RP-HPLC Determination of Three Anti-Hyperlipidemic Drugs in Spiked Human Plasma and in Dosage Forms

Sensitive, simple and accurate high performance liquid chromatographic (HPLC) methods for the determination of atorvastatin (AT), fluvastatin (FL) and pravastatin (PV) have been developed. The proposed methods involve the use of a 150 mmx4.6 mm Zorbax Extend-C18 column (5 μm particle size) and different chromatographic conditions for the separation of the three statins. Linearity range was 5-40, 5-30 and 10-60 μg mL-1 for AT, FL and PV respectively. The developed methods proved to be successful in the determination of all studied drugs in spiked human plasma samples.


Introduction
Statins lower cholesterol by inhibiting the synthesis of mevalonic acid, which is a precursor in cholesterol synthesis.Dropping mevalonic acid levels triggers the expression of more low density lipoprotein (LDL) receptors in the liver, which then removes LDL from blood stream 1 .
This work describes simple, fast and sensitive HPLC methods for the determination of atorvastatin, fluvastatin and pravastatin in bulk form, in pharmaceutical formulations and in spiked human plasma without sample pretreatment or time-consuming extraction prior to analysis.

Experimental
Agilent 1200 series isocratic quaternary pump HPLC instrument connected to 1200 multiple wavelength UV detector was used.Separation was performed on 150x4.6 mm Zorbax Extend-C18 column 5 µm particle size.Chromatographic peaks were electronically integrated and recorded using Chemstation software (Germany).pH/mv Meter with double junction glass electrode (Fisher, USA) and Hamilton syringes 25 µL were used

Reagent and chemicals
HPLC grade acetonitrile was purchased from Fisher scientific (UK).Phosphoric acid and potassium dihydrogen phosphate were purchased from Sigma-Aldrich Chemie (Germany).Demineralized water was further purified by filtering through a 0.45 µm Millipore filter (Gelman, Germany).

Materials
Atorvastatin calcium, fluvastatin sodium, pravastatin sodium, pyridoxine hydrochloride (PR) and ipubrofen (IB) authentic powders were kindly supplied by EPICO, Novartis Pharma, HI Pharm pharmaceuticals and Memphis Co., respectively.Plasma samples were purchased from the local hospital blood bank.

Pharmaceutical preparations
Ator ® tablets produced by EPICO, batch No. 083127, claimed to contain 20 mg AT.Lescol ® XL tablets packed by Novartis Pharma, batch No. 149/B5589, claimed to contain 84.24 mg fluvastatin sodium which corresponds to 80 mg fluvastatin free acid.Cholestate ® film coated tablets product of HI Pharm pharmaceuticals, batch No. 1012209, claimed to contain 10.5 mg pravastatin.

Solutions of atorvastatin
Accurately weighed amounts of standard AT were dissolved in 100 mL volumetric flask using methanol and diluted to volume with the same solvent to get 0.1 or 1 mg mL -1 solutions.

Solutions of fluvastatin
Accurately weighed amounts of standard FL were dissolved in 100 mL volumetric flask using acetonitrile -water mixture (50:50) and diluted to volume with the same solvent to get 0.1 or 1 mg mL -1 solutions.

Solutions of pravastatin
Accurately weighed amounts of standard PV were dissolved in 100 mL volumetric flask using acetonitrile -water mixture (50:50) and diluted to volume with the same solvent to get 0.1 or 2 mg Ml -1 solutions.

Solutions of pyridoxine HCl (internal standard)
Accurately weighed amounts of standard PR were dissolved in 100 mL volumetric flask using methanol and diluted to volume with the same solvent to get 0.5 or 10 mg mL -1 solutions.

Solutions of ipubrofen (internal standard)
Accurately weighed amounts of standard IB were dissolved in 100 mL volumetric flask using methanol and diluted to volume with the same solvent to yield 1 or 10 mg mL -1 solutions.Calibration

Atorvastatin
Aliquots of the standard drug solution (0.1 mg mL -1 ) in methanol equivalent to 0.04-0.4mg AT were transferred to a series of 10 mL volumetric flasks; 1 mL of pyridoxine internal standard solution (0.5 mg mL -1 ) was added to each flask then diluted to volume using the mobile phase consisting of acetonitile-50 mM KH 2 PO4 (60:40 v/v) adjusted to pH 3.5 by phosphoric acid.

