Simultaneous Estimation of Simvastatin and Ezetimibe in Bulk Drug and Tablet Formulation by High-Performance Liquid Chromatography and High-Performance Thin-Layer Chromatography

 

Chhalotiya UK*, Bhatt KK1 and Captain AD2

*1Indukaka Ipcowala College of Pharmacy, New Vallabh Vidhyanagar- 388121, Anand, Gujarat, India.

2A.R. College of Pharmacy and G. H. Patel Institute of Pharmacy, Vallabh Vidhyanagar- 388120, Anand, Gujarat, India.

*Corresponding Author E-mail:  usmangani84@gmail.com

 

ABSTRACT

Background and the purpose of the study: A suitable reverse phase high-performance liquid chromatography (RP-HPLC) and high-performance liquid thin layer chromatography (HPTLC) method for determination of Simvastatin (SIM) and Ezetimibe (EZE)   in bulk drug and tablet formulation. The present study describes a simple, rapid, sensitive, reliable, and economic RP-HPLC and HPTLC methods for determination of simvastatin and ezetimibe in bulk drug and tablet formulation which is more feasible than reported RP-HPLC and HPTLC assays.

Methods: The RP-HPLC separation was achieved on a Hypersil C-18 column (250 mm X 4.6 mm id, 5mm particle size) using acetonitrile– Water (80: 20, v/v) mobile phase at a flow rate of 1.0 mL/min at ambient temperature. The HPTLC separation was achieved on an aluminum-backed layer of silica gel 60F254 using hexane: toluene: ethyl acetate: acetic acid (3:1:6:0.2, v/v/v/v/v) mobile phase. Quantitation was achieved with UV detection at 235 nm over the concentration range 4–24 mg/mL for both drugs, with mean recoveries of 100.81 ± 0.28 and 100.27 ± 0.66% for SIM and EZE, respectively, using the HPLC method. Quantitation was achieved with UV detection at 235 nm over the concentration range of 1600–4800 ng/spot for both drugs, with mean recoveries of 100.34 ± 0.55 and 100.54 ± 0.83% for SIM and EZE, respectively, using the HPTLC method.

Conclusion: Proposed study describes new RP- HPLC and HPTLC methods for the estimation of simvastatin and ezetimibe in bulk drug and tablet formulation.  The method was validated and found to be simple, sensitive, accurate and precise.  Percentage of recovery shows that the method is free from interference of the excipients used in the formulation.  Therefore the proposed method can be used for routine analysis for estimation of simvastatin and ezetimibe in its bulk drug and pharmaceutical tablet formulation.

 

KEYWORDS: Validation; RP – HPLC; HPTLC; Simvastatin; Ezetimibe.

 

 


INTRODUCTION:

Simvastatin (SIM) and Ezetimibe (EZE) are Antihyperlipidemic drugs. Simvastatin is HMG-Co-A Reductase Inhibitor. Chemically it is 2, 2-Dimethyl butyric acid, 8-ester with (4R, 6R)-6-[2-[(1S, 2S, 6R, 8S, 8aR)-1, 2, 6, 7, 8a-hexahydro-4hydroxy-2, 6-dimethyl-1-naphthyl] ethyl] tetrahydro-4hydroxy-2H-pyran-2-one. It is used for the treatment of hyperlipidemia. It may be used alone or in combination with other antihyperlipidemic agents.  Ezetimibe is inhibiting the absorption of cholesterol in small intestine1. It is used as an anti-hyperlipidemic agent. Chemically it is 1-(4-fluorophenyl) - 3®-[3-(4-fluorophenyl)-3(S)-hydroxypropyl]-4(S)-(4-hydroxyphenyl)-2-azetidinone2–4.

 

The combination of SIM and EZE has been shown to be indicated as adjunctive therapy to diet for the reduction of elevated Total-Cholesterol, LDL-Cholesterol, and Apo B in patients with primary (heterozygous familial and non-familial) hypercholesterolemia5.

