Rapid and selective determination of pitavastatin calcium in presence of its degradation products and co-formulated drug by first-derivative micelle-enhanced and synchronous fluorimetric methods

Abstract

New spectrofluorimetric methods are presented for the rapid and selective determination of pitavastatin calcium (PIT) in the presence of its hydrolytic degradation products and the co-formulated drug ezetimibe (EZE). In the first method (method A), PIT was determined in the presence of its hydrolytic degradation products by measuring the first derivative (¹D) of its enhanced native fluorescence using sodium dodecyl sulfate (SDS) as an anionic micelle enhancer at a pH of 4.1, which was adjusted using Britton–Robinson buffer in aqueous media. PIT was subjected to stress conditions of 5 M hydrochloric acid and 5 M sodium hydroxide. The ¹D peak amplitude was measured at a λ_em of 415 nm using a λ_ex of 252 nm. The effect of different surfactants on the fluorescence of PIT was studied; the quantum yield of PIT was in the following order: SDS (0.30) > cetrimide (0.23) > Tween (0.02). A second method (method B) was developed for the simultaneous determination of PIT and the co-formulated drug EZE in the presence of degradation products of PIT. This method utilized synchronous fluorescence spectroscopy (SFS) in an acetonitrile medium. The wavelengths that were used were λ_ex = 260 nm and λ_em = 330 and 272 nm for PIT and EZE, respectively, using Δλ = 40 nm. Validation of the proposed methods was performed according to the ICH guidelines. The methods were employed for the determination of PIT (methods A and B) and EZE (method B) in bulk powder, pharmaceutical preparations of co-formulated drug substances and laboratory-prepared mixtures containing hydrolytic degradation products of PIT. Statistical analysis proved that the investigated assays were excellent. It was concluded that method A was inexpensive and environmentally friendly.

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1. Introduction

Pitavastatin calcium (PIT) is chemically designated as (3R,5S,6E)-7-[2-cyclopropyl-4-(p-fluorophenyl)-3-quinolyl]-3,5-dihydroxy-6-heptenoic acid calcium salt (Fig. 1). It is a novel fully synthetic statin, which has more potent cholesterol-lowering activity than other drugs in its class. PIT is an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. It is used in the treatment of hyperlipidemia and can reduce the risk of cardiovascular diseases in everyday medical practice.^1–3 On the basis of preclinical findings, PIT has been widely used as a first-line agent in lipid-modifying therapies.⁴

Fig. 1 Chemical structures of the investigated drugs.

Ezetimibe (EZE) has the systematic name 1-(4-fluorophenyl)-3-(3-(4-fluorophenyl)-3-hydroxypropyl)-4-(4-hydroxyphenyl)-azetidin-2-one (Fig. 1). It lowers levels of cholesterol by preventing its absorption from dietary and biliary sources via blocking the transport of cholesterol through the intestinal wall.^2,5 A previous study demonstrated that the co-administration of statins such as atorvastatin or PIT with EZE has an additive effect. The concomitant use of PIT and EZE, according to a European patent, has a remarkable blood cholesterol level lowering effect in comparison to that of the concomitant use of another HMG-CoA reductase inhibitor and EZE.^6,7

A literature survey revealed that few methods have been used for the simultaneous determination of PIT and EZE. These include thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC).^8,9 Several analytical methods such as spectrophotometry,^10,11 spectrofluorometry,¹² HPTLC,^13,14 HPLC,^15–20 stability-indicating HPLC,²¹ ultra-performance liquid chromatography (UPLC),²² LC-MS/MS^23,24 and LC-ESI-MS^25,26 have been reported for the determination of PIT in pharmaceutical dosage forms and/or biological samples.

There is no reported spectrofluorometric method for the determination of PIT in the presence of its degradation products or EZE. The aim of this study is to develop selective, sensitive, cheap and eco-friendly spectrofluorometric methods for the determination of the abovementioned drugs.

The first proposed method is a first-derivative micelle-enhanced spectrofluorometric process. This method was developed for the determination of PIT in the presence of its acid and base hydrolytic degradation products. Derivative spectrofluorimetry has been used to resolve spectral interference in emissions to enable the simultaneous determination of components of a mixture.^27–29 Derivatization of overlapping curves will enable the appearance of a zero-crossing point, where one of the curves gives a zero reading while the other has a pronounced value, which allows it to be determined.