Fluvastatin
Aliquots of the standard drug solution (0.1 mg mL -1 ) in acetonitrile -water mixture (50:50) equivalent to 0.05-0.3mg FL were transferred to a series of 10 mL volumetric flasks; 1 mL of pyridoxine internal standard solution (0.5 mg mL -1 ) was added to each flask then diluted to volume using the mobile phase consisting of acetonitile-50 mM KH 2 PO4 (60:40 v/v) adjusted to pH 3.5 by phosphoric acid.

Pravastatin
Aliquots of the standard drug solution (0.1 mg mL -1 ) in methanol equivalent to 0.04-0.4mg PV were transferred to a series of 10 mL volumetric flasks; 1 mL of ipubrofen internal standard solution (1 mg mL -1 ) was added to each flask then diluted to volume using the mobile phase consisting of acetonitile-50 mM KH 2 PO4 (40:60 v/v) adjusted to pH 3.5 by phosphoric acid.
Triplicate 20 µL of each drug solution were injected into the liquid chromatograph using the following chromatographic conditions: Detector wavelength: 210 nm for atorvastatin and 230 nm for fluvastatin and pravastatin.Flow rate: 1 mL min -1 .Column temperature: ambient temperature.The peak area ratios were plotted against drug concentration for calibration curve construction and the regression parameters were deduced.

Analysis of tablets
Five Ator ® , Lescol ® XL and Cholestate ® tablets were grounded to a homogenous fine powder, weighed and the average mass per tablet was determined.The amount of powder equivalent to 10 mg of AT, FL and PV was transferred accurately into a 100 mL volumetric flask containing 70 mL of methanol for AT or acetonitrile -water (50:50) for FL and PV.The content of the flasks was sonicated for about five minutes and then dilutions were made using methanol.Afterwards, the solutions were filtered to separate insoluble excipients.The obtained solution labeled to contain 0.1 mg mL -1 of each drug was analyzed by the corresponding HPLC method as detailed under calibration.

Atovastatin
Different aliquots (0.1 -0.7 mL) of the standard AT solution (1 mg mL -1 ), were added to 0.5 mL human plasma in a series of 10 mL centrifuge tube followed by 0.1 mL of the internal standard solution (10 mg mL -1 ) PR.

Fluvastatin
Different aliquots (0.1 -0.6 mL) of the standard FL solution (1 mg mL -1 ) were added to 0.5 mL human plasma in a series of 10 mL centrifuge tube followed by 0.1 mL of the internal standard solution (10 mg mL -1 ) PR.

Pravastatin
Different aliquots (0.1 -0.5 mL) of the standard PV solution (2 mg mL -1 ) were added to 0.5 mL human plasma in a series of 10 mL centrifuge tube followed by 0.2 mL of the internal standard solution (10 mg mL -1 ) IB.
For each drug, volume was completed to 2 mL with acetonitrile, then vortex for 3 min and centrifuged at 4000 rpm for 30 min.One mL of the clear supernatant was transferred to series of 10 mL volumetric flasks, completed to volume with the mobile phase to achieve the specific linearity range.The general procedure described under calibration was followed and each drug concentrations were calculated from the regression parameters.

Results and Discussion
Statins are rapidly absorbed from the gastro-intestinal tract.They have low absolute bioavailability ranging from 5 to 24% due to pre-systemic clearance in the gastro intestinal mucosa and/or first-pass metabolism in the liver, their primary site of action 40 .To assess the applicability of the method for determination of statins in biological fluids, drug spiked plasma samples were analyzed by the proposed method after deproteination with acetonitrile.

