Development of Simple Spectrophotometric method to Determinate Pitavastatin Calcium in Bulk and Pharmaceutical Preparations

 

Nazira Sarkis*1, Saad Antakli2, Rita Keshishian1

1Department of Analytical and Food Chemistry, Faculty of Pharmacy, Aleppo University, Syria.

2Department of Chemistry, Faculty of Science, Aleppo University, Syria.

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

 

ABSTRACT:

An accurate, precise, sensitive and direct extraction-free simple spectrophotometric method has been developed and applied to determine Pitavastatin calcium (PTV) in raw material and tablets preparations. This Spectrophotometric method is based on the formation of yellow colored ion-pair complex in chloroform by Bromocresol Green (BCG). The complex has maximum absorptivity at 416 nm. The variable factors affecting the reaction such as solvents, time, and temperature and reagent concentration were studied and optimized to achieve the best results. Beer's law is obeyed over concentration ranges (2 24) g/mL. Regression analysis showed a good correlation coefficient R2 = 0.9994. The limit of detection (LOD) and limit of quantification (LOQ) were to be 0.19g/mL and 0.59g/mL respectively and the molar absorptivity coefficient 61706 L/mole/cm. The average percent recovery was calculated and it was in range 98.59-101.97% for Pitavastatin calcium. All studied samples showed that the drug levels were confirmed due to Japanese pharmacopeia legislation.

 

KEYWORDS: Pitavastatin calcium, Bromocresol green, Spectrophotometric method, ion-pair.

 

 


INTRODUCTION:

Pitavastatin calcium (PTV) is Monocalcium bis{(3R,5S,6E)-7-[2-cyclopropyl-4-(4-fluorophenyl) quinolin-3-yl]-3,5-dihydroxyhept-6-enoate} with molar mass is 880.98g/mole[1]. The structure of PTV is shown in figure (1,a).

 

It is a novel member of the statins class. Like the other statins, it is an inhibitor of HMG-CoA reductase, the enzyme that catalyzes the first step of cholesterol synthesis. PTV is used for hypercholesterolemia treatment. It lowers total cholesterol and low density lipoprotein (LDL) and therefore, it is used for prevention of cardiovascular diseases (CVDs).[2,3]

 

Literature search reveals following methods Visible spectrophotometric methods by using ferric chloride[4], acidic potassium permanganate[5], 7,7,8,8-tetracyanoquinodimethane (TCNQ)[6], sulfonephthaleins dyes[7,8] and bromate-bromide mixture[9], spectrofluorometric method[10], UV spectrophotometric method[11], chromatographic methods[12-16], indirect titrimetric assay[17] and electrochemical methods.[18,19]

 

Many of the reported methods would require expensive and sophisticated instruments which are inaccessible to many laboratories. In contrast, spectrophotometry is considered as the most convenient analytical technique in most quality control. Accordingly, in this research simple direct extractive-free spectrophotometric method was developed by using Bromocresol green (Figure 1,b) for the estimation of PTV in pure drug and in pharmaceutical preparations.

 

Fig. 1: Structural formula of a- Pitavastatin calcium, b- Bromocresol Green

 

Many studies have been done using Bromocresol Green for determining active pharmaceutical ingredients by complex formation[20-23].

 

MATERIALS AND METHODS:

Instruments:

All spectral measurements were carried out using a T80+ UV/V spectrophotometer instrument Ltd (UK) connected to computer, quartz cells 1cm, Ultrasonic bath (Powersonic, model 405, Korea), Adjustable micro pipettes 100 to 1000L (LABGILLS, Germany) and analytical balance (Sartorius, model 2474, Germany).

 

Chemical regents:

Chloroform Emsure (Merck, Germany), standard synthetic of Pitavastatin calcium (99.5%), Bromocresol green from (BDH, ENGLAND).

 

Pharmaceutical tablet preparations:

The listed commercial products were subjected to analytical procedure:

Londalop tablets, Ultramedica Pharmaceutical company, Damascus- Syria, labeled 2, 4mg/tab. of Pitavastatin (as calcium).

