Determination of Pitavastatin Calcium by Analytical Spectrophotometry

Simple and rapid spectrophotometric method for the quantitative analysis of Pitavastatin calcium (PTV) in raw material and tablets pharmaceutical formulation has been described. The method is based on the formation of yellow ion-pair complex between Pitavastatin calcium and Bromocresol purple (BCP) in chloroform medium.
Different parameters affecting the reaction such as: effect of solvents, stability, reagent concentration, correlation ratio, etc. were optimized. The formed complex was quantified spectrophotometrically at absorption maximum 405 nm. Linearity range was 2.20 - 35.23 µg/mL, regression analysis showed a good correlation coefficient R2 = 0.9991. The limit of detection (LOD) and limit of quantification (LOQ) were to be 0.367 µg/mL and 1.112 µg/mL respectively. The average percent recovery was found to be (100.62 – 101.14) % for Pitavastatin Calcium. This study was applied on Syrian pharmaceutical trademark: (PAVACRIUM 4 & Londalop). The method was successfully applied for the determination of Pitavastatin calcium in tablets pharmaceutical formulation.
The proposed method is simple, direct, sensitive and do not require any extraction process. Thus, this method could be readily applicable for the quality control and routine analysis.


INTRODUCTION
Pitavastatin is the first synthetic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor which was discovered in Japan. It is chemically monocalcium bis (3R,5S,6E)-7-(2-cyclopropyl-4-[4-fluorophenyl]-3quinolyl-3,5-dihydroxy-6-heptenoate), used as the calcium salt in the treatment of hyperlipidaemia and can reduce the risk of cardiovascular diseases in everyday medical practice. Based on the preclinical findings, Pitavastatin significantly decreased the serum levels of total cholesterol and low-density lipoprotein cholesterol at doses of 1 mg/day or more. It also significantly decreased the serum levels of triglycerides within this dose range. There was no dose-dependence of the incidence of adverse reactions to Pitavastatin. One other characteristic of the agent is that Pitavastatin is minimally metabolized by the cytochrome P450 isozymes; it undergoes glucuronidation and is converted to the inactive lactone form, therefore, the incidence of any drug interactions is reduced. Due to the promising results observed in clinical trials, it has the potential to be an excellent addition to the worldwide lipid management market [1][2][3][4][5] .
The estimation of Pitavastatin calcium (PTV) from pharmaceutical formulations has been determining by several analytical methods. These include spectrophotometric method, based on ability of potassium permanganate to oxidize Pitavastatin in acidic medium 6 . UV spectrophotometric methods for the determination of Pitavastatin calcium 7,8 , Color reactions for spectrophotometric determination of Pitavastatin calcium 9,10. Spectrofluorometric method is based on measuring the native fluorescence of the drugs at their optimum excitation and emission wavelengths 11 , High performance thin layer chromatography (HPTLC) 12,13 . High performance liquid chromatography (HPLC) [14][15][16] . Ultra High performance liquid chromatography (UHPLC) method for the selective quantification of Pitavastatin calcium 17 . Reverse-phase high performance liquid chromatography (RP-HPLC) [18][19][20] . Liquid chromatography-tandem mass spectrometry (LC-MS/MS) 21,22 , Bromocresol purple (BCP) which is a brominated acid dye of the sulfonephthalein series derived from orthocresol that is obtained as a pinkish crystalline powder and is used as an acid-base indicator commonly used as indicator and spectrophotometric reagent.

Pitavastatin calcium stock solution
Stock solution 1×10 -3 M of Pitavastatin calcium (MW = 880.98 g/mol) was prepared by dissolving 8.85 mg of raw material equivalent to 8.894 mg (by taken the purity in consideration) in volumetric flask 10 mL with 2 mL methanol and completed to volume with Chloroform to give concentration 1 × 10 -3 M equivalent to 880.98 g/mL. The working standard solutions of Pitavastatin calcium were prepared by appropriate dilutions among (25 -400) L of 880.98 g/mL solution in volumetric flasks 10 mL and added to each one of BCP 10 -2 M equals to ten times of Pitavastatin calcium concentration, then completed to volume with Chloroform to give concentrations between (1.10 -35.24) g/mL of Pitavastatin calcium .

Reagent stock solution
Bromocresol purple 10 -2 M was prepared by dissolving 270.11 mg of BCP (Mw = 540.22 g/mol) in volumetric flask 50 mL and completing to volume with Chloroform.

Calibration Curve
To construct the calibration curve, five standard solutions for each concentration were prepared and the absorbance was measured of each solution five times.

Sample preparation
Two products were studied : Twenty tablets from PAVACORIUM 4 and Londalop Syrian products were weighed and finely powdered and an accurate weight equivalent to 4 mg (PTV) was accurately weighed, dissolved in volumetric flask 10 mL in Methanol, then 1 mL of the solution was taken to volumetric flask 10 mL and diluted to volume with Chloroform. 1 mL of the last solution was taken to volumetric flask 10 mL and added 1 mL of Bromocresol purple 10 -2 M, then diluted to volume with Chloroform, equivalent theoretically to 4.0 g/mL for (PTV).

RESULTS AND DISCUSSION
Pitavastatin calcium forms with Bromocresol purple at 25 ± 5 ºC yellow ion-pair complex. The result solution was scanned in the range of wavelengths 300 -550 nm against a blank of BCP solved in chloroform, then measured the absorbance at maximum wavelength 405 nm. We studied all the parameters of the colored result solutions to obtain the optimal conditions .

