Abstract
A rapid, stability indicating reverse phase liquid chromatographic method was developed for the determination of purity of Felodipine in active pharmaceutical substance form in the presence of its impurity and its degradation products. To develop the method which is also compatible to liquid chromatographic mass spectroscopic technique. The developed method is also used to determine the assay of Felodipine in bulk drug form. The drug is subjected to various stress conditions like acidic, basic, oxidation, UV light and thermal conditions. Considerable degradation was observed during base hydrolysis. Two degradation products were identified. The Waters Acquity UPLC BEH C18, 2.1 × 100 mm, 1.7 µm Column was used to achieve chromatographic separation. The gradient conditions, diluent and injection volume were optimized to achieve the acceptable resolution between impurities and its degradation products from Felodipine and to get good peak shapes. The masses were determined for main compound and its identified degradation products. Further, the characterization studies for main compound and its degradation products were performed using LCMSMS Q-TOF.
1 Introduction
Felodipine (FEL), a dihydropyridine derivative, chemically known as ethylmethyl-1,4-dihydro, 4-(2,3-dichlorophenyl)-2,6-dimethyl-3,5-pyridinedicarboxylate, acts as a calcium channel blocker and used to treat hypertension [1]. Felodipine reduces arterial smooth muscle contractility and subsequent vasoconstriction by blocking the influx of calcium ions through L-voltage-gated calcium channels. It can be used to treat mild to moderate essential hypertension [2]. There are several methods to determine impurities of felodipine in bulk drug and pharmaceutical dosage forms such as, Liquid chromatography, titration, spectroscopic techniques and liquid chromatography with amperometric detection [3–7]. The use of a liquid mobile phase with the ability to change mobilised polarity during chromatography and make other mobile phase alterations based on the characteristics of the substance being tested is a significant advantage in the separation process. The second component that allows for good separation is a larger selection of stationary phases. The separation line is linked to specific and sensitive detection systems, such as a spectrofluorometer, a diode detector, and an electrochemical detector, as well as other hyphenated systems, such as HPLC-MS and HPLC-NMR [8]. Liquid chromatography techniques have been widely utilised to determine trace levels of pharmacological active chemicals in biological samples and dosage forms in a rapid, accurate, sensitive, and selective manner. Both HPLC and UPLC utilize the basic concepts of liquid chromatography, however the former has been around since the 1960s, while the latter only surfaced in 2004, when Waters® developed their own UPLCTM devices. Both can be used for multiple LC modes, including normal and reverse phases, and can be linked with mass spectrometry systems for improved results, however the instrumentation required, and the performance produced by each system differ significantly [9–12]. Moreover, UPLC is a common laboratory technique that decreases the cost of designing and validating a method while increasing the efficiency of analysis. The speed of separation and efficiency improve with UPLC, resulting in the rapid development of techniques [13–19]. The goal of ultra-performance liquid chromatography (UPLC) drug analysis is to authenticate a drug's identity and provide quantitative data, as well as to follow the progress of a disease's treatment [8]. It was found in the literature search that there is no straightforward LC method that uses a simple, reasonably priced LC column and mobile phase that is compatible with LC-MS for determining purity and assaying for Felodipine drug substance and its degradations studies under different stress conditions. Therefore, the present study aimed to develop a simple, sensitive, and validated ultra-performance liquid chromatography which compatible with tandem mass spectroscopy (UPLC-MS/MS) method for the identification and characterization of forced degradation products of Felodipine. To identify the masses for FEL, process contaminants, and degradation products created under stress circumstances, the mobile phase employed for method development should be practical for LC-MS analysis. During the base hydrolysis of the medicinal molecule FEL, two degradation products (DP1 and DP2) were seen. The degradation products were subjected to LC-MS/Q-TOF analysis, which may aid future research studies in confirming the hypothesised structures.
