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Chromatographia

, Volume 82, Issue 1, pp 279–285 | Cite as

Development and Validation of RP-LC Method for the Simultaneous Determination of Simvastatin and Ezetimibe in Fixed-Dose Combination Tablets and in Rabbit Serum

  • Sevinc Kurbanoglu
  • Ozgur Esim
  • Cansel Kose Ozkan
  • Ayhan Savaser
  • Yalcin Ozkan
  • Sibel A. OzkanEmail author
Original
  • 77 Downloads
Part of the following topical collections:
  1. 50th Anniversary Commemorative Issue

Abstract

Combined drug therapy which is based on the co-administration of two or more drugs, have been used for a long time, to treat diseases. In this study, a simple, selective and rapid RP-LC method has been developed and validated for the sensitive and simultaneous determination of simvastatin (SMV) and ezetimibe (EZE) in fixed-dose combination tablets and in rabbit serum using a simple sample preparation procedure. The developed RPLC method for these lipid-lowering agents was completely validated and in the linear range of 0.05–50 µg mL− 1 EZE and 0.05–10 µg mL− 1 SMV. The calibration curves were obtained with limit of detection values of 0.013 µg mL− 1, 0.009 µg mL− 1—for EZE and SMV, respectively. The developed method was successfully applied to the analysis of EZE and SMV in fixed-dose combination tablets and in rabbit serum, and no interference was observed from any excipients and endogenous substances in the rabbit serum samples. Dissolution profiles of the pharmaceutical dosage form of these lipid-lowering agents were also studied.

Keywords

Simvastatin Ezetimibe Rabbit plasma Pharmaceutical dosage form Drug dissolution 

Introduction

For a long time, to treat diseases and increase patient compliance, drug combinations have been used [1, 2]. Combined drug therapy is based on the co-administration of two or more carefully selected drugs [3, 4, 5]. The application of drugs with different mechanisms or modes of action together can also directly affect the single target or a disease and treat it more effectively. This can be achieved by combining agents that either interfere with distinctly different mechanisms or effectively block counter regulatory responses. By combining two drugs in partial or complete, additivity can occur related to the combination degree to which their pharmacologic effects are diverse and complimentary [6, 7]. For these therapeutic benefits, drug combinations have been widely used and became the leading choice for treating the most chronic diseases [8, 9]. Also, by reducing the number of drugs taken daily especially in chronic diseases, the risk of omitted or delayed doses can be decreased.

Ezetimibe, ((3R,4S)-1-(4-fluorophenyl)-3-[(3S)-3-(4-fluorophenyl)-3-hydroxylpropyl)]-4-(4-hydroxy phenyl)-2-azetidinone, EZE is a novel lipid-lowering agent. Lipid-lowering agents are hypolipidemics that specifically inhibit the absorption of cholesterol and reduces the blood cholesterol level [10, 11]. EZE also decreases the unnecessary accumulation of cholesterol in blood vessels [12]. It is now compounded with several doses of with simvastatin, [2,2-dimethyl-,1,2,3,7,8,8a-hexahydro-3,7-dimethyl-8-[2 (tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl)-ethyl]-1-naphthalenylester, SMV] a lactone that inhibits the enzyme HMG Co Enzyme A as a single pill preparation. EZE as monotherapy lowers LDL-C by 17–19% in adults who have hypercholesterolemia [13, 14]. However, the combination therapy of ezetimibe (10 mg) and simvastatin (10–80 mg) was shown to be superior in lowering the LDL cholesterol from 46 to 61% compared with a 31–46% reduction with the same doses of simvastatin alone [14, 15]. Combination therapy also offers fewer dose-dependent adverse experiences. Combination drug products of ezetimibe and simvastatin are hence marketed and used in the treatment of primary hypercholesterolemia [16].

Analytical methods have been developed to quantitatively measure the plasma levels and quality control analysis of each drug alone and its combinations and to define whether or not a given drug combination would gain a synergistic effect. Because drug combinations are remarkably complex to determine, there have been several methods and approaches on drug combination analysis in many articles [17, 18, 19]. Likewise, there are several methods for determination of simvastatin and ezetimibe based on different techniques for its determination from pharmaceuticals [20, 21, 22]. The increasing need for rapid analysis to use time efficiently in the pharmaceutical field is a request for the development of faster higher throughput analytical procedures. Determination of drugs both in biological matrices and in vitro drug development tests are a challenge, which is often driven by the need for rapid and sensitive results from samples [23].

