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  • 1 Zydus Cadila Healthcare Limited, NH-8A, Moraiya-382213, Gujarat, India
  • 2 Institute of Pharmacy, Nirma University, Ahmedbad-382481, Gujarat, India
  • 3 Ramanbhai Patel College of Pharmacy, CHARUSAT, Changa, 388421, Gujarat, India
Open access

A novel, simple, robust, and rapid reversed-phased high-performance liquid chromatographic method has been developed for the separation and quantitative determination of the related substances of ezetimibe and simvastatin in combined dosage forms. Successful separation of the drug from the process-related impurities and degradation products formed under stress conditions was achieved on Inertsil ODS-3V (150 × 4.6 mm, 5.0 μm) column. The gradient liquid chromatography (LC) method employs solution A and solution B as mobile phase. The solution A contains 0.1% orthophosphoric acid solution in water, and solution B contains 0.1% orthophosphoric acid solution in acetonitrile. Flow rate was monitored at 2.0 mL/min, and the ultraviolet (UV) detection, at 238 nm. In forced degradation studies, the effect of acid, base, oxidation, UV light, and temperature was investigated, showing that good resolution between the peaks corresponds to process-related impurities and degradation products from both analyte. The performance of the method was validated according to the present International Conference on Harmonization (ICH) guidelines for specificity, limit of detection, limit of quantification, linearity, accuracy, precision, ruggedness, and robustness. To the best of our knowledge, a rapid LC method, which separates all the impurities of ezetimibe and simvastatin in combined dosage forms, disclosed in this investigation was not published elsewhere.

Abstract

A novel, simple, robust, and rapid reversed-phased high-performance liquid chromatographic method has been developed for the separation and quantitative determination of the related substances of ezetimibe and simvastatin in combined dosage forms. Successful separation of the drug from the process-related impurities and degradation products formed under stress conditions was achieved on Inertsil ODS-3V (150 × 4.6 mm, 5.0 μm) column. The gradient liquid chromatography (LC) method employs solution A and solution B as mobile phase. The solution A contains 0.1% orthophosphoric acid solution in water, and solution B contains 0.1% orthophosphoric acid solution in acetonitrile. Flow rate was monitored at 2.0 mL/min, and the ultraviolet (UV) detection, at 238 nm. In forced degradation studies, the effect of acid, base, oxidation, UV light, and temperature was investigated, showing that good resolution between the peaks corresponds to process-related impurities and degradation products from both analyte. The performance of the method was validated according to the present International Conference on Harmonization (ICH) guidelines for specificity, limit of detection, limit of quantification, linearity, accuracy, precision, ruggedness, and robustness. To the best of our knowledge, a rapid LC method, which separates all the impurities of ezetimibe and simvastatin in combined dosage forms, disclosed in this investigation was not published elsewhere.

Introduction

Simvastatin, chemically (1S,3R,7S,8S,8aR)-1,2,3,7,8,8a-hexahydro-3,7-dimethyl-8-{2-[(2R,4R)-tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl]ethyl}-1-naphthyl-2,2-dimethyl butyrate (Table 1), is obtained from the fermentation of Aspergillus terreus. After oral ingestion, simvastatin, which is an inactive lactone, is hydrolyzed to corresponding β-hydroxy acid, leading to the inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, responsible for catalyzing the conversion of HMG-CoA to mevalonate, which is an early and rate limiting step in cholesterol biosynthesis [13]. Administration of the highest approved statin dose offers only limited additional lowering of low-density lipoprotein (LDL) cholesterol at the expense of an increased incidence of side effects [4, 5]. Therefore, novel compounds that further reduce LDL cholesterol levels when added to statin therapy are of interest. Ezetimibe, (3R,4S)-1-(4-fluorophenyl)-3-[(3S)-(4-fluorophenyl)-3-hydroxypropyl]-4-4-hydroxyphenyl)-2-azetidi-none (Table 2), is a selective cholesterol absorption inhibitor, which potently inhibits the absorption of biliary and dietary cholesterol from the small intestine without affecting the absorption of fat soluble vitamins, triglyceride, or bile acids. Ezetimibe inhibits cholesterol absorption by binding to the Niemann–Pick C1-like 1 (NPC1L1) protein. The latter is located at the brush-border membrane of the enterocyte, where it contributes substantially to the intestinal uptake and cellular transport of cholesterols and noncholesterol sterols. Combined therapy of ezetimibe with a statin provides an incremental reduction in LDL cholesterol levels of 12–19%. Also, co-administration of ezetimibe with statins could significantly reduce the risk of coronary heart disease (CHD) events in patients with hypercholesterolemia [69].

Table 1.