 

Lipid Free tablets, Ibn-Roshed pharmaceutical company. Aleppo- Syria, labeled 2, 4mg/tab. of Pitavastatin (as calcium).

 

Standard preparation:

Pitavastatin calcium stock solution:

Stock solution of 1.13 x 10-3 M of Pitavastatin calcium (MW= 880.98 g/mole) was prepared by dissolving 25.1 mg of pharmaceutically raw material (after taking purity in consideration) equivalent to 25mg Pitavastatin calcium in Chloroform using 25mL volumetric flask which is equivalent to 1000g/mL Then 1mL of the solution was taken to volumetric flask 10mL and diluted with Chloroform to give concentration 100g/mL.

 

Bromocresol Green stock solution:

Stock solution of reagent BCG was prepared with a concentration of 1 10-3 M (MW = 698.04g/mole) by dissolving suitable weight of the reagent and diluting to mark with chloroform in 25ml volumetric flasks.

All stock solutions are stable for a period of 3 days when refrigerated (4 - 8C).

 

Working standard solutions:

The working standard solutions of the raw material samples were prepared by appropriate dilutions among (100 - 1200)𝜇L of 100𝜇g/mL Pitavastatin calcium in 5 mL volumetric flasks that contain optimum volume of BCG 1 10-3 M (CBCG = 10 CPTV). Working standard solutions containing (2-24μg/ml) of Pitavastatin calcium.

 

Preparation of pharmaceutical sample solutions:

20 tablets were powdered and mixed accurately. An amount of average weight theoretically equivalent to labeled content 2.09mg or 4.18mg of Pitavastatin calcium was taken and dissolved in 25ml of Chloroform and sonicated for 20 minutes. Then sample was filtered by using Bchner funnel. After that 0.5mL of the filtered solution diluted by chloroform in 5mL volumetric flasks after adding the reagent, dilutions were made to obtain an appropriate concentration (among the range of linearity). We obtained theoretically concentrations of 8.36𝜇g/mL and 16.72𝜇g/mL respectively.

 

RESULTS AND DISCUSSION:

Absorption Spectra:

Pitavastatin calcium does not have absorption in the visible region. Also, BCG in Chloroform has negligible absorbance. In contrast, when a solution of BCG is mixed with PTV in chloroform, an intense yellow color is immediately produced which has a maximum absorbance at 416 nm. (Figure 2)

 

 

Fig 2: a-Spectrum of PTV: BCG complex in chloroform (16 μg/mL of Pitavastatin Calcium)

b- spectrum of BCG in chloroform

 

Effect of organic solvents:

According to PTV and BCG dye solubility, few solvents (Acetone, Chloroform, Dichloromethane) were studied to select the best one which exhibits both of high molar absorptivity coefficient and negligible blank absorption. As a result, chloroform was the preferred solvent to be used, as it shown in (Figure 3)

 

Fig. 3: Effect of solvent on the PTV-BCG ion-pair complex formation (16μg/mL of Pitavastatin Calcium)

 

Effect of temperature:

Studies showed that the temperature has a negligible effect on the formation of PTV-BCG complex in the range of (20-30C).

 

Effect of reaction time and stability:

It was found that the formation PTV-BCG complex was direct after the mixing of drug and reagent. The absorbance of the complex was stable for 24 hours.

 

Effect of dye concentration:

The impact of the BCG concentration on the intensity of the color developed at the selected wavelength 416nm was studied adding different amounts (0.25 4) mL of BCG 1 10-3 M equivalent to (25-400) μM to constant concentration of PTV 20μM and completed to 10mL by Chloroform. The absorbance at 416nm for every added of BCG reagent was measured against the blank of Chloroform. It was found that the completed colored complex formation was 2mL of BCG 1 10-3 M, equal to 200μM BCG, which equivalent to ten times of Pitavastatin Calcium concentration.