For HPLC-based assays, the process allows the fast and instantaneous determination of many analytes in a single run. Besides, the high sensitivity of HPLC enables the use of low sample volumes and the determination of analytes in other matrices where drug concentrations can be low.

In the present study, it is aimed to rapid and sensitive simultaneous determination of SMV and EZE in their combined drug product and also in vivo biological samples. The major problem for simultaneous determination of combined dosage forms are degradation products of components. Thus, both drugs were applied to stress conditions and the separation of peaks was carefully identified. Hence, selectivity study can be realized. After obtaining degradation product response, the method was validated for all media. Dissolution test is one of the most important quality control parameters for biopharmaceutical point of view, the suitability of validated HPLC method was tested for dissolution of combined drug and release kinetics of both drugs were calculated. Also the suitability of validated method for in vivo was tested in rabbits after application of drugs orally.

Experimental

Chemicals

The chemicals that are used in the studies such as simvastatin, ezetimibe, hydrogen peroxide and ortho-phosphoric acid were obtained from Sigma-Aldrich (St. Louis, MO, USA). Methanol, acetonitrile, sodium hydroxide, and hydrochloric acid were gained from Merck. Pure Flex water system (ELGA, Wycombe, UK) was used to prepare all needed solutions. Unless otherwise it is stated, other chemicals and solvents were analytical reagent grade.

Instrumentation

The HPLC system consisted of Agilent Hewlett–Packard (Avondale, USA) Model 1100 series with UV detector. Thermo Scientific Benchtop pH meter (Orion 3 Star™ Plus, USA.) was utilized for pH measurements. Ultrasonic heating bath with Ultrasons J.P., S.A. (Barcelona) and NF 200 Centrifuge (NUVE, Turkey) were used for all preparations of standard and spiked rabbit serum samples.

Materials and Methods

Chromatographic Conditions

The chromatographic separations were performed using the mobile phase containing 0.1% H3PO4:methanol:acetonitrile (30:35:35) at pH 3.0 was used with 0.75 mL min− 1 flow rate. The chromatographic separations were performed at 30 °C using XTerra® C18 (250 mm × 3 mm ID × 5 µm, Waters, Milford, MA, USA) column. The samples were detected at 236 nm. The linearity of the detector response for ezetimibe and simvastatin was determined by plotting peak area ratios vs. concentration.

Preparation of Standard and Rabbit Serum Solutions

SMV and EZE stock solutions were prepared individually, by dissolving in methanol, followed by 10 min ultrasonication in ultrasonic bath. All the samples were arranged by diluting with mobile phase in their latter step of preparations. All stock solutions were kept at + 4 °C, saved from light and used within 24 h to avoid corruption. Sample solutions were prepared daily. 5 µg mL− 1 Indomethacin was used as internal standard in all experiments.

Rabbit serum samples were prepared using 2.5 mL of rabbit serum with 2.5 mL of ACN and required amount of SMV and EZE were added to prepare calibration curves. These solutions were centrifuged for 15 min with 3000 rpm. Supernatant part was diluted with mobile phase to related concentrations of SMV and EZE.

System Suitability Test Studies and Validation of the Analytical Method

According to USP criteria system suitability test parameters containing retention time, theoretical plate number, capacity factor, symmetry factor, selectivity, and RSD % of peak height or area for repetitive injections were reported. The analytical method refers to the performance of the analysis including creating of calibration curve, reference standard, evaluating accuracy, specificity and precision of the purposed method [24, 25, 26, 27]. Calibration curve was obtained by internal standard method where the area of the sample (SMV or EZE) was divided by the area of the internal standard (IS, 5 µg mL− 1 Indomethacin) and used as the y-axis of the graph. Moreover degradation studies were also performed in mild and hard conditions such as high temperatures (at 75 °C), alkaline and acidic hydrolysis and exposure to hydrogen peroxide, to assess the capability of the proposed method to separate EZE and SMV from their degradation products [28].