Chemical structure of simvastatin and simvastatin impurities

Compound nameStructuresIUPAC name
Simvastatin(1S,3R,7S,8S,8aR)-1,2,3,7,8, 8a-Hexahydro-3,7-dimethyl-8-{2-[(2R,4R)-tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl]ethyl}-1-naphthyl-2,2-dimet-hyl butyrate
Simvastatin EP impurity A (simvastatin hydroxyl acid USP)(3R,5R)-7-[(1S,2S,6R,8S,8aR) -8-[(2,2-dimethylbutanoyl) oxy]-2,6-dimethyl-1,2,6,7,8,8a-hexahydro naphthalen-1-yl]-3,5-dihydroxyheptanoic acid
Simvastatin EP impurity B (acetyl simvastatin USP)(1S,3R,7S,8S,8aR)-8-[2-[(2R,4R)-4-(acetyloxy)-6-oxotetrahydro-2H-pyran-2-yl]ethyl]-3,7-dimethyl-1,2,3,7,8,8a-hexahydro naphthalen-1-yl 2,2-dimethylbutanoate
Simvastatin EP impurity C (anhydro simvastatin USP)(1S,3R,7S,8S,8aR)-3,7-dimethyl-8-[2-[(2R)-6-oxo-3,6-dihydro-2H-pyran-2-yl]ethyl]-1,2,3,7,8,8a-hexahydronaphthalen-1-yl 2,2-dimethylbutanoate
Simvastatin EP impurity D (simvastatin dimer USP)(2R,4R)-2-[[(1S,2S,6R,8S,8aR)-8-[(2,2-dimethylbutanoyl)oxy]-2,6-dimethyl-1,2,6,7,8,8a hexahydronaphthalen-1-yl]ethyl]-6-oxotetrahydro-2H-pyran-4-yl (3R,5R)-7-[(1S,2S,6R,8S,8aR)
Simvastatin EP impurity E (lovastatin USP)R1 = R4 = CH3, R2 = R3 = H(1S,3R,7S,8S,8aR)-8-[2-[(2R,4R)-4-hydroxy-6-oxotetrahydro-2H-pyran- 2-yl]ethyl]-3,7-dimethyl-1,2,3,7,8,8a-hexahydro naphthalen-1-yl (2S)-2- methylbutanoate (lovastatin)
Simvastatin EP impurity F (epilovastatin USP)R1 = R3 = H, R2 = R4 = CH3(1S,3R,7S,8S,8aR)-8-[2-[(2R,4R)-4-hydroxy-6-oxotetrahydro-2H-pyran-2-yl]ethyl]-3,7-dimethyl-1,2,3,7,8,8a-hexahydro naphthalen-1-yl (2R)-2-methyl butanoate
Simvastatin EP impurity G (methylene simvastatin USP)R1 = R2 = CH3, R3 + R4 = CH2(1S,7S,8S,8aR)-8-[2-[(2R,4R)-4-hydroxy-6-oxotetrahydro-2H-pyran-2-yl]ethyl]-7-methyl-3-methylene-1,2,3,7,8,8a-hexahydro naphthalen-1-yl 2,2-dimethyl butanoate
Table 2.

Chemical structure of ezetimibe and ezetimibe impurities

Compound nameStructureIUPAC name
Ezetimibe(3R,4S)-1-(4-Fluorophenyl)-3-[(3S)-(4-fluorophenyl)-3-hydroxypropyl]-4-4-hydroxyphenyl)-2-azetidinone
Desfluoro ezetimibe(3R,4S)-1-Phenyl-3-[(3S)-3-(4-fluorophenyl)-3-hydroxypropyl]-4-(4-hydroxyphenyl)-2-azetidinone.
Diol of ezetimibe(3R,4S)-3-((S)-3-(4-Fluorophenyl)-3-hydroxypropyl)-4-(4-hydroxyphenyl)azetidin-2-ol
Benzylated ezetimibe(3R,4S)-4-(4-(Benzyloxy)phenyl)-1-(4-fluorophenyl)-3-((S)-3-(4-fluorophenyl)-3-hydroxypropyl)azetidin-2-one
m-Fluoroaniline analog1-(3-Fluorophenyl)-3-(3-(4-fluorophenyl)-3-hydroxypropyl)-4-(4-hydroxyphenyl)azetidin-2-one
Ezetimibe ketone impurity(3R,4S)-1-(4-Fluoro phenyl)-3-[3-(4-fluoro phenyl)-3-oxopropyl]-4-(4-hydroxy phenyl)-2-azeti dinone
Ezetimibe tetrahydropyran analog6-(4-Fluorophenyl)-3-((4-fluorophenylamino)(4-hydroxyphenyl)methyl)-tetrahydropyran-2-one

Monograph for ezetimibe and simvastatin is available in United States Pharmacopoeia (USP). In the literature, a number of analytical methods described for the determination of simvastatin in aqueous samples and human plasma including liquid chromatography (LC) [1012], liquid chromatography–tandem mass spectrometry (LC–MS/MS) [1316], gas chromatography–mass spectrometry (GC–MS) [17], micellar electrokinetic chromatography [18], cerimetric reaction based on redox and complex formation [19], and ultraviolet (UV) spectrophotometry [20]. Methods have also been reported for determination of ezetimibe in pharmaceutical formulations and biological fluids including LC [21, 22], LC–electrospray ionization (ESI)–MS [2325], in human plasma by LC–MS/MS, and a reversed-phase high-performance liquid chromatography (RP-HPLC) method for determination of the pharmaceutical form of the drug [26]. Related substances method for ezetimibe and simvastatin are reported individually. Literature survey reveals that there is no method reported for analysis of related substances simultaneously in ezetimibe and simvastatin tablets. Based on the facts, the study aimed to develop and validate a simple, economic, and rapid analytical method which can be easily applied in routine analysis for the determination of related substances in ezetimibe and simvastatin in combined dosage forms (i.e. tablets).