 

A literature survey revealed that different analytical methods involving for the determination of SIM include column high-performance liquid chromatography (HPLC), liquid chromatography-mass spectroscopy (LCMS) and Second derivative UV spectrometric6-9 in pharmaceutical preparation have been developed. Literature report concerning methods involving for the determination of EZE include HPLC, high-performance thin-layer chromatography (HPTLC) in pharmaceutical dosage forms10-13 as well as  simultaneous spectrophotometric determination, spectrofluorometric, LCMS and stability indicating assay14-16 have been published. Because of the absence of an official pharmacopoeial method for the simultaneous determination of SIM and EZE in pharmaceutical formulations, efforts were made to develop an analytical method for the estimation of SIM and EZE in their combined dosage form using HPLC and HPTLC methods.

 

Experimental:

Apparatus:

A Series 200 HPLC system (PerkinElmer, Shelton, CT) equipped with a Series 200 diode array detector, Series 200 quaternary gradient pump, Series 200 column oven, manual injector Rheodyne valve) with 20 µL fixed loop, Turbochrom navigator software (Version 6.1.1.0.0:K20), and Hypersil  C18 column (150mm× 4.6mmid, 5 µm particle size) was used. For HPTLC, a LinomatVautosprayer, TLC Scanner III with winCATS-4 software, twin-trough flat-bottom TLC developing chambers, and viewing cabinet with UV lamps (Camag, Muttenz, Switzerland) were used. HPTLC plates used were 10 × 10 cm silica gel with an indicator fluorescing at 254 nm, layer thickness 0.2 mm, aluminum-backing (E. Merck KGaA, Darmstadt, Germany).

 

Reagents and Materials:

SIM and EZE pure powder were procured as gratis samples from Comed chemicals Limited (Baroda, India) and Zydus cadila healthcare Limited (Ahmedabad, India). HPLC grade acetonitrile, methanol, and water were purchased from E. Merck (Mumbai, India). Ethyl acetate, hexane, toluene, and glacial acetic acid were procured from SD fine Chemical Ltd. (Ahmedabad, India) and were of analytical grade. Membrane filters (nylon 0.45 µm, 47 mm) were purchased from Gelman Laboratory (Mumbai, India). Tablets containing SIM (10 mg) and EZE (10 mg) of 1 brand, USV Pharmaceutical Ltd. (Mumbai, India) were purchased from the local market.

 

Chromatographic Conditions:

HPLC method:

The Hypersil C18 column (18) was used at ambient temperature. The mobile phase consisted of acetonitrile– water (80:20, v/v) and the flow rate was maintained at 1 mL/min. The mobile phase was passed through nylon 0.45 µm membrane filter and degassed before use. The elution was monitored at 235 nm, and the injection volume was 20 µL.

 

HPTLC method:

Solutions of SIM and EZE were applied to silica gel 60F254 HPTLC plates (10 × 10 cm) by means of a Linomat V automatic spotter equipped with a 100 µL syringe and operated with settings of band length, 6 mm; distance between bands, 5 mm; distance from the plate edge, 10 mm; and distance from the bottom of the plate, 10 mm. The plate was developed in a twin-trough chamber previously saturated for 30 min with the mobile phase hexane: toluene: ethyl acetate: acetic acid (3:1:6:0.2, v/v/v/v/v), for a distance of 8 cm. The spots on the air-dried plate were scanned with the Scanner III at 235 nm using the deuterium source.

Preparation of SIM and EZE Mixed Standard Stock Solutions:

For both the HPLC and HPTLC methods, a stock solution was prepared by weighing SIM (10 mg) and EZE (10 mg). Weighed powder of both drugs was accurately transferred to the same 100 mL volumetric flask and dissolved in, and then diluted to the mark with, methanol to obtain a mixed standard stock solution of SIM (100 µg/mL) and EZE (100 µg/mL).

 

Preparation of Sample Solutions:

Twenty tablets were weighed and finely powdered. A mass equivalent to 10 mg of both SIM and EZE was weighed and transferred in a 50 mL volumetric flask, and methanol (20 mL) was added. The solution was sonicated for 15 min, and the final volume was diluted to the mark with methanol to obtain a solution containing 200 µg/mL each of SIM and EZE. An aliquot of this solution (0.6 mL) was further diluted to 10 mL with methanol to obtain a solution containing 12 µg/mL each of SIM and EZE. The solution was filtered through a nylon 0.45 µm membrane filter.

