Characterization of novel stress degradation products of Bempedoic acid and Ezetimibe using UPLC–MS/MS: development and validation of stability-indicating UPLC method
Future Journal of Pharmaceutical Sciences volume 7, Article number: 234 (2021)
A receptive and easily comprehended technique was evolved for simultaneous assessment of Bempedoic acid and Ezetimibe and its impurities characterized by UPLC–MS/MS.
This technique involves chromatographic separation with a C18 column of water symmetry (150 mm × 4.6 mm, 3.5 µm). A mobile phase of 0.1% OPA (orthophosphoric acid) and acetonitrile in 50:50 v/v with 1 mL/min flow rate and ambient temperature was used. UV observation was taken at 230 nm. The recoveries, linearity, and quantification limits were found to be within the acceptable limit.
This technique was successfully tested with UPLC–MS to confirm the chemical structures of newly formed degradation products of Bempedoic acid and Ezetimibe and stress studies as per ICH Q2 (R1) guidelines.
Bempedoic acid is a pharmaceutical medicine utilized for the therapy of high cholesterol (high blood cholesterol levels) [1,2,3]. Bempedoic acid is approved for the treatment of hypercholesterolemia and therefore the highest tolerated statin therapy in adults with heterozygous , with hypercholesterolemia [5, 6], or with established atherosclerotic cardiovascular disorder [7, 8], who need additional lowering of LDL cholesterol [9, 10]. The most common adverse effects in clinical trials are muscle spasms, pain in the rear or within the limb, gout [11, 12], and gastrointestinal problems  like diarrhea [14, 15]. A less common but more serious effect was tendon rupture  within the structure of the shoulder, the biceps tendon, or the Achilles tendon .
Ezetimibe is a pharmaceutical drug unused and treats high blood cholesterol and certain other lipid abnormalities. Generally, it is used alongside dietary changes and a statin [18, 19]. It is preferred low in statin. It is taken orally. It is also available within the fixed combinations of Ezetimibe/Simvastatin, Ezetimibe/Atorvastatin, and Ezetimibe/Rosuvastatin. Usual consequences include upper respiratory infections, joint pain, diarrhea, and body exhaustion. Serious side effects include anaphylaxis [20, 21], liver problems, depression, and muscle breakdown. Its usage in pregnancy and breastfeeding [22, 23] is unsafe. Ezetimibe lowering the cholesterol involvement the intestines (Fig. 1). The experiments provided details on the conditions under which the drug was unstable to prevent possible instability, and suitable steps were taken during formulation.
Reagents and chemicals
Acetonitrile (HPLC mark), orthophosphoric acid (HPLC mark), and water (HPLC mark) were obtained from Merck India Ltd., Worli, Mumbai, India. APIs of Bempedoic acid (purity 99.8%) and Ezetimibe (purity 99.9%) were obtained from Cipla Pharmaceutical Company, Mumbai.
A chromatographic software of empower version 2 was used. Waters Acquity UPLC with a quaternary pump and PDA detector with empower 2.0 software was employed.
UPLC and MS/MS conditions
The chromatographic process involved the column of symmetry C18 (150 × 4.6 mm, 3.5µ) with ambient temperature. An isocratic elution containing 50% of 0.1% OPA and 50% of acetonitrile was used as mobile phase, and the flow rate of 1 mL/min with a dose volume of 20 µL was employed in UPLC.
In the forced degradation study, UPLC was connected to a mass spectrophotometer with the conditions and the splitter placed before the ESI source, allowing entry of only 35% of an eluent. The standard operating source conditions for MS scan of Bempedoic acid and Ezetimibe on positive ESI mode were optimized as follows: The fragmented voltage was set at 80 V, the capillary was set at 3000 V, the skimmer was set at 60 V, nitrogen was used as drying and nebulizing gas (45psi), and highly filtered nitrogen gas was used as collision gas.
Preparation of standard solution
Accurately weighed180 mg of Bempedoic acid and 10 mg of Ezetimibe were transferred into a 100-mL volumetric flask, and 70 mL of diluent was added and sonicated to dissolve it. Then, the volume was made up to the mark with diluent. Further, diluted 5 mL of the above solution was transferred into a 50-mL volumetric flask with diluent. And concentration of Bempedoic acid is 180 µg/mL and Ezetimibe is 10 µg/mL.