In the second suggested method, advantage is taken of the ability of synchronous fluorescence spectroscopy (SFS) to increase the selectivity and resolution of interfering spectra. An SFS method is presented for the simultaneous determination of both PIT and EZE in the presence of degradation products of PIT. Upon employing SFS, shifting and narrowing of the obtained emission spectra of components of a mixture occurs, which enables spectral resolution. These effects arise from permitting simultaneous emission and excitation scanning to take place with a difference in the values of λ used for scanning.^30,31

2. Experimental

2.1. Instrumentation

A Cary Eclipse fluorescence spectrofluorometer (Agilent Technologies) was used, which was operated with Cary Eclipse Scan Application software version 1.2 (147).

2.2. Materials and reagents

2.2.1. Pure samples. Pitavastatin calcium was kindly supplied by Mash Pharmaceuticals (B. N.: EL-03/L038/M/15001). Ezetimibe was kindly obtained from EVA Pharm Company (B. N.: AXBHH006645). Their purity was found to be 99.27 ± 1.49% and 99.48 ± 0.97%, respectively, in accordance with the manufacturer's methods.^32,33

2.2.2. Market samples. Livazo® tablets, which were labeled as containing 2 mg PIT per tablet, were manufactured by Algorithm S. A. L., Zouk Mosbeh, Lebanon, under license from Kowa Pharmaceuticals Europe Co. Ltd, UK (B. N.: C099). Choletimb® tablets, which were labeled as containing 10 mg EZE per tablet, were manufactured by Marcyrl Pharmaceutical Industries, Cairo, Egypt (B. N.: 1532471). Both formulations were obtained on the local market.

2.2.3. Solvents and reagents. All solvents used were of HPLC grade and chemicals were of analytical grade: acetonitrile, methanol (Macron Fine Chemicals, Poland), isopropanol (Sigma Aldrich, Germany), ethanol absolute (Fisher Scientific, UK), acetone (Alfa Chemicals, Egypt), sodium dodecyl sulfate (Adwic Co., Egypt, 0.4% w/v aq. soln), cetyltrimethylammonium bromide (Chadwell Heath, Essex, England, 0.4% w/v aq. soln), Tween 20 (MP Biomedicals, Inc., France, 0.4% w/v aq. soln), acetic acid (Adwic Co., Egypt, 0.04 M aq. soln), phosphoric acid (Adwic Co., Egypt, 0.04 M aq. soln), boric acid (Adwic Co., Egypt, 0.04 M aq. soln) and sodium hydroxide (Loba chemicals, India, 0.2 M aq. soln).

2.2.4. Buffer solutions. Britton–Robinson buffer (BRB) (pH 3.6 to 8.1) was prepared by mixing equal volumes of both 0.04 M phosphoric acid and 0.04 M boric acid, then adjustment of the pH was carried out with 0.2 M sodium hydroxide.³⁴

2.3. Solutions

2.3.1. Stock standard solutions (100.00 μg mL⁻¹). A stock standard solution of PIT was prepared by dissolving 10.00 mg PIT in 100 mL acetonitrile : water (50 : 50, v/v), whereas a stock standard solution of EZE was prepared by dissolving 10.00 mg EZE in 100 mL acetonitrile.

2.3.2. Working standard solutions (10.00 μg mL⁻¹). Two working standard solutions of PIT were prepared by diluting 5 mL of its stock standard solution to 50 mL either by using a solvent mixture of acetonitrile : water (50 : 50, v/v) or by using acetonitrile as the diluting solvent for methods A and B, respectively. On the other hand, a working standard solution of EZE was prepared by diluting 5 mL of its stock standard solution to 50 mL with acetonitrile.

2.3.3. Solutions of acid and base hydrolytic degradation products.
2.3.3.1 Stock solutions (1.00 mg mL⁻¹). Stock solutions of hydrolytic degradation products of PIT were separately prepared by heating an accurately weighed amount of 50.00 mg PIT with 50 mL of either 5 M hydrochloric acid or 5 M sodium hydroxide for 6 h at 100 °C. The solutions were neutralized and evaporated on a water bath. The residues were separately dissolved and diluted to 50 mL with acetonitrile : water (50 : 50, v/v).
2.3.3.2 Working standard solutions (10.00 and 100.00 μg mL⁻¹). Two working solutions were prepared for both acid and base degradation products. The first solutions were obtained by diluting 1 mL of the stock solutions of the degradation products to 100 mL to achieve a final concentration of 10.00 μg mL⁻¹. The second working solutions were prepared by diluting 5 mL of each of the stock solutions of the degradation products to 50 mL to achieve a final concentration of 100.00 μg mL⁻¹. Dilutions were performed using a solvent mixture of acetonitrile : water (50 : 50, v/v).