Analysis of Pharmaceutical Dosage Form

10 tablets of Inegy® containing excipient such as butylated hydroxyanisole, citric acid monohydrate, croscarmellose sodium, hypromellose, lactose monohydrate, magnesium stearate, microcrystalline cellulose, propyl gallate were weighed accurately, containing EZE and SMV. After that, the weighed tablets were moved to mortar. They were crushed and finely powdered to have mean value. From this powder, weight of one tablet is transferred into 100 mL flask, mixed with methanol and then filled to total volume with methanol.

Dissolution and Release Kinetics

Dissolution test of tablets were performed using potassium phosphate buffer (pH 6.8) as medium. Apparatus 2 (paddle) with a rotation speed of 75 rpm was chosen as test apparatus where these rotation speeds were fixed for each apparatus according to US Pharmacopeia (USP) 31 and the Food and Drug Administration (FDA) Guide. The 900 mL of dissolution medium was used and heated at 37 °C. The aliquots were withdrawn at 5, 10, 15, 20, 30, 45, and 60 min and same amount of fresh media was replaced. Further, drug stability in the dissolution medium for a period of 24 h and the influence of sample filtration were analyzed. KinetDS was used for the kinetic evaluation of the data obtained from dissolution studies [29].

In Vivo Experiments

New Zealand rabbits with a weight of 2.2 ± 0.5 kg were obtained from Gulhane Military Medical Academy Laboratory Animals Center (Ankara, TURKEY) (Gulhane Military Medical Academy Ethical Committee, Number: 2016/5). Rabbits were administrated simvastatin and ezetimibe orally. After 120 min from a single oral dose of 5 mg simvastatin and 5 mg ezetimibe administration, the blood samples were collected from the ear vein. Collected blood samples were centrifuged at 4000 rpm for 15 min. Plasma samples were collected and stored at − 40 °C until analysis.

Results and Discussions

Method Development

With the purpose of development an appropriate method for the simultaneous determination of EZE and SMV, HPLC parameters such as composition and pH of the mobile phase, temperature of the system, flow rate, of the system were optimized. Prior to the experiments, whole-spectrum analysis was performed and 236 nm was decided as finest for simultaneous analysis for EZE and SMV. Numerous diverse parameters such as mobile phases with different fraction of acetonitrile, methanol, in different pH were tried and strength values the mobile phase containing of ACN and phosphate [0.1% H3PO4:methanol:acetonitrile, (30:35:35) at pH 3.0] was found finest. System suitability test parameters were also reported in Table 1, indicating that the method is suitable for the simultaneous analysis of EZE and SMV.

Table 1

System suitability tests parameters

Compounds

Parameters

Recommended values

EZE

IND

SMV

Capacity factor

2.54

3.27

6.39

> 2

Selectivity to previous peak

1.29

1.96

> 1

Selectivity to next peak

1.29

1.96

> 1

Resolution to previous peak

7.48

2.96

9.50

> 2

Resolution to next peak

2.96

9.50

> 2

Tailing factor

1.10

1.11

1.05

< 2

Theoretical number of plates

3583

4381

5507

> 2000

Validation of the Method

Linearity

All the validation parameters were performed according to the guidelines [24, 25, 26, 27]. Using the optimized chromatographic method, compounds were determined in the linear range of 0.05–50 µg mL− 1 for EZE and 0.05–10 µg mL− 1 for SMV (Fig. 1 and figure SI. 1), with a correlation coefficient (r) of 0.999 for both compounds. LOD-LOQ values were calculated as 0.013–0.039 µg mL− 1 and 0.009–0.08 µg mL− 1for EZE and SMV, respectively. Other statistical parameters for the linearity results were reported in Table 2.

Fig. 1

Chromatograms of increasing concentrations of EZE and SMV in the presence of 5 µg mL− 1IS. a In mobile phase. b In Rabbit serum

Table 2

Statistical evaluation of the calibration data

Compounds

Mobile phase

Rabbit serum

EZE

SMV

EZE

SMV

Retention time(min)

2.25

4.69

2.25

4.69

Linearity range (µg mL− 1)

0.05–10

0.05–10

1.25–50

2.5–37.5

Slope (mAU µg− 1 mL)

0.115

0.184

0.024

0.038

Intercept (mAU)

2 × 10− 5

− 0.003

− 0.020

0.048

Correlation coefficient

0.9994

0.9992

0.9997

0.9990

LOD (µg mL− 1)