Optimization of chromatographic conditions
The composition of the mobile phase was studied by trying acetonitrile and KH 2 PO 4 (5x10 -2 M) in different ratios using isocratic and gradient elution.The best peak shape and adequate separation of the drug and internal standard was obtained by a final composition of acetonitrile-KH 2 PO 4 (60:40 v/v) for AT, FL and (40:60 v/v) for PV, respectively.All mobile phase pH was adjusted to 3.5 by orthophosphoric acid.Different flow rates (0.5-1.2 mL min -1 ) were tested; good resolution was obtained using 1 mL/min.Four wavelengths were tried (210, 230, 240 and 280 nm); much sensitive detector response was obtained at 210 nm for AT, 230 nm for FL and PV.System suitability parameters were calculated and the retention times were 2.4±0.05min for AT and 1.26 min for the internal standard PR (Figure 1).

Method validation
The procedure was intended for an assay which is described as a category I procedure in General Chapter (1225) Validation of Compedial methods of US Pharmacopeia.

Linearity, detection and quantitation limits
Calibration curves representing the relation between each drug concentrations and peak area ratio were constructed.Results show linear relationship in the range of 5-40, 5-30 and 10-60 µgmL -1 for atorvastatin, fluvastatin and pravastatin respectively; in triplicate run from which linear regression equations were calculated.Correlation coefficient, slope and intercept were listed in Table 1.Results indicate high sensitivity of the proposed methods.
According to ICH recommendation 41 , the approach based on the S.D. of the response and the slope was used for determining the detection and quantitation limits.The theoretical values were assessed practically and given in Table 1.

Accuracy
Accuracy of the measurements was determined using the calibration standards of the three drugs, where mean percentage of 100.58, 100.22 and 100.31 for AT, FL and PV, respectively, were obtained (Table 1).Accuracy was also assessed by the recovery of added standard, three concentrations each in duplicate to know concentration of commercial tablets using the proposed HPLC procedures.Results of mean % recovery for added standards in each tablet are reported in Table 2.

Injection repeatability
The RSD of drugs peak area ratio in five triplicate injections of standard drug solution determined each day of 3 consecutive days ranged from 0.14 -1.28% (Table 1).

Assay repeatability
Analysis of 3 concentrations of tablets was performed in triplicate each day of 3 successive days.Results for the intraday RSD and interday for each dosage form were recorded in Table 1 providing the high reproducibility and ruggedness of the proposed HPLC methods.

Specificity
Retention time of the peak in the chromatogram of tablets and spiked human plasma was the same as that of standard drugs without interference from excipients, additives or biological fluid components.Accordingly, specificity of the HPLC methods was evaluated by its successful application to determine drugs in their tablets with mean recovery of 99.25±0.73%for Ator ® tablets, 99.90±0.68 for Lescol ® XL tablets and 100.29±0.76 for Cholestate ® tablets.
A statistical comparison of the results obtained by the proposed methods and the reported or manufacturer HPLC methods is shown in Table 2.The values of the calculated 't' and 'F' are less than the tabulated ones, which reveals that there is no significant difference with respect to accuracy and precision between the proposed, reported and manufacturer methods.Moreover, the proposed methods were extended to analyze drugs in spiked human plasma by simple extraction and deproteination with acetonitrile, followed by centrifugation and the clear supernatant containing the drug was adjusted to volume by the mobile phase and analyzed directly by HPLC procedures.Nearly non-destructive extraction was obtained as obvious from a mean % recovery of 98.19 -98.87 from plasma (Table 3).

Conclusion
The proposed HPLC methods were shown to be specific, precise, linear and easy to perform allowing rapid determination of atorvastatin calcium, fluvastatin sodium pravastatin sodium in tablets and spiked human plasma.Validation of the proposed methods was carried out according to the ICH and USP guidelines.The short duration of the assay and its specificity were clear bonuses for routine analysis (batch analysis) and clinical application.

Table 1 .
Selected physical data for the determination of atorvastatin, fluvastatin and pravastatin by the proposed HPLC methods a LOQ and LOD were determined practically

Table 2 .
Statistical analysis of the results obtained by applying the proposed, reported and manufacturer methods for the analysis of AT, FL and PV in their tablets

Table 3 .
Determination of atorvastatin, fluvastatin and pravastatin in spiked human plasma * Average of three determinations