 

Stoichiometric Ratio:

Determination of the stoichiometric ratio of PTV-BCG complex formation was conducted by applying two methods; molar ratio and jobs continuous variation methods, both of them indicate that complex ratio is 1:2. (Figure 4)

 

Molar ratio method:

In this method the concentration of the reagent was constant 20μM and the drug concentrations were variable between the range (5 - 40) μM as it shown in (Figure 4, a)

 

Jobs continuous variation method:
In order to study the stoichiometric ratio of complex formation, drug and reagent stock solutions were prepared at the same concentrations 1 10-3 M and continuous variation volumes were taken from drug and reagent to have a resultant concentration of ([PTV]+ [BCG]= 40μM), as it shown in (Figure 4,b)

 
Fig. 4: a- Molar ratio plot, b- Jobs continuous variation plot PTV: BCG complex.

 


Mechanism of Reaction:

Anionic reagents like sulfonephthaleins dyes form ion pair complexes via electrostatic reaction between positively charged nitrogen of PTV and negatively charged sulfonic group of the BCG.[8,21] the dye is convert into an open quinoidal anionic derivative, which subsequently forms ion pair with PTV.

 

 

 

Method Validation:

The proposed method was validated for linearity, sensitivity, precision (reported as RSD %), accuracy (reported as recovery percentage and relative error), robustness and specificity according to International Conference on Harmonization guidelines (ICH)[24]

 

Linearity:

We studied the linearity of Pitavastatin calcium at the optimal reaction conditions where different standard concentrations (2 24) g/mL of PTV were prepared in a series of 5mL volumetric flasks, each one contains equivalent volumes of (BCG) reagent 1.13 x 10-3 M and (PTV) drug 1.13 x 10-4 M then the volume was made up to the mark with chloroform. The absorbance of yellow colored complex was measured at 416nm against the blank of (BCG), (Figures 5).

 

 
Fig. 5: Calibration curve of PTV-BCG complex.C1:2 g/mL, C2:4 g/mL, C3:8 g/mL, C4:10g/mL, C5:12g/mL, C6:14 g/mL, C7:16g/mL,C8:18 g/mL, C9:20g/mL, C10:24 g/mL
 

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

LOD and LOQ were calculated and presented in table 1 using the following equations:

 

LOQ = 10 SDand LOD = 3.3 S

m m

 

Where SD is the standard deviation of y-intercepts (a) of regression lines and (b) is the slope of the equitation of calibration curve, y = a + b x.

 

Table 1: Statistical data for calibration graphs.

Parameters

Value

λ max (nm)

416

Beers law limits (μg/mL)

2-24

Stability of the complex

24 hours

Temperature of solution

25 5 oC

Solvent

Chloroform

CBCG: CPTV, M

≥ 10

Molar absorptivity, ε (L/mol.cm)

61706

Sandells sensitivity SS (g/cm2)

0.014

Regression equation

Y = 0.076X - 0.034

Slope (b)

0.076

Intercept (C)

0.034

R2

0.9994

LOD (g/mL)

0.19

LOQ (g/mL)

0.59

 

Precision and Accuracy:

In order to assure that the developed method was precise and accurate, five replicate determinations were carried out on three different concentrations (10, 16, 20) μg/mL of standard PTV during of a period of three days. The analytical results were among the accepted range (98.70 - 101.85) % and RSD% was less than 2% summarized in table 2

 


Table 2: Accuracy and precision for PTV determination.

PTV taken 𝜇g/mL

Intraday Precision and Accuracy

Inter-day Precision and Accuracy

PTV found*

𝜇g/mL

Per %

SD 𝜇g/mL

RSD%

PTV found*

𝜇g/mL

Per %

SD 𝜇g/mL

RSD%

10

9.91

99.10

0.14

1.41

9.87

98.70

0.17

1.72

16

15.88

99.25

0.20

1.26

16.03

100.19

0.02

0.12

20

20.25

101.25

0.09

0.44

20.37

101.85

0.18

0.88

*n=5

 

Recovery:

The recovery was studied by three addition standards at levels (80% - 100% - 120%) for every product. Table 3 presents the recovery study results for the two Syrian trademark drugs.

 

Table 3: Recoveries study for accuracy estimation.