0.013

0.009

0.018

0.051

LOQ (µg mL− 1)

0.039

0.027

0.056

0.155

Within-day repeatabilitya (RSD %)

0.801

0.743

0.77

0.95

Between-day repeatabilitya (RSD %)

0.880

0.726

0.98

0.93

For spiked rabbit serum studies, the calibration curve was linear in the range of 1.25–50 µg mL− 1 for EZE and 2.5–37.5 µg mL− 1 for SMV, with a correlation coefficient (r) of 0.999 for both compounds. LOD–LOQ values were calculated as 0.018–0.056 µg mL− 1 and 0.051–0.155 µg mL− 1 for EZE and SMV in rabbit serum samples, respectively.

Precision Studies

For precision studies, within-day and between-days repeatabilities were performed to show how precise is the method and shortened in Table 2. Based on results of Table 2, there was no noteworthy difference among them for the evaluation of both standard solutions and spiked rabbit serum samples containing EZE and SMV.

Accuracy: Real Sample Applications and Recovery Studies

Inegy® tablets that contain EZE and SMV (15/20 mg) were analyzed with the proposed chromatographic method. Moreover, recovery analyses were also performed for both from rabbit serum and mobile phase as can be seen from Table 3. Reliable results were obtained which are in between 97 and 101% recovery levels.

Table 3

Results of recovery analysis

 

Mobile Phase

Rabbit Serum

 

EZE

SMV

EZE

SMV

Label claimed (mg)

15

20

Found

14.97

20.63

RSD (%)

0.36

0.81

Bias (%)

0.19

− 3.13

Added (mg)

5.00

5.00

5.00

5.00

Founda (mg)

5.01

5.00

4.90

4.97

Recovery (%)

100.30

100.02

98.08

99.43

RSD of recovery (%)

0.21

0.40

0.58

0.75

Bias (%)

− 0.30

− 0.02

1.92

0.57

Selectivity and Specificity: Forced Degradation Studies

The specificity of the method was utilized with hard and mild stress conditions for EZE and SMV. Acidic and alkaline hydrolysis, oxidation by hydrogen peroxide and heated-in-oven degradations studies were used. Using mild and hard conditions, at different times of the degradation were given in Table 4 as details. Peak purity test results were consequential for both SMV and EZE responses and it is observed that responses were successfully resolved from degradation products with the developed method.

Table 4

Results of stress conditions by RP-LC in terms of % degradation

Conditions

EZE

SMV

Retention time (min) of undefined degradation products

EZE

SMV

Mild conditions

    

 HCl (0.5 M)

10.78

59.81

1.12, 1.53, 1.73, 2.51

2.29, 2.46

 NaOH (0.5 M)

11.69

44.52

1.89

1.89, 3.75

 H2O2 (3%)

25.52

39.78

1.125, 1.95, 3.45

1.05, 3.69, 5.20

 UV light exposure (3 h at 254 nm)

29.84

4.15

1.85

2.44, 3.87

 Oven (3 h at 75 °C)

41.03

6.55

1.33

1.45, 5.20

Hard conditions

    

 HCl (1 M)

21.75

69.84

1.12, 1.53, 1.73, 2.51

1.05, 3.69, 5.20

 NaOH (1 M)

19.18

46.52

1.45, 1.79

1.11, 3.01

 H2O2 (30%)

25.40

76.38

1.11

1.11, 5.01

 UV light exposure (24 h at 254 nm)

48.24

16.35

1.85

1.91

 Oven (24 h at 75 °C)

43.80

64.95

1.33

3.84

Dissolution and Release Kinetics

The dissolution profile of EZE and SMV combination tablet was shown in Fig. 2. EZE showed slower dissolution behavior in dissolution media. The dissolution profile was almost the same for the two active substances. Although simvastatin and ezetimibe are fundamentally non-ionized over the physiological pH range, high rotation speed led the formulation to a higher dissolution rate and no precipitation, with almost complete dissolution of the dose within the time-frame of the experiment Taupitz et al. [30].

Fig. 2

Dissolution profiles of “filled circle” EZE and “filled square” SMV tablets, Inegy®

First-order, Hixson–Crowell cube root plot, Higuchi’s square root plot and Weibull models were investigated to comprehend the mechanism of ezetimibe and simvastatin release from the tablets. All calculated release kinetics, their rate constants and statistical parameters after fitting the cumulative release data are presented in Table 5. After fitting the release data to these kinetic models, best agreements were found with Weibull kinetic model according to the highest determination coefficient and lowest residual mean square values for samples [31].

