INTRODUCTION:

Pitavastatin [Figure 1] is (E)-7-[2-cyclopropyl-4-(4-fluorophenyl)qui-nolin-3-yl]-3,5-dihydroxy-hept-6-enoic acid. Pitavastatin (usually as a calcium salt) is a novel member of the medication class of statins. Like the other statins, it is an inhibitor of HMG-CoA reductase, the enzyme that catalyses the first step of cholesterol synthesis. It has been available in Japan since 2003, and is being marketed under license in South Korea and in India. It is likely that pitavastatin will be approved for use in hypercholesterolemia [1-2].

Figure 1. Chemical structure of Pitavastatin

Literature search revealed that methods like Review article on Analytical techniques for estimation of pitavastatin[3], validated stability indicating UPLC method [4], Simple LC–MS/MS methods for simultaneous determination of pitavastatin and its lactone metabolite in human plasma and urine [5], Determination of pitavastatin in human plasma via HPLC-ESI-MS/MS [6], Simultaneous determination of pitavastatin Ca and ezetimibe by liquid chromatography [7], HPLC determination of pitavastatin calcium in pharmaceutical dosage forms [8-9], Novel spectrophotometric method for the assay of pitavastatin calcium in pharmaceutical formulations [10], Quantification of pitavastatin calcium in Pharmaceutical Dosage Forms by HPTLC [11-13].However, these HPTLC method showing broad peak with complicated mobile phase . In the current work, we have developed and validated stability indicating HPTLC method for estimation of Pitavastatin Ca as per ICH guidelines [14-16] showing narrow peak with simplest mobile phase composition.

EXPERIMENTAL:

Chemicals and Materials

A request sample of Pitavastatin was received from Mylan Laboratories Ltd., Hyderabad. Methanol AR, toluene (AR) grade was purchased from Merck India limited. Pitavastatin (PIVASTA 4mg, zydus cadila) were procured from local market.

Instrumentation

HPTLC instrument consists of CAMAG Linomat-IV TLC applicator, CAMAG TLC-Scanner-III (version 1.4.1) supported with CAMAG Wincats^® software (version 1.4.1), CAMAG twin trough glass chamber (10 cm x 10 cm) for TLC development and Hamilton Syringe (100 μL). Chromatographic separation of the drugs was performed on Merck HPTLC plates precoated with silica gel 60F₂₅₄ (10 cm × 10 cm with 200mm layer thickness, catalogue no 1055480001, E. Merck, Germany). An electronic balance (ACCULAB Model ALC-210.4 Huntington valley, PA) a sonicator (EN 30 US, Entertech Fast-clean, Mumbai, India).

Preparation of standard solution

To prepare standard solution, accurately weighed 100 mg of Pitavastatin was transferred in to 100 mL volumetric flask, dissolved and diluted up to the mark with methanol to obtain stock solution containing 1.0 mg/mL of the drug.

Preparation of sample solution

The sample solution was prepared by accurately weighing twenty tablets containing Pitavastatin and finely powdered. Accurately weighed amount of powder equivalent to 100 mg of Pitavastatin was transferred to 100 mL volumetric flask and 50 mL of methanol was added. The mixture was sonicated for 30 min, diluted to mark with methanol and filtered through whattman filter paper no.41 from the stock solution 1.0 mL was accurately transferred to 10 mL volumetric flask and diluted with methanol to obtain a solution of 100 μg/mL of Pitavastatin, to applied on HPTLC plate.

Chromatographic Conditions

Toluene-methanol mixture in the ratio of 8:2 v/v was optimized for thin- layer chromatography plate development. The chamber was saturated with the mobile phase at room temperature for 15min. The run distance was kept at ~70mm and 10 mL of the mobile phase was used for a single development. The dosing speed of nitrogen applicator was kept 150 nL s with a pre-dosage volume of 5mL. Sample were applied as bands of 5mm width with gaps of 3mm in between. The developed plates were dried at room temperature for 5 min. detection was done at 245 nm using a deuterium lamp in absorption/reflectance mode. The slit dimension of the detector was kept at 4mm×0.45mm.

Forced Degradation Study of Pitavastatin

The degradation samples were prepared by transferring 10 mg of pitavastatin in to a 10mL volumetric flask and diluted to the mark with 0.1N HCL, 0.1N NaOH, water and 3% H₂O₂ for acidic, basic, neutral and oxidative degradation studies, respectively. Hydrolytic reactions were carried out in 0.1M HCL, 0.1M NaOH, water and 3% H2O2 at RT for 30 min respectively. Pure solid drug (1 mm thick layer in a dish) was exposed to dry heat at 60°C in an oven for 6h. Pure solid drug (1mm thick layer in a Petri dish) was kept in sunlight for 6 h. The samples were taken out and examined by HPTLC after suitable dilution.

METHOD VALIDATION:

The method was validated for the parameters listed below as per ICH guidelines

Linearity

Different concentrations of pitavastatin (50-400 ng/band) were applied on to the HPTLC plate and the peak areas were measured in the densitometer. The calibration curve was constructed by plotting the peak area versus concentration, and the regression equation was constructed. Each response was an average of five determinations.

Precision

Interday and intraday precision were evaluated by determining three corresponding responses, three times on same day and on three different days for pitavastatin and the results were reported in the terms of %RSD. Repeatability was carried out by applying a homogeneous sample of pitavastatin tablet powder solution (100µg/mL) five times on to the TLC plate and result were reported in terms of %RSD.