Materials and Methods

Chemicals and Reagents

Ezetimibe, simvastatin, and related impurities were obtained from Zydus Cadila Healthcare Limited, Gujarat, India. HPLC-grade acetonitrile, potassium dihydrogen phosphate, and orthophosphoric acid was obtained from Merck (Darmstadt, Germany). HPLC-grade water was prepared from Millipore Milli-Q water purification system from (Bedford, MA, USA). There are seven potential impurities in simvastatin named as impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, and impurity G (Table 1). There are six potential impurities in ezetimibe named as desfluoro ezetimibe, diol of ezetimibe, benzylated ezetimibe, ezetimibe ketone, m-fluoroaniline analog, and ezetimibe tetrahydropyran analog (Table 2). Ezetimibe ketone, m-fluoroaniline analog, and ezetimibe tetrahydropyran analog are degradation-related impurities of ezetimibe, and impurity A, impurity C, and impurity D are degradation-related impurities of simvastatin. All the process-related impurities are monitored during the validation study.

Chromatographic Conditions and Equipment

Chromatographic separation and quantification of analyte and impurities were performed using the Agilent HPLC system which consists of a quaternary solvent manager, a sample manager, and a diode array detector (DAD). The output signal was monitored and processed using Chromoleon Software (Dionex). Separation was achieved on Inertsil ODS 3V (150 × 4.6 mm, 5.0 μm) column (GL Sciences). The gradient LC method employs solution A and solution B as mobile phase. The solution A contains 0.1% orthophosphoric acid solution in water, and solution B contains 0.1% orthophosphoric acid solution in acetonitrile. The flow rate of the mobile phase was 2.0 mL/min. The gradient program was time (min)/sol A (v/v): sol B (v/v); T0.01/58:42, T10.0/58:42, T35.0/40:60, T45.0/05:95, T53.0/05:95, T55.0/58:42, and T60.0/58:42. The column temperature was maintained at 25 °C, and the detection was monitored at a wavelength of 238 nm. The injection volume was 40 μL. Standard and test solutions were prepared in 0.02 M phosphate buffer pH 3.2–acetonitrile in the ratio of 40:60, % v/v, and used as diluent.

Experimental

Standard solution for ezetimibe and simvastatin at concentration of 0.001 mg/mL and 0.002 mg/mL, respectively, was prepared by dissolving appropriate amount of standard material in diluent and used. Sample solution was prepared at concentration of 0.5 mg/mL of ezetimibe in diluent (0.02 M phosphate buffer pH 3.2–acetonitrile in the ratio of (40:60, % v/v)) by crushing tablets into fine powder, sonicated at 10 °C, and filtered through Millipore PVDF 0.45 μm syringe filter by discarding first 3 mL of solution. Sample solution was also spiked with all impurities of ezetimibe and simvastatin (desfluoro ezetimibe, diol of ezetimibe, benzylated ezetimibe, ezetimibe ketone, m-fluoroaniline analog, ezetimibe tetrahydropyran analog, simvastatin impurity A, simvastatin impurity B, simvastatin impurity C, simvastatin impurity D, simvastatin impurity E, simvastatin impurity F, and simvastatin impurity G) at their threshold level by dissolving impurities in diluent and used for method development (Figure 1).

Figure 1.
Figure 1.

Chromatogram of sample solution (spiked with all impurities)

Citation: Acta Chromatographica Acta Chromatographica 30, 2; 10.1556/1326.2017.00273

Calculation

All known degradation-related impurities of ezetimibe and simvastatin were calculated with respect to ezetimibe and simvastatin, respectively, by observing their relative retention time (RRT) (Table 3). All process-related impurities of ezetimibe and simvastatin are monitored during stress study and stability study based on their RRT (Table 4). All unknown impurities eluting between the regions of 0 to 20 min were calculated against ezetimibe as they were identified by their UV spectrum. All unknown impurities eluting after 20 min were calculated against simvastatin as their UV spectrum matches with simvastatin.

Table 3.

Relative retention time (RRT) and relative response factor (RRF) of degradation-related impurities of ezetimibe and simvastatin

NameRetention timeRRTRRF
m-Fluoroaniline analog11.101.141.09
Ezetimibe ketone impurity17.871.831.12
Ezetimibe tetrahydropyran analog16.021.641.12
Ezetimibe9.771.00Not applicable
Simvastatin impurity A20.800.701.10
Simvastatin impurity C40.591.361.10
Simvastatin impurity D49.371.661.05
Simvastatin29.741.0Not applicable
Table 4.