 

Method Validation:

Calibration:

Calibration graph (linearity of the HPLC method):

Calibration graphs were constructed by plotting peak areas vs concentrations of SIM and EZE, and the regression equations were calculated. The calibration graphs were plotted over 6 different concentrations in the range of 4–24 µg/mL for both drugs. Accurately measured mixed standard solution aliquots of SIM and EZE (0.4, 0.8, 1.2, 1.6, 2.0, and 2.4 mL) were transferred to a series of 10 mL volumetric flasks and diluted to the mark with mobile phase. Aliquots (20 µL) of each solution were injected under the operating chromatographic condition described above [number of replicates (n = 6)].

 

Calibration graph (linearity of the HPTLC method):

Calibration graphs were plotted over the concentration range of 1600–4800 ng/spot for both the drugs. Accurately prepared mixed standard solutions of SIM and EZE (16.0, 24.0, 32.0, 40.0, and 48.0 µL) were applied to the plate. The calibration graphs were developed by plotting peak area vs concentrations (n = 5) with the help of the winCATS software.

 

Accuracy (recovery):

The accuracy of the methods was determined by calculating recoveries of SIM and EZE by the standard addition method. Known amounts of mixed standard solution of SIM and EZE (6.0, 12.0, and 18.0 µg/mL for the HPLC method and 800, 1600, and 2400 ng/spot for the HPTLC method) were added to prequantitated sample solutions of tablet dosage forms. The amounts of SIM and EZE were estimated by applying values of peak area to the regression equations of the calibration graph.

 

Method precision (repeatability):

The precision of the instruments was checked by repeatedly injecting (n = 6) mixed standard solutions of SIM and EZE (12 µg/mL) for the HPLC method. Repeatability of HPTLC instruments was assessed by applying the same sample solution 6 times on a plate with the automatic spotter using the same syringe and by taking 6 scans of the sample spot for both SIM and EZE (3200 ng/spot) without changing the position of the plate.

 

Intermediate precision (reproducibility):

The intraday and interday precisions of the proposed methods were determined by analyzing mixed standard solution of SIM and EZE at 3 different concentrations (8.0, 12.0, and 16.0 µg/mL for the HPLC method and 2400, 3200, and 4000 ng/spot for the HPTLC method) 3 times on the same day and on 3 different days. The results are reported in terms of relative standard deviation (RSD).

 

Limit of detection (LOD) and limit of quantitation (LOQ):

The LOD with signal-to-noise (S/N) ratio of 3:1 and the LOQ with S/N ratio of 10:1 were calculated for both drugs using the following equations according to International Conference on Harmonization guidelines17:

LOD = 3.3 × σ /S

LOQ = 10 × σ /S

Where, σ  = The standard deviation (SD) of the response and S = The SD of the y-intercept of the regression line.

 

Specificity:

The excipients hydroxypropyl cellulose, mannitol, microcrystalline cellulose, polyethylene glycol 6000, and lactose monohydrate (Signet Ltd., Mumbai, India), and Methocel E5 Premium LV EP (Colorcon Asia Pvt. Ltd., Goa, India) were spiked into a reweighed quantity of drugs to assess the specificity of the methods. The peak area was measured to determine the quantity of the drugs.

 

Robustness:

Robustness of the methods was studied by changing the composition and the pH of mobile phase and determining the stability of the drugs in methanol for 24 h at ambient temperature. Spot stability was observed by performing 2-dimensional HPTLC development using the same mobile phase.

 

Analysis of SIM and EZE in Tablet Dosage Forms:

The responses of sample solutions were measured at 235 nm for quantitation of SIM and EZE by using the HPLC and HPTLC methods as described above. The amounts of SIM and EZE present in sample solution were determined by applying values of peak area to the regression equations of the calibration graph.

 

RESULTS AND DISCUSSION:

HPLC Method:

To optimize the HPLC parameters, several mobile phase compositions were tried. A satisfactory separation of SIM and EZE with good peak symmetry and steady baseline was obtained with the mobile phase acetonitrile–water (80: 20, v/v). Quantitation was achieved with UV detection at 235 nm based on peak area. Complete resolution of the peaks with clear baseline separation was obtained (Figure 1). The system suitability test parameters are shown in Table 1.