Table 5

Kinetic parameters of the model equations applied to the dissolution data of EZE, SMV tablets

Kinetic model

Rate constant (unit)

Statistics

EZE

SMV

First-order \({Q_t}={\text{ln}}{Q_0}\,+\,{{\text{k}}_{\text{1}}}t\)

k 1

 

0.234

0.233

\({r^2}\)

0.9106

0.7801

RMS

292.085

400.385

Weibull \({Q_t}={Q_0}(1 - {\text{exp}}[ - ({(t - {t_l})^ \wedge }A)/kW])\)

k W

 

9.09

7.41

A

 

1.10

1.11

\({r^2}\)

0.9384

0.8797

RMS

82.826

103.002

Higuchi \({Q_{t~}}=~{k_{\text{H}}}\surd t\)

k H

 

2.35

2.49

\({r^2}\)

0.7508

0.6344

RMS

329.907

323.434

Hixson Crowell \({Q_t}_{~}=~{[{k_{{\text{HC}}}}({\text{t}}~ - ~{t_{\text{l}}})\,+\,{Q_0}^{ \wedge }\left( {{\text{1}}/{\text{3}}} \right)]^ \wedge }3\)

k HC

 

0.0456

0.0434

\({r^2}\)

0.8244

0.6784

RMS

6747.876

10517.003

\({Q_t}\) is the amount of drug released in time t; Q0 is the initial amount of drug in the solution; k1 release rate constant of first-order kinetic; kw release rate constant of Weibull kinetic: kH release rate constant of Higuchi kinetic; kHC release rate constant of Hixson–Crowell equation; r2 determination coefficient; RMS residual mean square

In Vivo Experiments

The RP-HPLC method presented acceptable results for the instantaneous determination of EZE and SMV in rabbit plasma following an oral administration in rabbits. The plasma recovery analysis for EZE and SMV are presented in Table 3. The results can be providing essential information for reviewing the drug interaction in vivo and the therapeutic monitoring of EZE and SMV which is important for a novel EZE and SMV formulation development and safe remedy for clinical practice.

Conclusion

For the binary mixtures of EZE and SMV, the proposed method was successfully applied to the pharmaceutical dosage forms, and rabbit serum samples without any interference by the excipients of dosage form and endogenous substances from biological materials. In addition to this, the fully validated proposed method can be used for the determination of binary mixture of EZE and SMV dosage form for the monitoring their concentration for in vitro dissolution studies. Using the optimized chromatographic method, compounds were determined in the linear range of 0.05–50 µg mL− 1 for EZE and 0.05–10 µg mL− 1 for SMV. LOD-LOQ values were calculated as 0.013–0.039 µg mL− 1 and 0.009–0.08 µg mL− 1 for EZE and SMV, respectively. For spiked rabbit serum studies, the calibration curve was linear in the range of 1.25–50 µg mL− 1 for EZE and 2.5–37.5 µg mL− 1 for SMV, with a correlation coefficient (r) of 0.998 for both compounds. LOD–LOQ values were calculated as 0.018–0.056 µg mL− 1 and 0.051–0.155 µg mL− 1 for EZE and SMV in rabbit serum samples, respectively.

The obtained results can provide important data to examine the in vivo drug interaction and therapeutic monitoring of binary mixtures of EZE and SMV and to develop a novel combined EZE and SMV formulation for a more effective and safe solution for clinical application.

Notes

Acknowledgements

This paper is dedicated to Chromatographia 50th Anniversary commemorative issue.

Compliance with Ethical Standards

Conflict of Interest

All authors declare that he or she has no conflict of interest.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Supplementary material

10337_2018_3642_MOESM1_ESM.pdf (51 kb)
Figure SI. 1. Calibration graphs for EZE and SMV in A-C mobile phase and B-D rabbit serum (PDF 51 KB)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Analytical Chemistry, Faculty of PharmacyAnkara UniversityAnkaraTurkey
  2. 2.Department of Pharmaceutical Technology, Gulhane Faculty of PharmacyUniversity of Health SciencesAnkaraTurkey

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