2.4. Procedures

2.4.1. Method A: first-derivative (¹D) micelle-enhanced fluorescence method. Aliquots equivalent to 10.00 μg of each of PIT and its acid and base hydrolytic degradation products were accurately and separately transferred from their respective working solutions into 10 mL volumetric flasks. To each solution, 1 mL BRB buffer (pH 4.1) and 1 mL 400 μg mL⁻¹ SDS were added and the volumes were made up with water. The emission spectra of all of the prepared solutions were recorded in the range of 220–600 nm using a λ_ex of 252 nm against a blank solution, which was prepared similarly. The ¹D curves of the scanned spectra were computed using Δλ = 4 nm and a scaling factor of 10. The peak amplitude of the ¹D curves of PIT was measured at 392 nm.

2.4.2. Method B: synchronous fluorescence spectroscopy method (SFS). Aliquots equivalent to 10.00 μg of each of PIT, EZE, and the working solutions of the hydrolytic degradation products of PIT were accurately and separately transferred into 10 mL volumetric flasks and the volumes were made up with acetonitrile. The instrument was adjusted to synchronous mode using λ_ex = 260 nm for both PIT and EZE and Δλ = 40 nm, then emission spectra were recorded within the range of 220–600 nm against acetonitrile as a blank. The fluorescence intensities were measured at λ_em = 330 and 272 nm for PIT and EZE, respectively.

2.5. Method validation³⁵

2.5.1. Linearity.
2.5.1.1 Method A. Accurately measured aliquots equivalent to 0.10–10.00 μg mL⁻¹ PIT were transferred into a series of 10 mL volumetric flasks and then 1 mL BRB buffer (pH 4.1) and 1 mL 0.04% SDS were added. The volumes were made up with water to achieve a final concentration range of 0.01–1.00 μg mL⁻¹, and the procedure mentioned under method A was followed. The calibration graph was constructed by relating the peak amplitudes at 392 nm to the corresponding concentrations of PIT and the regression equation was computed.
2.5.1.2 Method B. Accurately measured aliquots equivalent to 2.50–30.00 μg mL⁻¹ PIT and 2.00–50.00 μg mL⁻¹ EZE were transferred into a series of 10 mL volumetric flasks. The volumes were made up with acetonitrile to obtain standard solutions in the concentration ranges of 0.25–3.00 μg mL⁻¹ for PIT and 0.20–5.00 μg mL⁻¹ for EZE, and then the procedure mentioned under method B was followed. The fluorescence intensities for each compound were plotted against the corresponding concentrations and the regression equations were computed.

2.5.2. Precision. For the evaluation of precision, the intra-day precision was determined by assaying freshly prepared solutions in triplicate with concentrations of PIT of 0.30, 0.50, and 0.90 μg mL⁻¹ and 0.40, 0.70, and 1.40 μg mL⁻¹ for methods A and B, respectively, and concentrations of EZE of 1.50, 2.00, and 2.50 μg mL⁻¹ for method B. The inter-day precision was calculated by assaying freshly prepared solutions in triplicate for three days and the RSD was calculated.

2.5.3. Selectivity.
2.5.3.1 Laboratory-prepared mixture with acid degradation products.
Method A. Aliquots equivalent to 0.80–0.99 μg mL⁻¹ PIT were separately transferred from its stock standard solution into a series of 10 mL volumetric flasks followed by aliquots equivalent to 0.20–0.01 μg mL⁻¹ of the acid degradation products. Then, 1 mL BRB buffer (pH 4.1) and 1 mL 0.04% SDS were added and the volumes were made up with water.
Method B. Aliquots equivalent to 2.10–2.97 μg mL⁻¹ of the binary mixture (3 μg mL⁻¹ PIT; 3 μg mL⁻¹ EZE) were transferred from the stock solutions into a series of 10 mL volumetric flasks followed by aliquots equivalent to 0.90–0.03 μg mL⁻¹ of the acid degradation products (1–30%). The volumes were made up with acetonitrile.

The procedure described under Linearity was then followed. The concentrations of the intact drugs were calculated from the corresponding regression equations.

2.5.3.2 Laboratory-prepared mixture with alkaline degradation products.
Method A. Aliquots equivalent to 0.80–0.99 μg mL⁻¹ PIT were separately transferred from its stock solution into a series of 10 mL volumetric flasks followed by aliquots equivalent to 0.20–0.01 μg mL⁻¹ of the base degradation products.
Method B. Aliquots equivalent to 2.10–2.97 μg mL⁻¹ of the binary mixture (3 μg mL⁻¹ PIT; 3 μg mL⁻¹ EZE) were transferred from the stock solutions into a series of 10 mL volumetric flasks followed by aliquots equivalent to 0.90–0.03 μg mL⁻¹ of the acid degradation products (1–30%). The volumes were made up with acetonitrile.