Relative retention time (RRT) of process-related impurities of simvastatin and ezetimibe

NameRetention timeRRT
Simvastatin impurity Ba39.981.34
Simvastatin impurity Ea24.730.83
Simvastatin impurity Fa25.330.85
Simvastatin impurity Ga27.360.92
Desfluoro ezetimibeb8.310.85
Diol of ezetimibeb4.440.45
Benzylated ezetimibeb36.153.70

RRT was calculated against simvastatin as these are process-related impurities of simvastatin.

RRT was calculated against ezetimibe as these are process-related impurities of ezetimibe.

Method Development

The main target of the chromatographic method is to achieve the separation of impurities and the main component ezetimibe and simvastatin with each other. A sample solution containing 0.5 mg/mL of ezetimibe spiked with all known impurities of ezetimibe and simvastatin (desfluoro ezetimibe, diol of ezetimibe, benzylated ezetimibe, ezetimibe ketone, m-fluoroaniline analog, ezetimibe tetrahydropyran analog, simvastatin impurity A, simvastatin impurity B, simvastatin impurity C, simvastatin impurity D, simvastatin impurity E, simvastatin impurity F, and simvastatin impurity G) at their threshold level was prepared in diluent (0.02 M phosphate buffer pH 3.2–acetonitrile in the ratio of 40:60, % v/v) and used for the method development. Desfluoro ezetimibe, diol of ezetimibe, and benzylated ezetimibe were spiked at level of 0.2% with respect to ezetimibe concentration of 500 μg/mL as these impurities are process-related impurities. Ezetimibe ketone, m-fluoroaniline analog, and ezetimibe tetrahydropyran analog were spiked at level of 0.5% with respect to ezetimibe concentration of 500 μg/mL as these impurities are degradation-related impurities. Simvastatin impurity A, simvastatin impurity B, simvastatin impurity C, simvastatin impurity D, simvastatin impurity E, simvastatin impurity F, and simvastatin impurity G were spiked at the level of 0.8% with respect to simvastatin concentration of 4000 μg/mL by considering their toxicity value.

Method Validation

The developed analytical method was validated for its acceptable performance to ensure suitability of indent purpose. The validation parameters like accuracy, precision, specificity, detection limit, quantification limit, linearity, range, ruggedness, and robustness experiments were executed. The proposed method was evaluated as per International Conference on Harmonization (ICH) guidelines [27, 28].

Linearity was studied by analyzing the mixed calibration standard solutions at six concentration levels. The linearity solutions were prepared by dissolving appropriate amount of impurity standard in diluent to achieve the concentration from limit of quantification (LOQ) to 150% of specification level. Linear regression equations were plotted with the least squares linear regression method. For each degradation-related impurity, the relative response factor (RRF) was calculated. The limits of detection (LODs) and LOQs of the all degradation-related impurities were estimated based on the signal-to-noise ratios of 3:1 and 10:1, respectively. Precision of the method was investigated taking into consideration of its intra-day and inter-day precision aspects. Intra-day precision was assessed by carrying out six independent solutions of ezetimibe and simvastatin tablet sample on the same day. In the intermediate precision study, six new solutions were made on the different day by different analyst. Both intra-day and intermediate precision were valuated according to the relative standard deviation (RSD) values. Accuracy of the method was measured by spiking pre-analyzed samples with four different concentration levels in triplicate, i.e., at LOQ, 50%, 100%, and 150% levels of the respective specifications of degradation impurities. Robustness of the method was verified by introducing small variations in the HPLC parameters, including the flow rate (1.8 and 2.2 mL/min), column temperature (20 °C and 30 °C), and percentage of mobile phase B of the initial gradient elution condition (37% and 47%).

Forced Degradation Studies

The capacity of the method to detect the responses of the impurities without interferences was determined employing a DAD. Forced degradation study was performed using acid, base, peroxide, thermal, humidity, and ultraviolet which may cause drug degradation to evaluate the specificity of the method. Ezetimibe and simvastatin tablet sample was exposed to acidic degradation with 5 mL of 1 N hydrochloric acid solution for 30 min at 70 °C, cooled and neutralized with 5 mL of 1 N sodium hydroxide solution, and then diluted to 0.5 mg/mL concentration of ezetimibe with diluent. Base degradation study was performed with 5 mL of 0.1 N sodium hydroxide solution for 10 min at 30 °C, cooled and neutralized, and then diluted to 0.5 mg/mL concentration of ezetimibe with diluent. The samples were exposed to light and UV radiation of 2,968,040 lx h and 103,959 Wh/m2 in a photolytic chamber. In the oxidative condition, the sample was exposed to 5 mL 10% hydrogen peroxide solution, heated at 70 °C for 30 min, and then dissolved to 0.5 mg/mL concentration of ezetimibe with diluent. As for the thermal degradation, tablet sample was heated at 80 °C for 5 days, and sample solution was prepared to 0.5 mg/mL concentration of ezetimibe with diluent.

