RELATED SUBSTANCES BY HPLC METHOD FOR THE DETECTION AND EVALUATION OF IMPURITIES IN EZETIMIBE DRUG MATERIAL

RELATED SUBSTANCES BY HPLC METHOD FOR THE DETECTION AND EVALUATION OF IMPURITIES IN EZETIMIBE DRUG MATERIAL

Durgababu Rapeti ^{* 1}, Gudibanda Chandra Sekhar Reddy ¹, Kapavarapu Maruthi Venkata Narayanarao ¹, Pulipaka Shyamala ² and Rallabhandi Murali Krishna ²

G. V. K. Biosciences Pvt Ltd, Hyderabad - 500076, Telangana, India.

Department of Physical Chemistry, Nuclear Chemistry and, Chemical Oceanography, Andhra University, Visakhapatnam - 530003, Andhra Pradesh, India.

ABSTRACT: The gradient HPLC method was developed to detect and quantify four related substances (FIP, FHO-2, STG-01, and desfluoro) in ezetimibe drug material. The efficient separation was carried out on Water X-Select CSH C18 column (4.6 mm × 100 mm, 2.5 μm). The impurities were separated in gradient mode of elution with 0.1% trifluoroacetic acid in water (mobile phase A) and 0.1% trifluoroacetic acid in acetonitrile (mobile phase B) at a flow rate of 1.0 ml /min. The effluents were detected at 254 nm. The method has been validated in compliance with the regulatory standards suggested by the International Conference on Harmonization. The parameters validated included precision, linearity, detection limit, quantification limit, specificity, accuracy, and robustness. The method showed good linearity from 0.03 μg/ml to 1.5 μg/ml for FIP, FHO-2, STG-01, and desfluoro. The developed method was applied to determine the studied impurities in three different batches of ezetimibe drug material. The method proposed in this work could be employed in quality control of ezetimibe bulk drug.

Ezetimibe, Related substances, Quality control, Gradient, HPLC, Analysis

INTRODUCTION: Ezetimibe is a lipid-reducing agent which prevents the absorption of cholesterol and associated phytosterol in intestine ^{1, 2}. Ezetimibe is approved as an adjunctive nutritional treatment to reduce cholesterol levels in mixed hyperlipidemia, primary hyperlipidemia sitosterol-emia, and familial hypercholesterolemia ^{3, 4}. Ezetimibe shows its cholesterol-lowering effect inthe blood without disturbing the absorption of other nutrients and fat-soluble vitamins in the intestine.

Chemically, ezetimibe is referred as (3 R, 4 S) – 1 - (4 - fluorophenyl) - 3 - [(3 S) - 3 - (4 -fluorophenyl) - 3 - hydroxypropyl] - 4 - (4 -hydroxyphenyl) azetidin - 2 - one Fig. 1A. Regulatory bodies such as the U.S. Food and Drug Administration (FDA) and International Conference on Harmonization (ICH) emphasize the requirements for purity and impurity identification in active pharmaceutical ingredient and formulation product ^5-7.

Impurities in pharmaceutical products are from reagents, catalysts, heavy metals, catalysts, charcoal, filter aids, degraded end products obtained during or after bulk drug production, enantiomeric impurity, etc. ⁸ As indicated by ICH Impurity Standards for new drug or drug products, the detection of impurities below 0.1% is not considered necessary unless impurities are predicted to be extremely potent or toxic. The overall acceptable daily dose level to be considered is 0.1%. A number of reports on the identification, development and characterization of ezetimibe-related impurities and their degradants were published ^9-15. Four process-related impurities 4-((4-fluorophenylimino)methyl)phenol (impurity-FIP), 3 - [5 - (4 - fluorophenyl) - 1, 5dioxaopentyl)] - 4 – phenyl (4S) - 2 - oxolidinone (impurity-FHO-2), 3 R, 4 S) – 3 - ((S) - 3 - (4 - fluorophenyl) - 3 –hydroxypropyl) - 4 - (4 - hydroxyphenyl) - 1 -phenylazetidin - 2 - one (impurity-desfluoro)  and S) - 3- ((S) - 5 - (4 - fluorophenyl) - 5 -hydroxypentanoyl) - 4 - phenyloxazolidin-2-one (impurity - STG - 01) were identified during the manufacturing of ezetimibe drug substance. The structures of these impurities are given in Fig. 1B, 1C, 1D, and 1E.