 

Figure 1: High-performance liquid chromatogram of SIM and EZE and corresponding retention times with detection at 235 nm

 

Table 1: System suitability test parameters for SIM and EZE for the proposed HPLC method

Parameter

SIM ± RSDa

  (nb = 6)

EZE ± RSDa

(nb = 6)

Retention time, min

9.43 ± 0.004

4.14 ± 0.001

Tailing factor

1.40 ± 0.01

1.67 ± 0.03

Resolution

---------

2.59 ± 0.05

Theoretical plate No.

2082.88 ± 0.30

3645.6 ± 0.65

a RSD = Relative standard deviation, %.

b n = Number of determinations.

 

 

HPTLC Method:

Several mobile phases were tried to accomplish good separation of SIM and EZE. Using the mobile phase , hexane: toluene: ethyl acetate: acetic acid (3:1:6:0.2, v/v/v/v/v) and 10 × 10 cm HPTLC silica gel 60F254 aluminum-backed plates, good separation was attained with retardation factor (Rf) values of 0.56 for SIM and 0.73 for EZE. A wavelength of 235 nm was used for the quantitation of the drugs. Resolution of the peaks with clear baseline separation was found (Figure 2). The system suitability test parameters are shown in Table 2.

 

Figure 2: HPTLC densitogram of SIM and EZE with scanning at 235 nm

Table 2. System suitability test parameters for SIM and EZE for the proposed HPTLC method

Parameter

SIM ± RSDa   (nb = 6)

EZE ± RSDa (nb = 6)

Rcf

0.56

0.73

Area (Average)

12370.9 ± 1.91

15255.7 ± 0.92

Pick purity

>0.9999

>0.9998

a  RSD = Relative standard deviation, %.

b  n = Number of determinations.

Rf = Retardation factor.

 

Validation of the Proposed Methods:

Linearity:

Linear correlation was obtained between peak areas and concentrations of SIM and EZE in the range of 4–24 µg/mL for both the drugs, respectively, for the HPLC method and 1600–4800 ng/spot for both the drugs, respectively, for HPTLC. Data of the regression analysis are summarized in Table 3.

 

Accuracy:

The recovery experiments were performed by the standard addition method. The recoveries obtained were 100.81 ± 0.28 and 100.27 ± 0.66% for SIM and EZE, respectively, by the HPLC method and 100.34 ± 0.55 and 100.54 ± 0.83% for SIM and EZE, respectively, by the HPTLC method (Table 4). The high values indicate that both methods are accurate.

 

Method precision:

The RSD values for SIM and EZE were found to be 0.248 and 0.579%, respectively, using HPLC and 0.423 and 0.917%, respectively, for HPTLC (Table 4). The RSD values were found to be <1%, which indicates that the proposed methods are repeatable.

 

Intermediate precision:

The RSD values were found to be <2%, which indicates that the proposed methods are reproducible (Table 4).

 

LOD and LOQ:

LOD values for SIM and EZE were found to be 0.355 and 0.200 µg/mL, respectively, for HPLC and 129.6 and 60.53 ng/mL, respectively for HPTLC. LOQ values for SIM and EZE were found to be 1.08 and 0.607 µg/mL, respectively, for HPLC and 392.4 and 183.42 ng/mL, respectively for HPTLC (Table 4). These data show that nanogram quantity of both drugs can be accurately determined.

 

Specificity:

Excipients used in the specificity studies did not interfere with the estimation of either of the drugs by the proposed methods. Hence, the methods were found to be specific for estimation of SIM and EZE.

 

Robustness:

Peak area and retention time variation were found to be <1%. Also, no significant change in peak area was observed during 24 h. No decomposition was observed in either the first or second direction of the 2-dimensional analysis for both drugs on the HPTLC plate. Hence, the methods were found to be robust for estimation of SIM and EZE.

Assay of the Tablet Dosage Form (SIM and EZE 10 mg/Tablet):

The proposed validated methods were successfully applied to determine SIM and EZE in their tablet dosage form (tablet A). The results obtained for SIM and EZE were comparable with the corresponding labeled amounts (Table 5).

 

Comparison of the Proposed Methods:

The assay results for SIM and EZE in their combined dosage form obtained using the HPLC and HPTLC methods were compared by applying the paired t-test. The calculated t-values of 0.70 for SIM and 0.081 for EZE were less than the tabulated t-value (2.13) at the 95% (P = 0.05) confidence level. Therefore, there was no significant difference in the determined content of SIM and EZE by the HPLC and HPTLC methods.