The procedure described under Linearity was then followed. The concentrations of intact drugs were calculated from the corresponding regression equations.

Synthetic mixtures of the binary mixture were prepared with different ratios (1 : 1, 1 : 2, and 1 : 5) of PIT and EZE, respectively. The procedure described under Linearity was followed. The concentration of each drug was calculated from the corresponding regression equations.

2.6. Application to pharmaceutical formulations

2.6.1. Method A. Ten tablets of Livazo® (2 mg) were accurately weighed and finely powdered. An accurately weighed amount of each powdered tablet equivalent to 5 mg PIT was transferred into a 50 mL volumetric flask. Then, 35 mL methanol was added and the solution was sonicated for 15 min and then left to cool to room temperature. The volumes were made up with methanol (100 μg mL⁻¹). The solution was then filtered through 0.45 μm filter paper and then 5 mL of the solution was evaporated, dissolved and made up to 50 mL with acetonitrile : water (50 : 50, v/v) to reach a final concentration of 10 μg mL⁻¹. Then, the procedure described under method A was followed.

2.6.2. Method B. Ten tablets of Livazo® containing 2 mg PIT and 10 tablets of Choletimb® containing 10 mg EZE were accurately weighed and finely powdered. An accurately weighed amount of each powdered tablet equivalent to 2 mg PIT or 10 mg EZE was accurately transferred and mixed into a 100 mL volumetric flask. Then, 35 mL methanol was added and the solutions were sonicated for 15 min and then left to cool to room temperature. The volumes were made up with methanol and the solutions were then filtered through 0.45 μm filter paper. Then, 5 mL of the solution was evaporated, dissolved and made up to 50 mL with acetonitrile in a 50 mL flask to reach concentrations of 2 μg mL⁻¹ PIT and 10 μg mL⁻¹ EZE. Then, the procedure described under method B was followed.

3. Results and discussion

Two rapid, sensitive and selective spectrofluorimetric stability-indicating methods were developed, optimized and validated for the determination of PIT in the presence of its hydrolytic degradation products (acid and alkaline) and the co-formulated drug EZE.

PIT is characterized by displaying native fluorescence owing to its fused aromatic rings and extended conjugated structure (Fig. 1). The excitation and emission spectra of PIT are shown in Fig. 2, which shows a λ_em of 418 nm after excitation at a λ_ex of 252 nm in water. Moreover, it is liable to degradation under both acidic and alkaline conditions. The acid and alkaline degradation products that were formed exhibited detectable fluorescence at the emission maximum of the drug.

Fig. 2 Excitation (-----) and emission (——) spectra of native fluorescence of pitavastatin calcium (0.8 μg mL⁻¹) in 1 mL BRB (pH 4.1), 1 mL SDS and water as solvent at a λ_em of 418 nm and a λ_ex of 252 nm.

The first stability-indicating method was based on measuring the amplitude of the first derivative (¹D) at a λ_em of 293 nm of the micelle-enhanced native fluorescence of PIT. The ¹D spectra of the hydrolytic degradation products display zero-crossing at the abovementioned value of λ_em. Forced degradation studies of PIT were carried out using 5 M hydrochloric acid and 5 M sodium hydroxide. Complete degradation was confirmed by HPLC. To increase the sensitivity of the method, advantage was taken of the enhancement of fluorescence by the addition of a surfactant at a concentration above its critical micellar concentration. This has resulted in an increase in the fluorescence quantum yield of a fluorophore in many cases.³⁶ This phenomenon has long been studied. It is suggested that in an organized micellar assembly enhancement of media occurs owing to the isolation and protection of the analyte from quencher molecules by micelles, as well as changes in the physical properties of the environment around it, e.g., increases in viscosity, polarity, etc. The enhancement depends on the type of micelle, the structure of the substrate and the interaction between them.^37,38 The fluorescence properties of PIT in various micellar media were studied. There was a fourfold increase in fluorescence intensity in the presence of SDS in comparison with that in aqueous solution. PIT has a pK_a of 4.1² and in an acidic medium it will be protonated; this would allow interaction to take place with the anionic surfactant SDS. Fig. 3 shows the excitation and emission spectra of PIT in water and upon adding 1 mL BRB buffer (pH 4.1) and 1 mL 0.04% SDS. The spectra of its acid and alkaline degradation products displayed considerable interference. The first derivative of the micelle-enhanced fluorescence spectrum was computed to cancel the interference of the acid and alkaline degradation products of PIT. Zero-crossing was observed at a λ_em of 293 nm and therefore this was selected as the wavelength for analysis (Fig. 4).