Preparation of sample solution
The samples were prepared by dissolving the finely ground tablets powder equivalent to 180 mg of Bempedoic acid and 10 mg of Ezetimibe sample, and they were transferred into a 100-mL volumetric flask, and 70 mL of diluents was added, ultrasonicated for 15 min, and diluted up to 100 mL mark with diluents. Further, diluted 5 mL of the sample stock solution was transferred into a 50-mL volumetric flask with diluents. Finally, the solution was filtered by utilizing a 0.45-µm syringe before injecting into the LC column.
The systematic technique UPLC was confirmed by evaluating the parameters such as system suitability, linearity, accuracy, the limit of detection, the limit of quantification, and robustness, and therefore, the results were found to be within the suitable range of ICH requirements.
To check the system performance, we used the parameters such as USP tailing, USP plate count, and percentage of relative variance.
Linearity and accuracy
Linearity was studied by using standard solutions of Bempedoic acid and Ezetimibe at several dilution levels (10%, 25%, 50%, 75%, 100%, 125%, 150%, and 200%). Accuracy was studied in three different dilution levels of 50%, 100%, and 150%. Finally, % of recovery and % of RSD were calculated.
Precision is of three types, namely
System Precision Reference standard solution of Bempedoic acid and Ezetimibe was injected six times and % RSD was calculated .
Method Precision Three levels of sample solutions of Bempedoic acid and Ezetimibe with concentrations of 90, 5 µg/mL (50%), 180, 10 µg/mL (100%), and 270, 15 µg/mL (150%) were injected and % recovery and % RSD were calculated.
Intermediate Precision Three levels of sample solutions of Bempedoic acid and Ezetimibe with concentrations of 90, 5 µg/mL (50%), 180, 10 µg/mL (100%), and 270, 15 µg/mL (150%) were injected in different days by using different columns. Then, % recovery and % RSD were calculated.
This technique was studied by changing the flow of ± 0.02%, organic phase of ± 10%, and wavelength of ± 5 nm.
LOD and LOQ
LOD means little quantity of analyte during a sample which will be detected, while LOQ explains the little quantity of analyte during a sample which will be observed with tolerable precision accuracy. The limit of detection and limit of quantification for Bempedoic acid and Ezetimibe were determined by injecting progressively low concentrations of ordinary solutions using the developed UPLC method. The limit of detection and limit of quantification were calculated as 3 s/n and 10 s/n, respectively, as per ICH guidelines where s/n indicates the signal-to-noise.
Stress degradation will not interfere between the peaks obtained for the chromatograms of forced degradation preparations. Stress degradation learnings were performed as reported by ICH guidelines Q1 (A) R2. The degradation peaks should be separated from one another, and therefore, the resolution between the peaks should be a minimum of 1.0. Therefore, the peak purity of the principle peak shape was passed. The forced degradation work was performed by different kinds of stresses to get the degradation of about 20%.
In acid degradation, the sample having 5 mL of 1 N HCl was transferred into a 100 mL volumetric flask and the flask was heated in a water bath at 60 °C for 30 min, allowed to cool to room temperature, and neutralized with 5 mL of 1 N NaOH. Then, it was made up to the mark with diluent. Further, diluted 5 mL of the above solution was transferred into a 50-mL volumetric flask with diluent and then filtered and injected into UPLC–MS system.
In alkali degradation, the sample having 5 mL of 1 N NaOH was transferred into a 100-mL volumetric flask and the flask was heated in a water bath at 60 °C for 30 min, allowed to cool to room temperature, and neutralized with 5 mL of 1 N NaOH. Then, it was made up to the mark with diluent. Further, diluted 5 mL of the above solution was transferred into a 50-mL volumetric flask with diluent and then filtered and injected into UPLC–MS system.
In peroxide degradation, sample having 5 mL of 30% hydrogen peroxide was transferred into a 100-mL volumetric flask. After that, the flask was heated in a water bath at 60 °C for 30 min. and allowed to cool to room temperature. Then, it was made up to the mark with diluent, and further, diluted 5 mL of the above solution was transferred into a 50-mL volumetric flask with diluent and then filtered and injected into UPLC–MS system.
In reduction degradation, sample having 5 mL of 10% sodium bisulfate solution was transferred into a 100-mL volumetric flask. The flask was heated in a water bath at 60 °C for 30 min and allowed to cool to room temperature. Then, it was made up to the mark with diluent, and further, diluted 5 mL of the above solution was transferred into a 50-mL volumetric flask with diluent and then filtered and injected into UPLC–MS system.