Results and Discussion

Optimization of Chromatographic Conditions

The main target of the chromatographic method is to resolve the impurities and the degradation products generated during stress studies from the ezetimibe and simvastatin. Optimization of chromatographic conditions was carried out by taking reference of simvastatin USP-related substances method. Impurities were co-eluted by using different stationary phases like C8, C18, CN, and phenyl with different particle sizes and lengths and different mobile phases containing buffers like phosphate and acetate with different pH (2–6) and using organic modifiers like acetonitrile and methanol in the mobile phase. Initially, a mixture of pH 2.0 buffer and acetonitrile was used in mobile phase gradient compositions on Kromasil C-18, 150 × 4.6 mm with 5 μm particles column. The mobile phase A contains 0.02 M phosphate buffer, pH 2.0, and mobile phase B contains acetonitrile. The flow rate was 1.7 mL/min. The LC gradient program was set as: time (t)/% solvent B: 0.01/35, 55/65, 65/95, 85/95, 86/35, and 92/35. The column temperature was maintained at 25 °C, and the detection was monitored at a wavelength of 238 nm. Impurity spiked sample was injected, and poor selectivity observed with these conditions was not considered for further analysis. Three impurities, namely, impurity F, impurity G, and simvastatin were eluting very close to each other in the conditions, and resolution between the impurities could not be improved in Kromasil C18 (100 × 4.6 mm), 3.5 μm column, and Kromasil C-18 (150 × 4.6 mm), 3.5 μm column. Later, 0.1% orthophosphoric acid in water was used as mobile phase A, and acetonitrile was used as mobile phase B which provides better selectivity and resolution. Ezetimibe and simvastatin tablet sample spiked with all process and degradation impurities in column, Purospher star RP18e, 150 × 4.6 mm, 3 μm particles, with mobile phase A as 0.1% orthophosphoric acid in water and 0.1% orthophosphoric acid in acetonitrile as mobile phase B at a flow rate of 1.5 mL/min and LC gradient program was set as: time (t)/% solvent B: 0.01/45, 35/65, 45/85, 65/95, 70/95,71/45, and 77/45 with UV 238 nm. Three impurities, namely, impurity F, impurity G, and simvastatin, were eluting very close to each other in the conditions, and then, Inertsil ODS 3V, 150 × 4.6 mm, 5 μm, was taken for better separation and better selectivity. Further experimentation was continued by changing only the flow rate and gradient composition, respectively. As the simvastatin impurity D is a late eluting impurity because of its bulky group, mobile phase B was kept as 0.1% orthophosphoric acid in acetonitrile and flow rate was kept 2.0 mL/min to elute this impurity rapidly when gradient program reaches to 95% composition of mobile phase B. The peak shapes and the resolution were very good between all impurities, ezetimibe, and simvastatin with 2.0 mL/min flow rate, and the gradient set as: time in minutes per % solution B: 0/42, 10/42, 35/60, 45/95, 53/95, 55/42, and 60/42. The peaks were identified by injecting and comparing the retentions with standards. This was selected based on the observations that the detector response was optimum, when compared to the determinations were made at other wavelengths for all compounds.

Method Validation

Stress Studies

Stress studies of a drug substance can help in identifying likely degradation products, which can, in turn, help in establishing degradation pathways and the intrinsic stability of the molecule. The method can be used to validate the stability-indicating power of the analytical procedures used so that the method can be successfully used as a tool for establishing the shelf life of the product. Appropriate degradation was obtained in the acidic (5.9%), alkaline (9.6%), oxidative (6.3%), thermal (22.2%), humidity (3.1%), and ultraviolet (1.5%) conditions. Simvastatin degrades into impurity A during acid and alkaline hydrolysis. Ezetimibe degrades into m-fluoroaniline analog during alkaline hydrolysis. During peroxide oxidation, simvastatin impurity A was observed which is a metabolite of simvastatin. Ezetimibe degrades into tetrahydropyran analog and ezetimibe ketone impurity during thermal degradation. Simvastatin impurity A and impurity C were observed when the sample was applied for thermal stress. No significant degradation was observed during humidity conditions and photolytic degradation. Photodiode array (PDA) detector was employed to check and ensure the homogeneity and purity of ezetimibe and simvastatin peak in all the stressed sample solutions and peak purity found passing with more than 990 (Figures 2–4). Mass balances were calculated by adding the assay value and the amounts of the impurities to the unstressed assay value in all stress conditions. Results were 98.2%, 99.5%, 99.1%, 98.2%, 98.4%, and 98.3% for acidic, alkaline, oxidative, thermal, humidity, and ultraviolet conditions, respectively. Therefore, it can be concluded that the method is specific with a good mass balance. The results of forced degradation studies are summarized in Table 5.

