable reactions are shown in Fig 1&2 respectively.

 

Table-III Optical characteristics of pravastatin:

Parameters	Method 1	Method 2	Method 3
λmax (nm)	234.0	610.0	616.0
Beer’s law limits (µg/ml)	2-12	2-12	2-10
Molar absorptivity (1/mol.cm)	--	2.5263x104	3.08x105
Sandell’s sensitivity (µg.cm2/0.001 Au)	-	0.1538	0.0126
Regression equation (y=a+bc) Slope (b) Intercept (a)	14481 7880	0.066 -0.008	0.079 -0.01
Correlation coefficient (r)	0.9990	0.9992	0.9990
Relative standard deviation (%)	0.1367	0.2906	0.8474
% Range of error (confidence limit) 0.05 level 0.01 level	0.1143 0.1691	0.2429 0.3594	0.7085 1.0483
Amount of drug (mg/tab)	16.453	18.84	19.01
% Recovery	97.20	94.22	95.05
confidence limit  ( μ) 0.05 level 0.01 level	1308456±1725.853 1308456±2553.387	0.5848±1.421×10-3 0.5848±2.103×10-3	0.5546±3.929×10-3 0.5546±5.814×10-3

 

CONCLUSION:

The methods developed for the estimation of pravastatin were simple, precise and accurate for the routine analysis. The RP-HPLC method was found to be most sensitive and accurate then other developed method and gives reproducible result.

 

ACKNOWLEDGEMENT:

The authors are thankful to NITTE University, Mangalore for the support given in carrying out this work.

 

REFERENCES:

1.     Indian Pharmacopoeia – 2010, Vol 3 pp 1945-47.

2.     Onal, Shagirli O; Development of stability indicating HPLC method for pravastatin sodium, Journal of chromatographia,2006, vol-64,pp157-162.

3.     Steffen B, et al., Quantification of pravastatin in human plasma and urine after solid phase extraction using high performance liquid chromatography with ultraviolet detection. Journal of chromatography, Analytical technologies in the biomedical and life sciences 818(2), 2005, pp257-62.

4.     Chaudhari B G., Detrmination of simvastatin, pravastatin sodium and rosuvastatin calcium in tablet dosage forms by HPTLC, Indian J Pharm science,2007,69,130-132.

5.     Zhu Z .,High performance liquid chromatography coupled with negative ion tandem mass spectrometry for the determination of pravastatin in human plasma. Journal of chromatography B, 2003, vol 783(1), 133-140

6.     Kircali K, Determination of pravastatin by capillary electrophoresis, Framaco, 2004,59(3),241-244

7.     Balaji.S, et al., Development of spectrophotometric method for determination of Pravastatin sodium in bulk and tablet formulation, International Journal of PharmTech Research, Volume 1 (4), 2009, pp1017-1019.

8.     ICH-Guidelines Q2B Validation of Analytical Procedure: Methodology (CPMP/ICH/281/95), Geneva, Switzerland.

 

 

 

Accepted on 02.08.2013         © RJPT All right reserved