In thermal degradation, 1gm sample powder was weighed in a Petri dish and exposed to dry heat at 105 °C for 6 h. After that, equivalent weight of 180 µg/mL of Bempedoic acid and 10 µg/mL of Ezetimibe sample was weighed, transferred into a 100-mL volumetric flask, and dissolved in a diluent. Then, it was made up to the mark with diluent. Further, diluted 5 mL of the above solution was transferred to a 50-mL volumetric flask with diluent.
In photolytic degradation, tablets were ground finely into powder form and 1gm sample was exposed to photo-light UV 200 W-hrs and fluorescence light 1.2 million lux-hours. After that, equivalent weight of 180 µg/mL of Bempedoic acid and 10 µg/mL of Ezetimibe sample was weighed, transferred into a 100-mL volumetric flask, and dissolved in a diluent. Then, it was made up to the mark with diluent. Further, diluted 5 mL of the above solution was transferred into a 50-mL volumetric flask with diluent.
An isocratic elution of Bempedoic acid and Ezetimibe involved symmetry C18 column with a flow rate of 1 mL/min, and ambient temperature was maintained within the column. A mobile phase of 0.1% OPA and acetonitrile in 50:50 v/v was used. UV observation was taken at 230 nm.
The standard solution of Bempedoic acid (180 µg/mL) and Ezetimibe (10 µg/mL) was injected into the UPLC system, and the chromatogram of UPLC is shown in Fig. 2. %RSD was calculated by using the peak areas, and the results were found to be within the acceptable limit. Results of system suitability are shown in Table 1.
Specificity was not used to test the power of the assay of the method but to eliminate the consequences of all interfering substances in Bempedoic acid and Ezetimibe peak results, specifically by comparing the chromatograms of the blank samples presented in Fig. 3. The justified technique exhibited that the selected drugs were eluted without the involvement of peaks that occurred by the excipients in the market products.
Linearity of the developed test method was proven by preparing a series of linearity of solutions containing Bempedoic acid and Ezetimibe at eight different concentrations ranging from Bempedoic acid 18–360 µg/mL (18, 45, 90, 135, 180, 225, 270, and 360 µg/mL) and Ezetimibe 1–20 µg/mL (1, 2.5, 5, 7.5, 10, 12.5, 15, and 20 µg/mL). The calibration curves were linear throughout the concentration series of Bempedoic acid and Ezetimibe. The values of linearity are listed in Table 2 and Fig. 4. The coefficient of correlation values of both analytes Bempedoic acid and Ezetimibe were 0.9997 and 0.99964 in the calibration curve, respectively .
Accuracy of Bempedoic acid and Ezetimibe depends on recovery studies, which were administered at three different dilution levels (50%, 100%, and 150%). APIs with concentrations of 90, 180, 270 µg/mL of Bempedoic acid and 5, 10, 15 µg/mL of Ezetimibe were prepared. According to the test procedure, the test solutions were injected as three preparations of each spike level and therefore the assay was performed. The shared recovery values were observed to be within the range of 98%–102%, and the results are shown in Table 3.
The precision of this analysis was assessed in terms of method and intermediate variations. The intraday studies were calculated by executing three levels of sample solutions of Bempedoic acid and Ezetimibe with concentrations of 90, 5 µg/mL (50%), 180, 10 µg/mL (100%), and 270, 15 µg/mL (150%) in an equivalent day under the equivalent experimental conditions. Intermediate precision of the tactic was administered within the same laboratory by studying the analysis with different days and different columns. The tactic was very precise, and RSD values were found to be < 2%. Good recoveries (98 to 102%) of the selected drugs were obtained at each attached concentration and showed that the tactic was accurate. The results are given in Table 4.
LOD and LOQ
LOD and LOQ were separately determined by the calibration curve method; LOD and LOQ of the compounds were calculated by injecting continuous lower accumulation of standard solutions using the developed UPLC method. The LOD values for Bempedoic acid and Ezetimibe were observed as 0.225 µg/mL and 0.013 µg/mL and s/n values were 7 and 4, respectively. LOQ values were 0.743 µg/mL and 0.043 µg/mL and 27 and 21 were the s/n values, respectively.