Fig. 3 Emission spectra of native fluorescence of pitavastatin calcium in media enhanced by sodium dodecyl sulfate micelles at a pH of 4.1: BRB and water as diluting solvent (——), pitavastatin calcium in water (-----), acid degradation products (……), alkaline degradation products (-.-.-.-.-) and blank reagent (_.. _) at a λ_em of 418 nm and a λ_ex of 252 nm (1 μg mL⁻¹).

Fig. 4 First-derivative spectra of: (A) pitavastatin calcium () and its acid () and alkaline () degradation products at a λ_em of 392 nm and a λ_ex of 252 nm (1.0 μg mL⁻¹). (B) Different concentrations of pitavastatin calcium (0.01–1.00 μg mL⁻¹) at a λ_em of 392 nm and a λ_ex of 252 nm. Inset: calibration curve correlating the peak amplitudes of the first-derivative fluorescence intensities of pitavastatin calcium determined using method A with the corresponding concentrations (0.01–1.00 μg mL⁻¹).

In the second method, a synchronous fluorescence spectroscopy technique (SFS) was employed for the simultaneous determination of both PIT and EZE in co-formulated preparations and in the presence of acid and alkaline degradation products of PIT. SFS plays an important role in the simultaneous determination of compounds in multicomponent mixtures owing to its remarkable advantages of spectral simplification, reduction in light scattering, and improvement in selectivity over that of conventional fluorescence spectroscopy.^39–41 Taking advantage of the spectral resolution achieved by the shifting and narrowing of the emission spectra upon adding SFS and increasing the sensitivity by the enhancement of fluorescence using organized micellar media has enabled the analysis of multicomponent mixtures of polycyclic aromatic hydrocarbons.^42,43

An emphasis is made in this work on the development of a rapid and simple synchronous fluorescence-based method for the determination of a binary mixture of PIT and EZE in the presence of hydrolytic degradation products of PIT. Fig. 5 shows the normal emission spectrum of a binary mixture of PIT and EZE in acetonitrile. Both PIT and EZE exhibit native fluorescence with λ_maximum for PIT and EZE occurring at values of λ_em of 373 nm and 310 nm, respectively, after excitation of both at 260 nm, where no complete separation was observed (Fig. 5). Therefore, the SFS technique was performed, which resulted in complete spectral resolution and enabled the simultaneous determination of both PIT and EZE in their drug formulations and pharmaceutical preparations, as well as in the presence of degradation products of PIT. Fig. 6 shows the emission spectra of a mixture of PIT and EZE obtained by employing SFS with the optimum chosen parameters, in which complete resolution of the spectra of PIT and EZE was observed.

Fig. 5 Intensity of the native fluorescence spectrum of a mixture of pitavastatin calcium and ezetimibe at values of λ_em of 373 nm and 310 nm, respectively, after excitation of both at 260 nm in acetonitrile (3 μg mL⁻¹ each).

Fig. 6 Intensity of the synchronous fluorescence spectra of a synthetic mixture of pitavastatin calcium, ezetimibe and 30% of the degradation products of pitavastatin (acid and alkaline ) at Δλ = 40 nm at values of λ_em of 330 nm and 272 nm () for both drugs, respectively, in acetonitrile (3 μg mL⁻¹ each).

3.1. Optimization of experimental conditions

Several factors were found to affect the fluorescence. These factors were studied and optimized; for example, the effect of different diluting solvents including water, methanol, ethanol, acetonitrile, isopropanol and acetone was studied. It was observed that by using water and acetonitrile, the strongest fluorescence was obtained for PIT and EZE, respectively. However, upon using acetonitrile : water (50 : 50, v/v) and employing SFS (method B), a considerable shift in the synchronous spectrum of EZE to longer wavelengths occurred, which caused interference with the spectrum of PIT; therefore, acetonitrile alone was used as the diluting solvent.

Different model surfactant systems were tested, whereby the maximum fluorescence signals were obtained using SDS as an anionic surfactant, whereas little enhancement was observed upon using Tween 80 as an example of a non-ionic surfactant. The cationic surfactant cetrimide caused a considerable enhancement of fluorescence, but this was still less than that for SDS. The quantum yields [QY]^44,45 upon adding different surfactants were calculated and the order of the quantum yields for the surfactants was SDS > cetrimide > Tween (Table 1).