2 Experimental
2.1 Drugs and reagents
Felodipine (FEL) and Impurity 1 (Imp 1) samples were procured from USP India Private Limited, Hyderabad (Fig. 1A and B). Both samples were in the form of active pharmaceutical ingredients (API), which were employed in this study. The high-quality grade reagents and solvents like ammonium formate, acetonitrile, sodium hydroxide, hydrochloric acid and hydrogen peroxide were used from Merck corporation, Mumbai, India. The Milli-Q-Water was obtained from Millipore water system (Millipore Corporation, Billerica, MA, USA).
Chemical structures A) Felodipine; B) Impurity 1
Citation: Acta Chromatographica 36, 2; 10.1556/1326.2022.01106
2.2 Instrumentation
The chromatographic analysis was performed by using Acquity H class UPLC connected with Photodiode Array (PDA) detector system. The Acquity BEH C18 100 × 2.1 mm, 1.7 µm system (Waters®) was used for separation of FEL and its impurities through chromatographic system. The chromatographic data was collected using the Empower 3 programme. Sartorius balances were used for measuring weights (CPA225D). The Suntest Atlas XLS + photolytic chamber (Atlas Material Testing, USA) was used for photolytic studies. The humidity degradation study was done by Espec SH-642 Temperature and Humidity Benchtop Chamber (AT Equipment Corporation, CA, USA). A Memmert hot air oven was used to conduct the stress study for thermal conditions (100- 800 Model, Make: Memmert KG, Germany). The Synapt G2 Q-ToF mass spectrometer with Waters® UPLC Acquity system linked to PDA detector was utilized for LCMS and MSMS research. This study was performed to identify the masses of FEL, Imp 1 and its degradation products. Further, the characterization studies for FEL and its degradation products helped to draw the fragmentation pathway from MSMS spectra. In addition, mass accuracy study for proposed structure of degradation product obtained as supporting document. The parameters set for MS studies were as follows: cone voltage: 3.0 v, capillary voltage: 3.2 v, source temperature: 100 °C, desolvation temperature: 250 °C and gas flow: 800 L h−1. Masslynx 4.1 software was used for data acquisition [15–19].
2.3 Chromatographic conditions
For mobile phase A, 10 mM Ammonium formate adjusted to pH 3.0 with formic acid and for mobile phase B, 100% acetonitrile was used. The flow rate maintained at 0.2 mL per minute throughout the study. The gradient program was kept as time (min)/Solution A (%) as follows: 0/70, 2/70, 10/30, 14/30, 14.1/70 and 18/70. The 2.0 µL injection volume was used. The column temperature was maintained at 25 °C and auto sampler was maintained at ambient temperature. The wavelength detection was done at 254 nm. The diluent used in the ratio of water and acetonitrile (2:8 v/v). The conditions were same for both UPLC and LCMSMS studies.
2.4 Assay sample preparation
The FEL sample, which weighed about 10 mg, was put into a 100 mL volumetric flask (VF), dissolved in enough diluent, and then diluted to volume with diluent. The final concentration of sample in this solution was 100 μg mL−1.
2.5 Organic impurities sample preparation
About 25 mg of FEL sample was weighed and transferred into 50 mL VF, dissolved in sufficient amount of diluent and then diluted to volume with diluent. The final concentration of sample in this solution was 500 μg mL−1.
2.6 Preparation of impurity stock solution
About 10 mg of Impurity 1 sample was weighed and transferred into 100 mL VF, dissolved in sufficient amount of diluent and then diluted to volume with diluent. The final concentration of impurity 1 in this solution was 100 μg mL−1.
2.7 Organic impurities standard solution preparation
Transferred each 1.0 mL of Assay sample preparation and impurity stock solution into 100 mL volumetric flask containing little amount of diluent and then diluted to volume with diluent.
3 Analytical method validation
The developed LC method was validated according to International Conference for Harmonization (ICH) [20].
3.1 System suitability
The system suitability was established using replicated injections of both assay sample preparation and organic impurities standard solution. The tailing factor, percentage RSD and column efficiency were checked for both FEL and impurity 1 peaks and observed the tailing factor is less than 1.0, percentage RSD is less than 2.0% and column efficiency is greater than 3,000 for both peaks. The obtained resolution between FEL and impurity was greater than 2.0.