No method has been published for quantification of the impurities FIP, FHO-2, desfluoro, and STG-01 in ezetimibe drug material. In this report, a sensitive and reliable gradient HPLC method has been developed and validated to monitor and quantify FIP, FHO-2, desfluoro, and STG-01 simultaneously in the ezetimibe drug material.

FIG. 1: STRUCTURE OF [A] EZETIMIBE [B] FIP [C] FHO-2 [D] DESFLUORO [E] STG - 01

MATERIALS AND METHODS:

Impurities, Drug and Chemicals and Solvents: GVK Biosciences Private Limited (Hyderabad, India) provided the gift reference samples of FIP, FHO-2, desfluoro, and STG-01 and ezetimibe. Trifluoro acetic acid and acetonitrile were purchased from Merck (Mumbai, India). Milli Q water produced from the Milli Qpurification system was used.

Apparatus and Conditions for Chromato-graphy: Waters Acquity Arc HPLC System and Water X-Select CSH C18 column (4.6 mm i.d. × 100 mm, 2.5 µm) were used in this current investigation. Chromatographic separation of FIP, FHO-2, desfluoro, and STG-01 was carried out at 40 ^oC temperature. The impurities and ezetimibe were separated in gradient mode of elution with 0.1% trifluoroacetic acid in water (mobile phase A) and 0.1% trifluoroacetic acid in acetonitrile (mobile phase B) at a flow rate of 1.0 ml /min with the sample injection volume of 10 μl. The effluents were detected at 254 nm wavelength. The mobile phase A and B gradient programme was set as shown in Table 1.

TABLE 1: GRADIENT PROGRAMME FOLLOWED IN THIS INVESTIGATION

Standard Solutions: The primary stock solution (1 mg/ml) was prepared in a 20 ml volumetric flask by accurately weighing 20 mg each of FIP, FHO-2, STG-01, and desfluoro, dissolved in 10 ml diluent (acetonitrile) and diluted to volume using dilent. The secondary stock solution (20 μg/ml) was prepared by diluting 2 ml of primary stock to 10 ml with diluent.

Procedure for Calibration Curves: The second-dary stock solution (20 μ/ml) was diluting appropriately with diluent to get calibration solutions with concentrations ranging from 0.03 μ/ml to 0.15 μ/ml for FIP, FHO-2, STG-01, and desfluoro.

For each concentration, 10.0 μl injections are made and chromatographer under the above-mentioned conditions. In order to produce the calibration graph, the peak area obtained at each concentration was plotted against the corresponding concen-tration. The data from peak area and concentration were then applied to determine the regression equation.

Procedure for Monitoring Impurities in Test Sample (Ezetimibe Drug Material): Accurately weighed 50 mg of the test sample was transferred to a volumetric flask of 50 ml capacity, dissolved in 10 ml of diluent and diluted with diluent to mark. 10.0 μl injections are made and chromatographed under the above-mentioned conditions. The peak areas of FIP, FHO-2, STG-01, and desfluoro obtained were then used to quantify the content of impurities in the ezetimibe drug-using correspond-ding regression equation or calibration graph.

RESULTS AND DISCUSSION:

Method Optimization: The HPLC method was developed with an aim to make available a specific procedure for the simultaneous analysis of FIP, FHO-2, STG-01, and desfluoro impurities in ezetimibe drug material. A different range of analytical columns, isocratic, and gradient mobile phase systems with different flow rates have been tried in order to find the best HPLC conditions for the separation and analysis of examined impurities. Good separation was achieved using a Water X-Select CSH C18 column (4.6 mm × 100 mm, 2.5 µm). Gradient elution mode provided better results than isocratic elution mode. Therefore, gradient elution was chosen. The mobile phase composition was 0.1% trifluoroacetic acid in water (mobile phase A) and 0.1% trifluoroacetic acid in acetonitrile (mobile phase B) at a flow rate of 1.0 ml /min. The FIP, FHO-2, desfluoro, STG-01 were detected and quantified at 254 nm where the sensitivity is good Fig. 2.