Table 1 Quantum yield of pitavastatin calcium with different surfactants using quinine sulphate as a standard in 0.05 M H₂SO₄, as calculated according to ref. 44

Surfactant	Abs	Integrated emission	QY^a
a QY = Ys(Fu/Fs)(As/Au), QY = quantum yield, As = absorbance of standard, Au = absorbance of unknown, Ys = quantum yield of standard, Fu = integrated emission of unknown, Fs = integrated emission of standard.
Quinine sulphate (standard)	0.018	30 480	—
SDS	0.012	24 383	0.30
Cetrimide	0.014	16 597	0.23
Tween	0.014	12 490	0.02

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For testing the effect of pH, a universal buffer (Britton–Robinson buffer) with various pH values ranging from 3.1 to 8.1 was used. The optimum pH was found to be 4.1. The influence of pH values above 4.1 caused a gradual decrease in FI. As the fluorescence intensity for protonated species is always higher than that for neutral species, it can be inferred that the protonated form of a species interacts more strongly with anionic micelles of SDS than the neutral form of the drug. The influences of the concentration and volume of SDS on the FI were studied using different volumes of 0.04% SDS (w/v). It was found that an increase in the volume of the SDS solution resulted in a corresponding increase in FI up to 1 mL, after which no further increase was observed. Therefore, 1 mL SDS was chosen as the optimum volume for PIT. The synchronous parameters investigated for method B included the scanning range and the effect of Δλ on SFS. The synchronous wavelength range of 220–600 nm was scanned while changing Δλ. The effect of different values of Δλ on the resolution of the emission spectra of PIT and EZE from Δλ = 10 nm to Δλ = 70 nm is shown in Fig. 7, in which the optimum value of Δλ was found to be 40 nm with λ_em values of 330 nm and 272 nm for PIT and EZE, respectively, using a λ_ex of 260 nm. Fig. 8 and 9 show the intensity of synchronous fluorescence for different concentrations of PIT in the presence of EZE and vice versa.

Fig. 7 Intensity of synchronous fluorescence at different Δλ values of 10–70 nm (), with the best spectrum at Δλ = 40 nm (), of a synthetic mixture of pitavastatin calcium and ezetimibe (each at 2 μg mL⁻¹) at λ_em values of 330 nm and 272 nm, respectively, in acetonitrile.

Fig. 8 Intensity of synchronous fluorescence for different concentrations of pitavastatin calcium (0.25–3.00 μg mL⁻¹) and ezetimibe (3.00 μg mL⁻¹) at Δλ = 40 nm and λ_em = 330 nm and 272 nm for both drugs, respectively, in acetonitrile. Inset: calibration curve correlating the intensities of the synchronous fluorescence of pitavastatin calcium with the corresponding concentrations.

Fig. 9 Intensity of synchronous fluorescence for different concentrations of ezetimibe (0.20–5.00 μg mL⁻¹) and pitavastatin calcium (3.00 μg mL⁻¹) at Δλ = 40 nm and λ_em = 272 nm and 330 nm for both drugs, respectively, in acetonitrile. Inset: calibration curve correlating the intensities of the synchronous fluorescence of ezetimibe to the corresponding concentrations.

3.2. Method validation

Calibration curves were constructed, which represented the relationship between the fluorescence intensities and the corresponding concentrations in the ranges of 0.01–1.00 μg mL⁻¹ and 0.25–3.00 μg mL⁻¹ for PIT using methods A and B, respectively, whereas the range was 0.20–5.00 μg mL⁻¹ for EZE using method B (Fig. 8 and 9). The characteristic parameters for the regression equations were computed. The LOD and LOQ were calculated and the results are presented in Table 2. The developed methods were found to be accurate for the determination of PIT, where values of the recovery percentage ± RSD of 100.25 ± 1.026% and 100.22 ± 1.091% for methods A and B, respectively, were obtained, as shown in Table 2.

Table 2 Results of validation of assay obtained by employing the proposed first-derivative micelle-enhanced fluorescence and synchronous fluorescence methods for the determination of pitavastatin calcium (PIT) and ezetimibe (EZE) in drug formulations

Parameter	Method A	Method B
	PIT	PIT	EZE
*Mean ± SD, n = 5.a is the intercept,b is the slope.
Linearity range (μg mL^−1)	0.01–1.00	0.25–3.00	0.20–5.00
LOD (μg mL^−1)	0.0022	0.047	0.049
LOQ (μg mL^−1)	0.0075	0.155	0.165
Accuracy* (mean ± SD)	99.68 ± 0.828%	99.73 ± 1.548%	100.12 ± 1.366%
Slope^b	254.699	93.110	97.411
SE of slope	2.146	0.824	1.211
Confidence limit of the slope	249.623 to 259.774	90.993 to 95.227	94.448 to 100.373
Intercept^a	21.719	9.337	36.234
SE of intercept	1.064	1.487	3.199
Confidence limit of intercept	19.202 to 24.236	5.515 to 13.159	28.407 to 44.060
Correlation coefficient (r)	0.9994	0.9996	0.9991
SE of estimation	2.237	2.068	5.707