3.2 Linearity
The different concentrations at 0.00025 mg per mL to 0.00075 mg per mL of both FEL and impurity 1 were prepared from stock solutions and injected for organic impurities method. For Assay, the different levels of concentration 0.05 mg per mL to 0.15 mg per mL of FEL were used. For both procedures, the linearity curve was shown for peak area responses against concentration.
3.3 Accuracy
The accuracy studies were performed using different level concentration of FEL and Impurity 1 sample preparations for both assay and chromatographic purity (CP) methods at 50, 100 and 150 percent levels. This study helps to understand the closeness of value obtained from the test results and true value.
3.4 Sensitivity
The series of Impurity 1 diluted solution were injected to estimate the Limit of detection (LOD) and Limit of quantification (LOQ) at a signal to noise ratio of 3:1 and 10:1 respectively.
3.5 Robustness
Intentionally changed the experimental conditions to test the robustness of developed method. The resolution between the FEL and impurity 1 was checked at different conditions. The flow rate kept as 0.18 and 0.22 mL min−1 in place of 0.2 mL min−1. The column temperature maintained at 20 and 30 °C instead of 25 °C. The pH of solution A was changed to 2.8 and 3.2 instead of 3.0. The other chromatographic conditions were kept constant for all above varied conditions.
3.6 Specificity
The analyte peak response was measured in the presence of its potential impurities. The possible degradation products were identified by carrying out the stress testing for drug substance. It helps to build the degradation pathways and the characteristic stability of the molecule. Degradation studies were carried out in accordance with ICH recommendations under various stressful conditions in order to determine the specificity and stability of the method [20–22]. The various degradation procedures were conducted to determine the stress conditions as follows: (1) 0.1 M HCl for 12 h at room temperature to provide acidic conditions, (2) 0.1 M NaOH for 12 h at room temperature to provide basic condition, (3) 6% peroxide for 24 h at room temperature for oxidation, (4) exposed at 105 °C for 48 h to obtain thermal condition, (5) exposed to 85 °C and 85% RH for 3 days to deliver humidity and (6) for photolytic effect, 1.2 million lux hours followed by 200 Wh m−2 was provided.
4 Results and discussion
It was noted that resolution between Imp 1 and the FEL major compound was crucial. Various adjustments were performed to the buffer concentration, PH of Solution A, column temperatures, and other factors in order to produce a resolution between the primary component and its Imp 1 greater than 2.0. From a linearity analysis employing multiple concentration level solutions containing the primary ingredient and Imp 1, the correlation coefficient (R2) was higher than 0.999 and shown in Fig. 2. The LOD and LOQ of FEL and its Imp.1 were observed as 50 and 150 ng mL−1 respectively and estimated based on signal to noise ratio (Table 1). The precision analysis found that the FEL and impurity 1 peak response's percent RSD at LOQ concentration was under 2%.
Linearity curves A) FEL; B) Impurity 1
Citation: Acta Chromatographica 36, 2; 10.1556/1326.2022.01106
Linearity, LOD and LOQ results
| Parameter | FEL | Impurity 1 |
| Linearity (y = ax + b) | ||
| Slope | 1 + E07 | 6 + E06 |
| Intercept | 4.7174 | 14.8726 |
| Correlation-coefficient | 0.99995 | 0.99980 |
| LOD (ng mL−1) | 50 | 50 |
| LOQ (ng mL−1) | 150 | 150 |
The LC-MS detector was used to scan FEL and its impurities and to calculate their m/z values. However, the PDA detector was used for LC method development and validation. The system suitability from organic impurities solution showed the retention time for Imp 1 and Felodipine was 11.73 and 12.04 respectively. The relative retention time for both the compounds was 0.97 (Imp 1) and 1 (FEL). The USP resolution between Imp 2 and FEL was 2.45. The relative standard deviation percentage was 0.28 for Imp 1 and 0.51 for Felodipine. The m/z value from MS was 382.0628 for Imp 1 and 384.0714 for Felodipine. System suitability from assay standard retention time and USP tailing were 12.13 and 1.04 respectively. The USP plate count and % RSD were 146,014 and 0.12 respectively. However, the m/z value was similar to organic impurities solution. The devised analytical method's accuracy was confirmed by the obtained percentage RSD values, which were less than 2% (Table 2). The chromatogram of organic impurities standard solution is shown in Fig. 3.