Recovery

Average %

RSD %

SD 𝜇g/mL

Recovery%

Total found*

g/mL

Added

g/mL

Sample

g/mL

Product

101.97

0.59

0.60

102.29

11.03

4.8

6.12

Londalop

2 mg

1.94

1.96

100.83

12.17

6.0

1.82

1.87

102.78

13.52

7.2

98.59

0.62

0.61

98.75

10.62

4.8

5.88

Lipid Free 2 mg

0.76

0.75

98 .00

11.76

6.0

0.35

0.34

99.03

13.01

7.2

98.59-101.97%

Range of Recovery

Average

Recovery% =(total found concentration sample concentration)/add concentration 100

* n=5

 


Robustness:

In order to demonstrate the robustness of the developed method, five replicates determinations were carried out on two different concentrations changing variable parameters of measurement (wavelength, scanning interval and scanning speed) the obtained results are showed in table 4:

 

Table 4: Robustness study results

Parameter

Change

PTV (μg/mL)

RSD %

Per %

λ max

(416nm)

+2 nm

10.03

0.20

100.30

12.10

0.62

100.83

-2 nm

10.03

0.29

100.30

12.13

0.19

101.08

Scanning Interval

1 nm

10.013

0.16

100.13

12.09

0.12

100.75

2 nm

10.01

0.12

100.10

12.09

0.14

100.80

Scanning Speed

Fast

10.05

0.55

100.50

12.10

0.16

100.83

Slow

10.07

0.31

100.70

12.11

0.90

100.92

Specificity:

To assure that the developed method was specific for PTV determination in pharmaceutical products without interfering the excipients in these formulations. The method was applied in Syrian products and the obtained results were compared with a validated reference method [11]. The results showed that no interference was exhibited by excipients. Table 5:

 

Application:

PTV estimation in Syrian products (Londalop 2, 4 mg/tab and Lipid Free 2, 4mg/tab):

The developed method was applied for quantitative determination of (PTV) in four Syrian tablets formulations. The samples were prepared as described in the section of samples preparation and analyzed. Quantitative analysis was done by using calibration curve. The results are showed in table 5:

 


Table 5: Estimation of (PTV) in pharmaceutical products

Product

Claim Amount (g/mL)

Average recovery % SD

t- test

F-test

 

Reference method[11]

Developed method

Londalop 2

8.36

104.68 0.55

10.3.9 0.70

1.96

1.63

Londalop 4

16.72

101.92 0.55

101.08 0.96

1.71

3.06

Lipid Free 2

8.36

96.78 0.59

96.09 0.66

1.40

2.48

Lipid Free 4

16.72

102.6 1.06

102.28 1.30

0.43

1.50

The value of 𝑡 (tabulated) at 95% confidence level and for four degrees of freedom is 2.78.

The value of 𝐹 (tabulated) at 95% confidence level and for four degrees of freedom is 6.39.

n=5

 


The dosages of (PTV) were conformed to JP legislation. [1] (the tablets must contain not less than 95.00 percent and not more than 105.00 percent of labelled amount).

 

CONCLUSION:

Pitavastatin calcium was estimated in Syrian pharmaceutical products Londalop 2 mg, 4 mg and Lipid Free 2 mg, 4 mg by simple spectrophotometric method based on forming yellow complex by BCG in chloroform. This complex exhibits maximum absorptivity at 416 nm.

 

The developed method of (PTV) determination is accurate, simple, sensitive, easy and directly applicable in quantitative analysis at room temperature with high molar absorbance (61706 L/mol.cm) in comparison to previous spectrophotometric methods without certain chemical treatment or using expensive solvents.

 

Recoveries study assures that the excipients in the studied pharmaceutical products didn't interfere into the analysis of the active pharmaceutical compound in tablets formulations. Therefore, the method is practical and valuable for routine application in quality control laboratories for the assay of (PTV).

 

ACKNOWLEDGEMENT:

The Ministry of High Education in Syria financially and technically supported this work through department of analytical and food Chemistry, Faculty of Pharmacy, University of Aleppo, Syria.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 04.12.2019 Modified on 11.02.2020

Accepted on 28.03.2020 RJPT All right reserved

Research J. Pharm. and Tech. 2020; 13(12):6206-6211.

DOI: 10.5958/0974-360X.2020.01082.3