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The repeatability and intermediate precision of the proposed methods were determined for three concentration levels (0.30, 0.50, and 0.90 μg mL⁻¹ and 0.40, 0.70, and 1.40 μg mL⁻¹ for methods A and B, respectively, for PIT and 1.50, 2.00, and 2.50 μg mL⁻¹ for EZE) by assaying three times on the same day and in triplicate on three successive days using the developed methods (n = 9). The results in Table 3 indicate the satisfactory precision of the proposed methods. For testing the selectivity of the methods, different laboratory-prepared mixtures containing different percentages of the studied drugs and their different degradation products were prepared and analyzed by the proposed methods. The methods were found to be selective, as indicated by the results in Table 4. The presence of 1–20% and 1–30% of hydrolytic degradation products of PIT, respectively, did not affect the recovery of the pure drug substances by both methods. Moreover, a set of laboratory-prepared mixtures of the two investigated drugs in variable proportions were analyzed to determine the specificity of the suggested methods (Table 4).

Table 3 Repeatability and intermediate precision of the proposed first-derivative micelle-enhanced fluorescence and synchronous fluorescence methods for the determination of pitavastatin calcium (PIT) and ezetimibe (EZE) in drug formulations

Method A
Taken (μg mL^−1)	Intra-day		Inter-day
	Found^a (μg mL^−1) ± RSD	Precision (RSD%)	Found^a (μg mL^−1) ± SD	Precision (RSD%)
a Mean of three determinations.
0.3	0.29 ± 0.577	0.196	0.30 ± 1.041	0.340
0.5	0.50 ± 1.232	0.246	0.49 ± 1.528	0.310
0.9	0.90 ± 1.528	0.169	0.89 ± 0.171	0.190

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Method B
PIT					EZE
Taken (μg mL^−1)	Intra-day		Inter-day		Taken (μg mL^−1)	Intra-day		Inter-day
	Found^a (μg mL^−1) ± SD	Precision (RSD%)	Found^a (μg mL^−1) ± SD	Precision (RSD%)		Found^a (μg mL^−1) ± SD	Precision (RSD%)	Found^a (μg mL^−1) ± SD	Precision (RSD%)
0.40	0.40 ± 0.001	0.25	0.41 ± 0.004	0.86	1.50	1.51 ± 0.020	1.32	1.51 ± 0.013	0.84
0.70	0.74 ± 0.006	0.91	0.70 ± 0.007	0.96	2.00	2.03 ± 0.036	1.78	2.03 ± 0.025	1.24
1.40	1.41 ± 0.012	0.86	1.41 ± 0.022	1.56	2.50	2.50 ± 0.047	1.26	2.50 ± 0.006	0.23

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Table 4 Specificity of the proposed first-derivative micelle-enhanced fluorescence and synchronous fluorescence methods for the determination of pitavastatin calcium (PIT) in the presence of its degradation products and ezetimibe (EZE) in laboratory-prepared mixtures

Method A
Acid degradation products% (w/w)	Recovery%^a of intact drug	Alkaline degradation products% (w/w)	Recovery%^a of intact drug
a Mean of three determinations.
1	100.55	1	100.95
5	98.23	5	100.51
10	99.54	10	100.38
20	98.81	20	98.54
Mean ± RSD%	99.28 ± 1.008	Mean ± RSD%	100.38 ± 1.061

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Method B
Acid degradation products% (w/w)	Recovery%^a of intact drugs		Alkaline degradation products% (w/w)	Recovery%^a of intact drugs
	PIT	EZE		PIT	EZE
1	99.47	99.50	1	98.82	100.50
5	99.98	100.80	5	100.33	98.90
10	99.53	98.53	10	99.17	99.62
20	98.87	99.74	20	98.99	101.20
30	99.95	101.45	30	99.92	100.80
Mean ± RSD%	99.58 ± 0.52	100.04 ± 1.14	Mean ± RSD%	99.45 ± 0.65	100.20 ± 0.93

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Synthetic mixture (μg mL^−1)		Recovery%^a
PIT	EZE	PIT	EZE
1	1	99.58	98.54
1	2	99.46	99.72
1	5	98.89	101.22
Mean ± RSD		99.31 ± 0.37	99.83 ± 1.35

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The accuracy of the developed spectrofluorimetric methods was also determined by spiking previously analyzed drug products with extra concentrations of PIT and EZE. Satisfactory recoveries were obtained indicating no interference from tablets' excipients. The results are presented in Table 5.