Figure 2.
Figure 2.

Chromatogram of sample solution (acid degradation and alkali degradation)

Citation: Acta Chromatographica Acta Chromatographica 30, 2; 10.1556/1326.2017.00273

Table 5.

Results of forced degradation study

Stress typeOptimized stress conditionMajor impurity observedTotal impurities
Acid hydrolysis1 N Hydrochloric acid solution at 70 °C for 30 minSimvastatin impurity A (5.4%)5.9%
Alkali hydrolysis0.1 N Sodium hydroxide solution 30 ° C for 10 minSimvastatin impurity A (7.1%), m-fluoroaniline analog of ezetimibe (2.1%)9.6%
Peroxide oxidation10% Hydrogen peroxide at 70 °C for 30 minSimvastatin impurity A (5.9%)6.3%
Thermal degradation80 °C for 5 daysSimvastatin impurity A (7.1%), Simvastatin impurity C (4.1%), ezetimibe ketone (5.3%), ezetimibe tetrahydropyran analog (4.9%)22.2%
Humidity degradation10 days 40 °C/75% RHSimvastatin impurity A (2.1%)3.1%
Photolytic degradation2,968,040 lx h, 103,959 Wh/m2Simvastatin impurity A (1.0%)1.5%
Figure 3.
Figure 3.

Chromatogram of sample solution (peroxide degradation and thermal degradation)

Citation: Acta Chromatographica Acta Chromatographica 30, 2; 10.1556/1326.2017.00273

Figure 4.
Figure 4.

Chromatogram of sample solution (humidity degradation and UV degradation)

Citation: Acta Chromatographica Acta Chromatographica 30, 2; 10.1556/1326.2017.00273

Limit of Detection and Limit of Quantification

LOD is defined as the lowest amount of analyte that can be detected, but not necessarily quantitated as an exact value, whereas limit of quantification is defined as the lowest amount of analyte that can be determined with suitable precision and accuracy [27, 28]. The LOD and LOQ for impurities were determined by measurable response at signal to noise ratio of 3:1 and 10:1, respectively, by injecting progressively known concentrations of the standard solutions using the developed HPLC method. The precision study was also carried out at the LOQ level by injecting six individual preparations of impurities and calculating the % RSD of the impurity peak areas. The LOD and LOQ values of ezetimibe, simvastatin, and their known impurities are mentioned in Table 6.

Table 6.

Regression and precision data

ParameterName of compound
SimvastatinSimvastatin impurity ASimvastatin impurity CSimvastatin impurity D
LOD (μg/mL)0.1340.1390.1330.139
LOQ (μg/mL)0.4010.4160.3980.417
Correlation coefficient0.99350.99190.99680.9932
Intraday precision (%RSD)2.43.23.22.7
Intermediate precision (%RSD)2.53.02.72.5
Precision at LOQ (%RSD)3.44.12.43.2
ParameterName of compound
Ezetimibem-Fluoroaniline analogEzetimibe ketone impurityEzetimibe tetrahydropyran analog
LOD (μg/mL)0.0340.0340.0340.033
LOQ (μg/mL)0.1020.1030.1010.099
Correlation coefficient0.99370.99340.99100.9923
Intraday precision (%RSD)2.63.13.12.6
Intermediate precision (%RSD)2.92.52.62.9
Precision at LOQ (%RSD)2.92.73.33.4

Linearity

The linearity of an analytical procedure is its ability to obtain test results that are directly proportional to the concentration of the analyte in the sample [27, 28]. The detector response of all degradation impurities, ezetimibe, and simvastatin was evaluated by analyzing by injecting different concentrations of the standard solutions. Linearity was checked for m-fluoroaniline analog of ezetimibe (limit NMT 0.5%), ezetimibe ketone (limit NMT 0.5%), ezetimibe tetrahydropyran analog (limit NMT 0.5%), simvastatin impurity A (limit NMT 0.8%), simvastatin impurity C (limit NMT 0.8%), and simvastatin impurity D (limit 0.8%) with respect to the concentration of ezetimibe (500 μg/mL) and simvastatin (4000 μg/mL) at not less than six different concentration levels ranging from LOQ to 150%. Ezetimibe was found linear in the range of 0.102 to 1.530 μg/mL, and simvastatin was found linear in the range of 0.401 to 48.120 μg/mL. Ezetimibe m-fluoroaniline analog, ezetimibe ketone analog, and ezetimibe tetrahydropyran analog were found linear in the range of 0.103 to 3.863 μg/mL, 0.101 to 3.788 μg/mL, and 0.099 to 3.713 μg/mL. Simvastatin impurity A, simvastatin impurity C, and simvastatin impurity D were found linear in the range of 0.416 to 49.200 μg/mL, 0.398 to 48.880 μg/mL, and 0.417 to 48.260 μg/mL. The Y-intercept, slope, and correlation coefficient were calculated for each of the analyte from linear regression equation. The cross-validated r2 was also calculated and found to be more than 0.99 (Table 6). Response factor from the linearity of each impurity was determined by dividing the simvastatin slope by impurity slope. The response factor for m-fluoroaniline analog, ezetimibe ketone, and ezetimibe tetrahydropyran analog was 1.09, 1.12, and 1.12 respectively. The response factor for simvastatin impurity A, impurity C, and impurity D was 1.10, 1.10, and 1.05, respectively. The result showed that an excellent correlation existed between the peak area and concentration of the analyte. This confirmed the linear relationship between peak areas and concentrations.