Accuracy

Accuracy was determined by calculating the recovery of pitavastatin by the standard addition at different levels (80, 100 and 120%) of the labelled claim. A known amounts of a standard of pitavastatin were added to preanalyzed sample of tablet powder. Each solution was applied in triplicate and recovery was calculated by measuring the peak areas and fitting these values in to the regression equation of the calibration curve.

Limit of Detection and Limit of Quantitation

The limit of detection (LOD) and limit of quantitation (LOQ) of the drug were calculated using following equations as per ICH guideline.

LOD=3.3(σ/s), LOQ=10 (σ/s)

Where σ is the standard deviation of the response and S is the slope of the calibration curve.

Specificity

It is performed using the typical constituted placebo, blank solvent used for the analysis of active pharmaceutical ingredient (API), and placebo degraded under same conditions as applied for API.

Robustness

It was performed by total amount of mobile phase, and chamber saturation time.

RESULT AND DISCUSSION:

Optimization of the mobile phase

HPTLC was performed on aluminium-backed Silica Gel 60 GF₂₅₄ HPTLC plate from Merck, India. Sample solution was applied as 5mm band and 3mm spacing at a constant application rate of 4 sec μl^-1 by means of Linomat IV sample applicator fitted with a 100-μl syringe. The Linear ascending development was performed in a CAMAG twin trough glass chamber with mobile phase consisting of toluene: methanol (8:2 v/v). The optimum time for saturation of the chamber with mobile phase vapour was 15 min at ambient temperature (25 ± 2 °C) and relative humidity 60 ± 5%. The plates were allowed to develop at distance of 7.0 cm. Subsequent to development, the plates were dried in current of dry air by using compact hot air dryer. Plates were evaluated by densitometry at 245 nm by means of a CAMAG TLC scanner III with Wincat software version 1.4.1 Figure 2. displays the corresponding chromatogram of standard Pitavastatin in optimized HPTLC condition. The retardation factor of pitavastatin was about 0.50 ±0.02.

Figure 2. Densitogram of Pitavastatin standard (500ng/band)

Validation of the method

The response for the drug was found to be linear in the concentration range 50-400 ng/band for pitavastatin with correlation coefficient of 0.997. The regression equation obtained was y=535.887+16.64*x, where y is the peak area and x is the concentration (ng/band). The %RSD values were 1.45-1.70% and 1.57-1.81% for intraday and interday precision, respectively and the repeatability was found to be 1.26%, which confirms that the method is precise. The recovery was in the range 99.27-101.87%, indicating the method accuracy. The LOD and LOQ were found to be 1.05 and 3.21ng/band, respectively. The specificity of the method was ascertained by absence of any other peak of the placebo, degradation products of the placebo, and the solvent. The method was found to be robust.

The summary of the validation parameters is given in Table 1.

Table 1. Validation parameters for determination of Pitavastatin by HPTLC methods.

Sr.No.	Validation Parameters	Pitavastatin
1	Regression equation Correlation coefficient	Y= 535.887+16.64*x 0.997
2	Linearity	50-400 ng/band
3	Precision
	Intraday	1.45-1.70%RSD
	Interday	1.57-1.81%RSD
4	Recovery	99.27-101.87%
5	Limit of Detection	1.05ng/band
6	Limit of Quantitation	3.21ng/band
7	Specificity	Specific
8	Robustness	Robust

SD = standard deviation, RSD = relative standard deviation, y = concentration of Pitavastatin in µg/band, x = peak area of Pitavastatin

Degradation Studies

Forced degradation studies of pitavastatin were carried out under various stress conditions and the resultant chromatogram is shown in Figures 3. The percentage degradation was calculated, which is listed in Table 2.

Figure 3. Densitogram showing mixed degradation behavior of Pitavastatin

Deg1-thermal condition, deg2-acidic condition, deg3-basic condition, deg4-sunlight condition

1mL of each degraded sample mixed and applied on the TLC plate showing above densitogram.

Table 2. Degradation behaviour

Stress Condition	% Degradation
Acid 0.1N HCl (RT for30min)	15%
Basic 0.1N NaOH (RT 30min)	8%
Oxide 3%H₂O₂(RT30min)	Negligible
Neutral H₂O (RT 30min)	Negligible
Photostability (Sunlight 6h)	20%
Thermal (60°C,6h)	5%

Showing % degradation as compared with area of standard

Analysis of Formulation

In PIVASTA tablet the mean content of Pitavastatin was found to be 99.12% with a % RSD of 1.58%, which indicated that the method is suitable for routine analysis of Pitavastatin in its formulation.

CONCLUSION:

A new analytical method has been developed to determine Pitavastatin in a pharmaceutical dosage form. In this study, stability of Pitavastatin was established according to ICH-recommended stress condition. The developed method was proven to be linear, precise, accurate and specific. There was no interference from any degradation products or excipients in the determination of Pitavastatin.

ACKNOWLEDGMENT:

Authors acknowledge the Mylan Laboratories Ltd, Hyderabad for gift sample of Pitavastatin.

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Received on 06.02.2018 Accepted on 05.03.2018

Asian J. Pharm. Ana. 2018; 8(1): 49-52.

DOI: 10.5958/2231-5675.2018.00009.1