FIG. 2: UV SPECTRUM OF EZETIMIBE AND RELATED SUBSTANCES

The FIP, desfluoro, ezetimibe, FHO-2, STG-01, and their corresponding peaks were separated at the Rt 2.578 min, 12.51 min, 13.032 min, 14.446 min, and 16.163 min, respectively under the mentioned HPLC conditions optimized Fig. 3.

Validation: The optimized procedure was validated in agreement with ICH guidelines ¹⁶.

Specificity: The specificity was evaluated by injecting blank diluent (acetonitrile), standard solution (FIP-0.1 μg/ml, FHO-2 - 0.1 μg/ml, des-fluoro - 0.1 μg/ml, STG-01- 0.1 μg/ml) and ezetimibe drug sample solution spiked with impurities (FIP-0.1 μg/ml, FHO-2 - 0.1 μg/ml, desfluoro - 0.1 μg/ml, STG-01- 0.1 μg/ml) and recorded the chromatograms Fig. 4A, 4B and 4C. Interferences by acetonitrile and ezetimibe were not observed at the Rt of FIP, FHO-2, STG-01, and desfluoro impurities and thus proved the specificity of the method.

FIG. 3: CHROMATOGRAM OF FIP, DESFLUORO, EZETIMIBE, FHO-2, STG-01 OBTAINED AFTER METHOD OPTIMIZATION

FIG. 4A: CHROMATOGRAM OF DILUENT BLANK

FIG. 4B: CHROMATOGRAM OF IMPURITIES STANDARD SOLUTION (FIP - 2.918 MIN, DESFLUORO - 13.131 MIN, EZETIMIBE - 13.656 MIN, FHO -2- 15.164 MIN, STG-01- 16.782 MIN)

FIG. 4C: CHROMATOGRAM OF EZETIMIBE (13.032 MIN) DRUG SAMPLE SPIKED WITH IMPURITIES (FIP – 2.578 MIN, DESFLUORO – 12.51 MIN, FHO -2- 14.446 MIN, STG-01- 16.163 MIN)

Specificity was further assessed by determining the peak purity of ezetimibe, FIP, FHO-2, STG-01, and desfluoro. Table 2 contains a summary of peak purity results. The low values for the purity angle of ezetimibe peak and impurities peaks than purity threshold values in standard solution and ezetimibe drug sample with impurities indicated the method specificity.

Limit of Detection and Limit of Quantification: The limit of detection (LD) and limit of quantification (LQ) were measured as the concentrations of impurities with a signal-to-noise ratio of ≥3 and ≥10, respectively.

Table 3 contains the LD and LQ results of FIP, FHO-2, STG-01, and desfluoro. LQ values of FIP, FHO-2, STG-01, and desfluoro were further confirmed through analysis of standard solution at LQ concentration level six times.

The relative standard deviation values for the peak areas of impurities were checked in Table 4. The relative standard deviation for the peak areas of FIP, FHO-2, STG-01, and desfluoro at LQ concentration was less than 10.0% indicated the preciseness of the method at LQ concentration level.

The chromatograms at LQ and LD concentration levels are shown in Fig. 5A and 5B.

Linearity: Linearity was assessed from LQ to 150% of the specification level with respect to test concentration (0.1 μg/ml). The method showed good linearity from 0.03 μg/ml to 1.5 μg/ml for FIP, FHO-2, STG-01, and desfluoro. The details of the linearity study are shown in Table 5. The correlation coefficient values for FIP, FHO-2, STG-01, and desfluoro are greater than 0.99, indicating the good linearity of the proposed method.