Stability of stock solution
Time effect on stability standard stock solution of Pitavastatin calcium in Chloroform was studied in three different concentrations 1×10 -5 , 2 ×10 -5 and 3 × 10 -5 M. We did not notice any significant absorption changes within one month.

Effect of reagent concentration
To study the effect of reagent concentration on the colored complex solution, we made a series of 10 mL of separated volumetric flasks, by adding 0.25 mL of Pitavastatin calcium 25 × 10 -6 M equivalent to 25 µM and added between (0.0125 -0.300 mL) of (BCP) 1×10 -2 M, equivalent to (12.5 -300 µM) after completing the volume to 10 mL by chloroform. The absorbance at 405 nm for every added (BCP) reagent was measured against the blank of chloroform. It was found that the completed colored complex formation in the best condition was 250 µM of (BCP) equivalent to 0.250 mL of (BCP) which equal to ten times of Pitavastatin calcium concentration, as it is shown in Fig. 2.

Calculation of formation constant for the (PTV:BCP) complex
The conditional stability constants ( ) of the ion-pair complexes were calculated from molecular ratio and the continuous variation curves.
Data using the following equation 23

METHOD'S VALIDATION
The validity and suitability of the proposed method was assessed by linearity (evaluated by regression equation), limit of detection (LOD), limit of quantification (LOQ), accuracy (reported as percent %), precision (reported as RSD %), robustness, and Sandall's sensitivity.

Linearity
We studied the linearity of Pitavastatin calcium concentrations at the optimal conditions, where we made a series of 10 mL of separated volumetric flasks , each one contains concentration of BCP equals to ten times of Pitavastatin calcium concentration, where the variable concentrations of PTV stock solution 1 ×10 -3 M and the concentrations of BCP stock solution 1 ×10 -2 M, then the volumetric flasks completed to 10 mL with Chloroform, finally we measured the absorbance at 405 nm for each concentration against the blank of BCP in chloroform.

Limit of Detection (LOD) and Limit of Quantification (LOQ)
In spite of the measurement LOD and LOQ, five concentrations were analyzed in five replicates.
LOD and LOQ for Pitavastatin Calcium were calculated by using the following equations: Where SD, is the standard deviation of y intercepts of regression lines and m is the slope of the calibration curve. The limit of detection (LOD) and limit of quantification (LOQ) were to be 0.367 g/mL and 1.112 g/mL respectively.

Accuracy
To determine the precision and accuracy of the proposed method, five replicates determinations were carried out on five different concentrations of standards (PTV).

Precision
In order to demonstrate the precision of the proposed method, intra-day and inter-day variability studies were performed at three different concentrations (4.40, 13.21, and 22.02) g/mL for Pitavastatin Calcium at the same day in two hour's time interval and also at three different days. Method efficiency was tested in terms of RSD % for both intra-day and inter-day precisions .
The precision was ascertained by carrying out five replicates of standard Pitavastatin calcium under study and the mean was calculated. The results are showed in Table 2. The RSD % results were not more than 2.91 % during the determination in one day or three days, where the method is considered very precise.

Robustness
The robustness of an analytical procedure is a measure of its capacity to maintain unaffected results by a very small variation of some parameters and provides an indication of its reliability during normal usage. The studied variables parameters were slit scan speed and the wavelength, which performed at concentration (13.21 g/mL) for Pitavastatin calcium Table 3.

Sensitivity Sandell's and molar absorptivity
Sensitivity of the proposed method for Pitavastatin Calcium was determined by calculating Sandell's sensitivity (SS), it was to be SS = 0.0259 µg/cm 2 . The mean molar absorptivity ε was found equal to 67830.11 L/mol.cm.

Recovery
The recovery was studied by three addition standards (80 %, 100 %, and 120 %) for every product. Table 4 presents the recoveries results for the two Syrian products (PAVACORIUM 4 and Londalop).

Estimation of Pitavastatin Calcium in PAVACORIUM 4 and Londalop products
The developed method was applied for quantitative determination and identification Pitavastatin Calcium in two Syrian pharmaceutical products (PAVACORIUM 4 and Londalop) for three different batches for each one. The samples were prepared as described in the section of samples preparation and analyzed. Quantitative analysis was done by using calibration curve. The obtained results are summarized in table 5. In general, the concentrations of the detected Pitavastatin Calcium compounds in the two products were within the allowed limits under USP legislation 26 , The tablets must contain not less than 90.00 % and not more than 110.00 % of labeled amount. So the obtained results are conformed to USP legislation 26 . The relative standard deviations RSD % (n = 5) of the quantitative results were in the range of 0.80 -2.90 % for PAVACORIUM 4 and 2.59 -3.41% for Londalop. Mean for five replicates.

CONCLUSION
We developed a new method which is suitable for the identification and quantification of Pitavastatin Calcium in raw material and Syrian tablets formulation. A good percentage of recovery shows that the method can be successfully used in pharmaceutical quality control and routine analyses. The proposed method is simple, sensitive, rapid, specific, a little cost. It could be applied for quality control of Pitavastatin Calcium in pharmaceutical factories. The levels of Pitavastatin Calcium compounds in the analyzed preparations were found to be within the permissible limits set by the USP legislation 26 .