 

The literature describes HPLC method for determination of SIM and HPLC methods for determination of EZE in tablet dosage forms. The assay results obtained by these methods were used for statistical comparison to evaluate the validity of developed HPLC and HPTLC methods. For SIM, the calculated F-value was found to be 0.27 (for HPLC), which is less than the tabulated F-value (3.89) at the 95% (P = 0.05) confidence level. For EZE, the calculated F-values were found to be 0.95 (for HPLC) and 2.84 (for HPTLC), which were less than the tabulated F-value (3.48) at the 95% (P = 0.05) confidence level. Therefore, there were no significant differences among the methods.

 

CONCLUSIONS:

Thus, the objective of project work was development and comparison of analytical method of SIM and EZE in their combined dosage form. The developed and validated HPTLC method for SIM and EZE was found to be simple specific and cost effective and can be routine applied for analysis of SIM and EZE in their combined dosage form. HPLC method is more sensitive giving precise results (interday, intraday) for both the drugs.

 

The proposed methods have the advantages of simplicity and convenience for the separation and quantitation of SIM and EZE in combination and can be used for the assay of their dosage form. Also, the low solvent consumption and short analytical run time lead to environmentally friendly chromatographic procedures. The results were compared statistically, and both methods were found to be precise and accurate. The additives usually present in the pharmaceutical formulations of the assayed analytes did not interfere with determination of SIM and EZE. The methods can be used for the routine simultaneous analysis of SIM and EZE in pharmaceutical preparations.

 

ACKNOWLEDGMENTS:

We are grateful to Comed chemicals limited and Zydus cadila healthcare limited for the gratis samples of pure SIM and EZE and thankful to SICART at vallabh vidyanagar for providing instrumental facility.


Table 3. Regression analysis of calibration graphs for SIM and EZE for the proposed HPLC and HPTLC methods

Parameter

HPLC

HPTLC

SIM

EZE

SIM

EZE

Concn range

4 - 24 µg/ml

4 - 24 µg/ml

1600 – 4800 ng/spot

1600 – 4800 ng/spot

Slope

50622

42598

3.6699

4.7650

SDa of the slope

485.13

206.53

0.0595

0.0306

Intercept

12521

5226.7

535.7

232.6

SD of the intercept

3245.67

2433.37

122.42

97.6

Correlation coefficient

0.9984

0.9996

0.9941

0.9986

SD = Standard deviation.

 

Table 4. Summary of validation parameters for the proposed HPLC and HPTLC methods

Parameter

HPLC

HPTLC

SIM

EZE

SIM

EZE

LODa

0.355 µg/ml

0.200 µg/ml

129.6 ng/spot

60.53 ng/spot

LOQb

1.08 µg/ml

0.607 µg/ml

392.4 ng/spot

183.4 ng/spot

Accuracy, %

100.37 – 102.43

100.67 – 102.86

99.35 – 101.84

99.73 – 101.13

Repeatability, (RSDc, %, n = 6)

0.743

0.553

1.047

0.572

Precision (RSD, %)

Interday (n = 3)

Intraday (n = 3)

 

0.21 – 0.40

0.29 – 0.67

 

0.21 – 0.75

0.42 – 0.62

 

0.20 – 1.63

0.31 – 1.94

 

0.64 – 1.63

0.78 – 1.46

a LOD = Limit of detection, b LOQ = Limit of quantitation, c RSD = Relative standard deviation.

 

Table 5. Assay results for the combined dosage form using the proposed HPLC and HPTLC methods.

Tablet

SIM ± SDa (nb = 5), %

EZE ± SD (n = 5), %

HPLC

HPTLC

HPLC

HPTLC

A

100.4 ± 0.27

100.8 ± 0.17

100.3 ± 0.16

100.1 ± 0.084

a SD = Standard deviation, b n = Number of determinations.

 

 


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Received on 12.11.2009                             Modified on 15.01.2010

Accepted on 18.02.2010                            © RJPT All right reserved

Research J. Pharm. and Tech. 3(2): April- June 2010; Page 507-511