System suitability results
| System suitability from Organic impurities solution | |||||
| Compound name | RT* | RRT# | USP Resolution | % RSD | m/z value |
| Imp 1 | 11.73 | 0.97 | — | 0.28 | 382.0628 |
| Felodipine | 12.04 | 1.00 | 2.45 | 0.51 | 384.0714 |
| System suitability from assay standard | |||||
| Compound name | RT* | USP Tailing | USP Plate count | % RSD | m/z value |
| Felodipine | 12.13 | 1.04 | 146,014 | 0.12 | 384.0714 |
*RT: Retention time; # RRT: Relative Retention time.
Chromatogram of organic impurities standard solution to check the system suitability
Citation: Acta Chromatographica 36, 2; 10.1556/1326.2022.01106
4.1 Stress studies and degradation behaviour of the drug
The degradation behavior of felodipine was studied under the following stress settings: acidic and basic hydrolysis, oxidation, photolysis, thermal, ultrasonic, and humidity conditions. For 12 h, the medication significantly degraded in base hydrolysis at room temperature. The chromatogram obtained from base degradation sample solution illustrates the distinct separation between FEL, DP 1 and DP 2 as well as the two degradation products that were identified, DP1 and DP2 (Fig. 4). Other stress conditions, such as acidic hydrolysis, oxidation, photolysis, heat, ultrasonic, and humidity, did not cause any appreciable degradation (Table 3) and the overlaid chromatograms obtained from all degradation studies were shown in Fig. 5.
Chromatogram of base degradation sample
Citation: Acta Chromatographica 36, 2; 10.1556/1326.2022.01106
Study of forced degradation data
| Degradation conditions | % degraded | Purity angle | Purity threshold | Mass balance (%) |
| 0.1 M HCl for 12 h at room temperature | No Degradation | 0.223 | 0.352 | 99.6 |
| 0.1 M NaOH bench top for 12 h at room temperature | 4.0 Unknown 1 (DP 1) 14.0 Unknown 2 (DP 2) | 0.225 0.252 | 0.335 0.352 | 99.6 |
| Stressed with 6% H2O2, 24 h kept on bench top at Room temperature | No degradation | 0.211 | 0.309 | 99.7 |
| Thermal at 105 °C for 48 h | No degradation | 0.225 | 0.356 | 99.9 |
| Exposed to Visible light for about 1.2 Million Lux-hours and UV light for about 200 Wh m−2 | No degradation | 0.227 | 0.314 | 99.8 |
| Ultrasonic for 1 h | No degradation | 0.238 | 0.356 | 99.9 |
| Humidity 85% RH and 85 °C for 3 days | No degradation | 0.265 | 0.378 | 99.8 |
Overlaid chromatograms for all stressed samples, (a) Acid Hydrolysis, (b) Base Hydrolysis, (c) Peroxide, (d) Thermal, (e) Photolytic and (f) Humidity
Citation: Acta Chromatographica 36, 2; 10.1556/1326.2022.01106
4.2 Characterization of FEL and its degradation products
Three pieces in all were discovered and identified by characterization investigations and MS/MS. To identify the source of each fragment ion and to depict the predicted fragmentation pathway of FEL, DP1, and DP2, a multistage (MSn) mass fragmentation study was conducted. The MS spectrum of the molecular ion ([M + H]+) of FEL at m/z 384 was investigated to better understand the distinct FEL degradation patterns.