Intraday Precision and Intermediate Precision

Intraday precision of the related substance method was checked by injecting six individual preparations (n = 6) of ezetimibe and simvastatin tablet sample spiked with 0.2% of ezetimibe m-fluoroaniline analog, ezetimibe ketone, and ezetimibe tetrahydropyran analog and spiking 0.5% of simvastatin impurity A, simvastatin impurity C, and simvastatin impurity D, calculated % RSD for (in % w/w) each impurity. The % RSD for simvastatin impurity A, simvastatin impurity C, and simvastatin impurity D was observed 3.2%, 3.2%, and 2.7%. The % RSD for ezetimibe m-fluoroaniline analog, ezetimibe ketone, and ezetimibe tetrahydropyran analog was observed 3.5%, 3.9%, and 2.4% (Table 6). The % RSD of area for both intra-day precision and intermediate precision was found within 5%. The results proved that the proposed method provides acceptable precision.

Accuracy

Accuracy of an analytical procedure expresses the closeness of agreement between the value, which is accepted either as a conventional true value or an accepted reference value and the value found [27, 28]. In order to evaluate accuracy of the method for related substances, determination was checked by spiking pre-analyzed samples with four different concentration levels in triplicate, i.e., at LOQ, 50%, 100%, and 150% levels of the respective specifications of degradation-related impurities. The mixtures were analyzed by the proposed method (n = 3), and results are compared (Table 7). All results are within an acceptable limit. We can say the method is accurate.

Table 7.

Accuracy study at different levels

Recovery level% of Recoverya
Simvastatin impurity ASimvastatin impurity CSimvastatin impurity D
LOQ84.5 ± 3.588.9 ± 3.486.1 ± 3.9
50%94.8 ± 2.396.3 ± 2.194.5 ± 2.4
100%95.1 ± 2.897.1 ± 2.896.2 ± 2.6
150%96.7 ± 2.297.3 ± 2.997.4 ± 3.2
Recovery level% of Recoverya
m-Fluoroaniline analogEzetimibe ketone impurityEzetimibe tetrahydropyran analog
LOQ85.6 ± 3.189.1 ± 3.184.1 ± 3.3
50%92.6 ± 2.591.3 ± 3.193.4 ± 2.9
100%93.4 ± 3.294.1 ± 3.294.3 ± 2.8
150%94.1 ± 2.595.1 ± 3.195.4 ± 2.6

Mean ± % RSD for three determinations at each level.

Robustness

The robustness of an analytical procedure provides an indication of its reliability during normal use. To determine the robustness of the developed method, the experimental conditions were purposely altered and resolution between nearby impurities was evaluated. The flow rate of the mobile phase was 2.0 mL/min. To study the effect of flow rate, it was changed by ±10% from 2.0 to 2.2 and 1.8 mL/min. Column oven temperature (T) was 25 °C, and to study the effect of column oven (T), it was changed by 5.0 °C units from 25 °C to 30 °C and 20 °C. Gradient program was changed to observe the organic phase sensitivity. Percentage of mobile phase B of the initial gradient elution condition was altered from 42% to 37% and 47% to observe the effect of organic phase in the selectivity of the method. In all altered condition (flow rate, column temperature, organic composition), all analyte peaks were well separated and having resolution more than 2.5 between each nearby impurity peak and there was no change in elution order of impurities and main components. The results confirmed that the proposed method is robust.

Ruggedness

To prove the method ruggedness, the precision of test method was performed by a different analyst by using different column and different HPLC system (Shimadzu LC-2010, quaternary solvent manager) on different day. The % RSD of intermediate precision was found within 5%; this proves method ruggedness (Table 6).

Solution Stability

The solution stability of ezetimibe and simvastatin tablet solutions was examined at different time intervals; the cumulative RSD was calculated for each process and degradation impurity response to check the consistency in the cumulative RSD. Mobile phase composition and preparation were kept constant during the study period. Chromatograms were reviewed for the formation of any unknown peak due to the interaction of formulation matrix and analyte in analytical solution. The cumulative % RSD was found, and it was concluded that sample solution and standard solution were found stable at 10 °C for 14 h and 94 h, respectively.

Conclusion

A validated RP-HPLC method was developed for the separation and quantitative determination of related substances of ezetimibe and simvastatin in combined dosage forms. All the degradation products were well separated from the drug substance, demonstrating the stability-indicating nature of the proposed method. The RP-HPLC method is simple, robust, accurate, and selective. The method was completely validated as per ICH guide lines, and results from validation confirm that the method can be used for its intended purpose.