Table 5 Results of assay and application of standard addition technique for the determination of pitavastatin calcium (PIT) by the proposed first-derivative micelle-enhanced fluorescence and synchronous fluorescence methods for the simultaneous determination of a binary mixture of PIT and ezetimibe (EZE) in drug products

Method A
Drug product	Recovery^a of claimed amount ± RSD%	Amount taken (μg mL^−1)	Standard added (μg mL^−1)	Recovery%^b of standard added
a Mean ± RSD of five determinations.b Mean of three determinations.
Livazo 2 mg PIT per tablet	92.83 ± 1.402	0.20	0.05	101.05
			0.10	99.63
			0.20	100.26
			0.60	98.49
			0.80	101.21
			Mean ± RSD%^a	100.13 ± 1.11

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Method B
Drug product	Recovery^a of claimed amount ± RSD%	Amount taken (μg mL^−1)	Standard added (PIT) (μg mL^−1)	Recovery%^b of standard added	Standard added (EZE) (μg mL^−1)	Recovery%^b of standard added
Livazo 2 mg PIT per tablet	94.33 ± 1.381	0.40 PIT and 2.00 EZE	0.40	101.046	2.00	99.44
Choletimb 10 mg EZE per tablet	91.43 ± 0.906		0.50	99.64	2.50	99.55
			0.60	101.21	3.00	101.6
			Mean ± RSD%	100.63 ± 0.858	Mean ± RSD%	100.19 ± 1.214

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The results obtained by employing the proposed methods for the analysis of the studied compounds in bulk powders and pharmaceutical preparations were statistically compared with those obtained using the manufacturer's methods. The calculated values of t and F were less than the theoretical values, which revealed that there was no significant difference with respect to accuracy and precision,⁴⁶ as indicated in Table 6.

Table 6 Statistical comparisons between the results obtained by employing the proposed first-derivative micelle-enhanced fluorescence and synchronous fluorescence spectroscopy and the manufacturer's methods for the determination of pitavastatin calcium (PIT) and ezetimibe (EZE) in drug formulations

Parameter	Drug formulations
	Method A		Method B
	Proposed method	Manufacturer's^a method	Proposed method		Manufacturer's method
			PIT	EZE	PIT^a	EZE^b
a Manufacturer's HPLC method, using Intersil C18 (5 μm, 250 × 4.6 mm) as the column, phosphate buffer (pH 3.5), acetonitrile in the ratio of 50 : 50 v/v as the mobile phase with a flow rate of 1.5 mL min^−1, an injection volume of 20 μL and detection at 245 nm.b Manufacturer's HPLC method, using Intersil C18 (5 μm, 250 × 4.6 mm) as the column, phosphate buffer (pH 4.5), acetonitrile in the ratio of 65 : 35 v/v as the mobile phase with a flow rate of 1 mL min^−1, an injection volume of 20 μL and detection at 240 nm.c The values in parentheses are the theoretical values of t and F.
Mean	99.68	99.27	99.73	100.12	99.27	99.48
SD	0.828	1.490	1.548	1.366	1.490	0.971
N	5	5	5	5	5	5
Variance	0.686	2.220	2.396	1.866	2.220	0.943
SE	0.372	0.666	0.692	0.611	0.666	0.434
t value (2.306)^c	0.538	—	0.479	1.121	—	—
F Value (6.400)^c	3.236	—	1.079	0.96	—	—

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The proposed spectrofluorimetric methods have various advantages over published chromatographic methods that are employed to determine PIT and EZE in pharmaceutical preparations, being less tedious and less time-consuming. Moreover, they are stability-indicating methods and can determine PIT in the presence of its degradation products either alone or in a binary mixture with EZE.

4. Conclusion

Two proposed spectrofluorimetric methods are presented. The first is a stability-indicating method based on the determination of pitavastatin calcium in the presence of its acid and alkaline hydrolytic degradation products using first-derivative micelle-enhanced spectrofluorimetry. The method is environmentally friendly and cost-saving as a result of using inexpensive and less toxic reagents. The second is also a stability-indicating method for the determination of pitavastatin calcium and is, moreover, used for the simultaneous determination of pitavastatin calcium and ezetimibe, a co-formulated drug, using synchronous spectrofluorimetry. The proposed methods are characterized by being simple, sensitive, specific and having a shorter analysis time in comparison to other methods, so they can be employed for routine analysis in quality control laboratories.

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