As per ICH norms, deliberate variations were made within the method parameters such as change in flow (± 0.02%), organic content in the mobile phase (± 10%), and wavelength of detection (± 5 nm). So there is no tactic capacity to stay unaffected by system suitability. Table 5 shows the robustness of the tactic evaluated by observing the result of the modified parameters on retention time, tailing factor, and content percentage using UPLC. The degree of reliability of the consequences which were obtained by small deliberate variations showed that the tactic was strong.
To assess the steadiness of the sample, a solution was analyzed initially for 24 h at different intervals of time. No significant degradation was observed during this era, and therefore, the mean deviation and mean were not quite 5.0%, suggesting that the solutions were stable for a minimum period of 24 h, which was sufficient for the entire analytical procedure for UPLC.
Forced degradation studies of Ezetimibe and Bempedoic acid
According to ICH stability guidelines, there are various types of forced conditions, i.e., thermal, basic, acidic, oxidative, photolytic, and reductive forced degradation studies were conducted by using the sample brand name Nexlizet (containing 180 mg of Bempedoic acid and 10 mg of Ezetimibe) (Fig. 5). Seven numbers of DPs, DP1–DP7, were observed and characterized by UPLC–MS. The studies provided information about the conditions in which the drug is unstable to avoid potential instabilities; proper measures were often taken during formulation. Tables 6 and 7 represent the degradation results and validation parameters of Bempedoic acid and Ezetimibe.
In acid degradation, the selected samples were hydrolyzed with 1 N HCl for 3 h at 60 °C, 16.1% of Bempedoic acid and 12.4% Ezetimibe degradation was observed using HPLC, and 16.4% of Bempedoic acid and 11.6% of Ezetimibe degradation was observed using UPLC, and three degradation products, namely DP1, DP2, and DP5, were formed.
Alkali degradation of selected samples was initiated with 1 N NaOH, 15.2% of Bempedoic acid and 13.5% Ezetimibe degradation was observed using HPLC, and 17.7% of Bempedoic acid, 13.6% of Ezetimibe was observed using UPLC, and three degradation products, namely DP1, DP2, and DP5, were formed.
Peroxide decomposition of selected drug sample was studied in 30% hydrogen peroxide, 18.7% of Bempedoic acid and 15.8% of Ezetimibe degradation was observed using UPLC, and four degradation products, namely DP1, DP2, DP5, and DP7, were formed.
Reduction degradation of selected drugs was studied in 30% sodium bisulfate solution, 18.5% of Bempedoic acid and 16.4% of Ezetimibe degradation was observed using UPLC, and one DP1 degradation product was formed.
The thermal degradation sample was exposed at 105 °C for 6 h, 16.3% of Bempedoic acid and 16.6% of Ezetimibe degradation was observed in UPLC, and two degradation products, namely DP6 and DP2, were formed.
The sample was exposed to sunlight for 12 h, 16.2% of Bempedoic acid and 16.8% of Ezetimibe degradation was observed using UPLC, and three degradation products, namely DP1, DP3, and DP4, were formed.
Collision-induced dissociation of Bempedoic acid and Ezetimibe
DP1: Scheme 1 shows the fragmentation mechanism of DP1, and the ESI spectrum showed the most intense [ M+H]+ ion of m/z-449, which was observed under acid, alkali, peroxide, and photolytic degeneration conditions. The MS/MS spectrum of DP1 displayed abundant product ions at m/z-361 (loss of C4H8O2), m/z-273 (loss of C4H8O2 from m/z 361), and m/z-157 (loss of C6H12O6 from m/z 273). The MS/MS experiments combined with accurate mass measurements have confirmed the proposed scheme. Figures S6 and S7 represents collision induced dissociation of Bempedoic acid and Ezetimibe and MS spectral data.
DP2: Scheme 2 shows the fragmentation mechanism of Ezetimibe DP2, and the MS/MS spectrum showed more intense [M + H] ion of m/z-373, which was noticed under acid, alkali, thermal, and peroxide conditions. The spectrum displayed abundant product ions at m/z-295 (loss of benzene), m/z-217 (loss of benzene from m/z 292), m/z-123 (loss of phenol from m/z 217), and m/z-63 (loss of C3H8O from m/z 123). The MS/MS experiments combined with correct mass evaluations have confirmed the proposed scheme.
DP3: Scheme 3 shows the fragmentation mechanism DP3 of m/z 427 with molecular formula C24H23F2NO4, which was noticed under photolytic conditions. The MS spectrum displays abundant product ions at m/z-274 (loss of C9H11OF), m/z-179 (loss of m/z C6H5F from m/z 274), m/z-93 (loss of C3H8O from m/z 153), and m/z-85 (loss of phenol from m/z 179). The MS/MS measurements combined with correct mass evaluations have confirmed the proposed scheme.