Compliance with Ethical Standards

This article does not contain any studies with human participants or animals performed by any of the authors.

Acknowledgment

The authors wish to thank the management of Zydus Cadila Healthcare Limited for supporting this work. They also acknowledge support from colleagues, Analytical Research and Development Laboratories Ltd., and Zydus Cadila Healthcare Limited.

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If the inline PDF is not rendering correctly, you can download the PDF file here.

  • 1.

    Desager, J.-P.; Horsmans, Y. Clin. Pharmacokinet. 1996, 31, 348371.

  • 2.

    O'Neil Marryadele, J. The Merck Index an Encylopedia of Chemiclas, Drugs and Biologicals. Marck research laboratories: 2006.

  • 3.

    Stancu, C.; Sima, A. J. Cell. Mol. Med. 2001, 5, 378387.

  • 4.

    Armitage, J. The Lancet 370, 17811790.

  • 5.

    Grigore, L.; Norata, G. D.; Catapano, A. L. Vasc. Health Risk Manage. 2008, 4, 267.

  • 6.

    Darkes, M. J. M.; Poole, R. M.; Goa, K. L. Ezetimibe. Am. J. Cardiovasc. Drugs 2003, 3, 6776.

  • 7.

    Davis, H. R.; Hoos, L. M.; Tetzloff, G.; Maguire, M.; Zhu, L.-J.; Graziano, M. P.; Altmann, S. W. Arterioscler., Thromb., Vasc. Biol. 2007, 27, 841.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8.

    Kastelein, J. J. P.; Sankatsing, R. R. Int. J. Clin. Pract. 2005, 59, 14641471.

  • 9.

    Mauro, V. F.; Tuckerman, C. E. Ann. Pharmacother. 2003, 37, 839848.

  • 10.

    Carlucci, G.; Mazzeo, P. Il Farmaco (Pavia) 1992, 47, 817823.

  • 11.

    Kim, B. C.; Ban, E.; Park, J. S.; Song, Y. K.; Kim, C. K. J. Liq. Chromatogr. Relat. Technol. 2004, 27, 30893102.

  • 12.

    Ochiai, H.; Uchiyama, N.; Imagaki, K.; Hata, S.; Kamei, T. J. Chromatogr. B: Biomed. Sci. Appl. 1997, 694, 211217.

  • 13.

    Jemal, M.; Ouyang, Z.; Powell, M. L. J. Pharm. Biomed. Anal. 2000, 23, 323340.

  • 14.

    Miao, X.-S.; Metcalfe, C. D. J. Chromatogr. A 2003, 998, 133141.

  • 15.

    Yang, H.; Feng, Y.; Luan, Y. J. Chromatogr. B 2003, 785, 369375.

  • 16.

    Zhang, N.; Yang, A.; Rogers, J. D.; Zhao, J. J. J. Pharm. Biomed. Anal. 2004, 34, 175187.

  • 17.

    Morris, M. J.; Gilbert, J. D.; Hsieh, J. Y. K.; Matuszewski, B. K.; Ramjit, H. G.; Bayne, W. F. Biol. Mass Spectrom. 1993, 22, 18.

  • 18.

    Srinivasu, M. K.; Raju, A. N.; Reddy, G. O. J. Pharm. Biomed. Anal. 2002, 29, 715721.

  • 19.

    Basavaiah, K.; Devi, O. Z. Eclética Química 2008, 33, 2128.

  • 20.

    Chavhan, V.; Reddy, K.; Ahhirao, K. J. App. Pharm. 2014, 6, 5564.

  • 21.

    Doshi, A. S.; Kachhadia, P. K.; Joshi, H. S. Chromatographia 2008, 67, 137142.

  • 22.

    Singh, S.; Singh, B.; Bahuguna, R.; Wadhwa, L.; Saxena, R. J. Pharm. Biomed. Anal. 2006, 41, 10371040.

  • 23.

    Dash, R. N.; Habibuddin, M.; Humaira, T.; Patel, A. A. J. Liq. Chromatogr. Relat. Technol. 2015, 38, 874885.

  • 24.

    Li, S.; Liu, G.; Jia, J.; Li, X.; Yu, C. J. Pharm. Biomed. Anal. 2006, 40, 987992.

  • 25.

    Oswald, S.; Scheuch, E.; Cascorbi, I.; Siegmund, W. J. Chromatogr. B 2006, 830, 143150.

  • 26.

    Sistla, R.; Tata, V. S. S. K.; Kashyap, Y. V.; Chandrasekar, D.; Diwan, P. V. J. Pharm. Biomed. Anal. 2005, 39, 517522.

  • 27.

    Guideline, I. H. T. Stability testing of new drug substances and products. Q1A (R2), current step 2003, 4.

  • 28.

    Guideline, I. H. T. Validation of analytical procedures: text and methodology. Q2 (R1) 2005, 1.

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