TABLE 2: PEAK PURITY DATA FOR EZETIMIBE AND IMPURITIES STUDIED

TABLE 3: LD AND LQ DATA FOR IMPURITIES STUDIED

Concentration; W.r.t - with respect to; S/N - signal to noise ratio; LD - limit of detection; LQ - limit of quantification

TABLE 4: CONFIRMATION AT LIMIT OF QUALIFICATION LEVEL

Avg - average; STD Dev - standard deviation; RSD - relative standard deviation

FIG. 5A: CHROMATOGRAMAT LQ CONCENTRATION LEVEL (FIP - 2.898 MIN, DESFLUORO - 13.118 MIN, EZETIMIBE - 13.644 MIN, FHO -2- 15.150 MIN, STG-01- 16.778 MIN)

FIG. 5B: CHROMATOGRAMAT LQ CONCENTRATION LEVEL (FIP - 2.903 MIN, DESFLUORO - 13.115 MIN, EZETIMIBE - 13.640 MIN, FHO -2- 15.150 MIN, STG-01- 16.770 MIN)

TABLE 5: LINEARITY AND REGRESSION DATA FOR IMPURITIES STUDIED

RRF - relative retention factor

Precision: The precision of the method has been established by the analysis of six replicates of the test sample (ezetimibe drug material) spiked with FIP, FHO-2, STG-01, and desfluoro at 0.1 μg/ml concentration level. The percentage relative standard deviation values for peak areas of FIP, FHO-2, STG-01, and desfluoro were calculated in Table 6. Relative standard deviation was less than 10.0%, indicated the preciseness of the method.

Accuracy: The accuracy was determined by analyzing test sample (ezetimibe drug material) spiked with studied impurities at LQ level, 50%, 100%, 150% of specification level concentration (0.1 μg/ml) in three replicates. As response difference was observed, the relative retention factor values for FIP, FHO-2, STG-01, and desfluoro was established and applied in recovery calculation. The recovery percentage of FIP, FHO-2, STG-01 and desfluoro obtained at each level was given in Table 7. The percentage recoveries for studied impurities at each level ranged from 70% to 130% (approved limit). The percentage recoveries indicated the method’s accuracy. Robustness: In order to test the effect of slight and deliberate change in column temperature and flow rate on the analysis, a robustness study conducted on the test sample (ezetimibe drug material) spiked with FIP, FHO-2, STG-01 and desfluoro at a concentration of 0.1 μg/ml. The percent difference in relative retention time of FIP, FHO-2, STG-01, and desfluoro were determined Table 8. The difference value was less than 15% (acceptable limit). This study showed that the method is reasonably insensitive to variations in the conditions studied.

Batch analysis: The method developed was applied in three different batches of ezetimibe drug material to evaluate the content of FIP, FHO-2, STG-01, and desfluoro. The impurities were below the detection limit (0.03 μg/ml) in all three successive plant batches.

TABLE 6: PRECISION DATA FOR IMPURITIES STUDIED

Avg - average; Std Dev - standard deviation; RSD – relative standard deviation

TABLE 7: ACCURACY DATA FOR IMPURITIES STUDIED

RRF – relative retention after

TABLE 8: ROBUSTNESS DATA FOR IMPURITIES STUDIED

RRT - relative retention time

TABLE 9: BATCH ANALYSIS OF EZETIMIBE FOR FIP, FHO-2, STG-01 AND DESFLUORO

CONCLUSION: The gradient HPLC method was developed to detect and evaluate FIP, FHO-2, STG-01, and desfluoro in ezetimibe drug material. The estimation of FIP, FHO-2, STG-01, and desfluoro in ezetimibe was found to be specific, precise, robust, and accurate. Applying the method to three successive batches of ezetimibe showed that these impurities were well below the specification limit (0.03%). Hence, these impurities (FIP, FHO-2, STG-01, and desfluoro) are omitted from the regular analysis of ezetimibe.

ACKNOWLEDGEMENT: The authors thank the management of GVK Biosciences Private Ltd, Hyderabad, for facilities, co-operation, and support during the study.

CONFLICTS OF INTEREST: Nil

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    How to cite this article:

    Rapeti D, Reddy GCS, Narayanarao KMV, Shyamala P and Krishna RM: Related substances by HPLC method for the detection and evaluation of impurities in ezetimibe drug material. Int J Pharm Sci & Res 2021; 12(1): 217-25. doi: 10.13040/IJPSR.0975-8232.12(1). 217-25.

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