DP4 : Scheme 4 shows the fragmentation mechanism for DP4 of m/z-499, which was noticed under photolytic degradation condition. The spectrum displays abundant product ions at m/z-346 (loss of C9H11OF), m/z-173 (loss of m/z C6H5F from m/z 346), m/z-93 (loss of m/z C3H8O from m/z-153), and m/z-93 (loss of C7H8O from m/z 173). The MS/MS experiments combined with correct mass evaluations have confirmed the proposed scheme.
DP5: Scheme 5 shows the fragmentation mechanism for DP5 of m/z-393.4, which was noticed under acid, alkali, and peroxide degradation conditions. The spectrum displays abundant product ions at m/z-137 (loss of C9H11F), m/z-95 (loss of C6H5F from m/z 256), and m/z-94 (loss of C6H5OH from m/z 161). The MS/MS experiments combined with correct mass evaluations have confirmed the proposed scheme.
DP6: Scheme 6 shows the fragmentation mechanism for DP6 of m/z-154, which was noticed under thermal degradation condition. The spectrum displays abundant product ions at m/z-72 (loss of C6H12) and m/z-84 (loss of C5H12). The MS/MS experiments combined with correct mass evaluations have confirmed the proposed scheme
DP7: Scheme 7 shows the fragmentation mechanism of degradation product 7 of m/z-493, which was noticed under peroxide degradation condition. The spectrum displays abundant product ions at m/z-399 (loss of C6H5F), m/z-359 (loss of C8H8O2 from m/z-493), m/z-265 (loss of C6H5F from m/z-359), m/z-205 (loss of C11H14FO2 from m/z-399), and m/z-71 (loss of C11H14FO2 from m/z-265). The MS/MS experiments combined with correct mass evaluations have confirmed the proposed scheme.
We have developed a responsive, robust, and fast UPLC process. The factors influencing the efficiency of the system were optimized, and the resulting method displayed high sensitivity and selectivity. A literature survey found that little attention was paid to the structural elucidation of the degradation products (DPs) of Bempedoic acid and Ezetimibe. A few attempts have been made for major impurities. According to the ICH stability guidelines [24,25,26,27,28], there are different forms of forced conditions, i.e., thermal, basic, acidic, oxidative, photolytic, and reductive forced degradation studies have been conducted [29,30,31,32,33,34]. Thus, in continuation of our previous efforts [35, 36], seven DPs (DP1–DP7) were observed and characterized by UPLC–MS, and few articles were mentioned in the last few years for quantification and analysis of Bempedoic acid and Ezetimibe in various chemical and biological matrices by using HPLC, UPLC, and characterization of its degradation products [37,38,39,40,41,42,43,44]. In the present study, we intended to explore a specific, sensitive, and new UPLC method toward the analysis of Bempedoic acid, Ezetimibe, and characterization of its new degradation products by UPLC–MS.
In this study, a unique, simple, rapid, economical, sensitive, and simply available UPLC technique was developed for the coincident determination of Bempedoic acid and Ezetimibe in bulk and tablet dosage form. The advantages of this method are shorter run time, low price, accessibility, reliability, sensitivity, and reproducibility. The degradation actions of the drugs were examined under hydrolysis (acid, base, and neutral), oxidation, and photolytic and thermal stress conditions. The drugs were found to be stable in thermal hydrolysis and unstable in acid, alkali, and oxidative conditions. The degradation products were identified [M + H]+ ion, and the proposed structures were supported by UPLC–MS/MS experiments combined with correct mass evaluations. The UPLC method was supported as per ICH guidelines and finally applied to the marketed formulations.
Availability of data and materials
The data for verification are provided with a supplementary file, and the rest of the data, if required, will be available upon request.
Ultra-performance liquid chromatography
Limit of detection
Limit of quantization
International Council for Harmonization
Ultra-performance liquid chromatography–mass spectrometry
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Vejendla, A., Talari, S., Ramu, G. et al. Characterization of novel stress degradation products of Bempedoic acid and Ezetimibe using UPLC–MS/MS: development and validation of stability-indicating UPLC method. Futur J Pharm Sci 7, 234 (2021). https://doi.org/10.1186/s43094-021-00381-6