Based on the observed [M + H]+ ions, two degradation product peaks were found in the LC-ESI-MS spectrum. Then, molecular ions were chosen for MSn investigations in order to characterize DPs. Accurate mass measurements were carried out to verify the molecular composition of FEL and its decomposition products. The spectra from HRMS study for all the FEL, DP1 and DP2 are shown in positive mode (Fig. 6).
MSMS Spectra of FEL and base degradation sample peaks A) FEL; B) 356 and C) 370
Citation: Acta Chromatographica 36, 2; 10.1556/1326.2022.01106
The accurate mass data for protonated molecules was determined and calculated error in parts per million (ppm) between exact and accurate masses are tabulated in Table 4 and the proposed formulae of fragments are shown in Table 5.
Mass accuracy table for FEL and its degradation products DP1 and DP2
| Compound name | Retention time (Min) | Molecular formula | Calculated m/z | Observed m/z (M + H) | Error (mDa) |
| Felodipine | 10.22 | C18H20Cl2NO4 | 384.0769 | 384.0714 | 14.3 |
| DP1 | 6.73 | C16H16Cl2NO4 | 356.0456 | 356.0429 | 7.58 |
| DP2 | 5.71 | C17H18Cl2NO4 | 370.0613 | 370.0630 | −4.59 |
MSMS data of FEL, DP 1 and DP 2 in ESI positive mode
| Compound name | Retention time (Min) | Molecular formula | Observed m/z | Molecular formula for Fragment ions (m/z) | Fragment ions (m/z) | RDB (Ring Plus Double Bond) |
| Felodipine | 10.22 | C18H20Cl2NO4 | 384.0714 | C17H16Cl2NO3+ | 352 | 9.5 |
| C16H14Cl2NO3+ | 338 | 10.5 | ||||
| C12H16NO4+ | 238 | 5.5 | ||||
| DP 1 | 6.73 | C16H16Cl2NO4 | 356.0429 | C16H14Cl2NO3+ | 338 | 10.5 |
| C15H12Cl2NO3+ | 324 | 10.5 | ||||
| C10H12NO4+ | 210 | 5.5 | ||||
| DP 2 | 5.71 | C17H18Cl2NO4 | 370.0630 | C17H16Cl2NO3+ | 352 | 10.5 |
| C15H12Cl2NO3+ | 324 | 10.5 | ||||
| C11H14NO4+ | 224 | 5.5 |
4.3 FEL (m/z: 384)
The resultant ions at m/z 238 (loss of C6H4Cl2) from m/z 384, m/z 338 (loss of C2H6O) from m/z 384, and m/z 352 (loss of CH4O) from m/z 384 are visible in the MS/MS spectrum (Fig. 6A). The hypothesized fragmentation pathway was constructed using chemdraw software from the MS spectrum acquired from the investigations for FEL, DP1 and DP2. Figure 7 depicts the anticipated degradation mechanism for FEL.
Mass fragmentation pathway of Felodipine
Citation: Acta Chromatographica 36, 2; 10.1556/1326.2022.01106
4.4 DP1 (m/z: 356)
The chemical ion at m/z 356 is confirmed to be from DP1 by the LC/MS/MS and HRMS data (Fig. 6B). The chemical name for C15H12Cl2NO3 that results from the loss of CH4O in the product ion at m/z 324 is (5-carboxy-4-(2,3-dichlorophenyl)2,6-dimethyl-1,4-dihydropyridin-3-yl) (oxo)methylium. The chemical name of the product ion at m/z 338 is (4-(2,3-dichlorophenyl)-5-(methoxycarbonyl)-2,6-dimethyl-1,4-dihydropyridin-3yl) (oxo)methylium. It was produced when H2Cl2O was lost. The chemical name of the product ion at m/z 210 was 3-carboxy-5(methoxycarbonyl)-2,6-dimethyl-1,4-dihydropyridin-4-ylium. It was produced when C6H4Cl2 was lost. From the analysis of the fragmentation of m/z 356, three product ions in total were found. The molecular formulas of C16H16Cl2NO4 was visible in the ESI-QTOF spectrum with a 5.00 ppm error. (4-(2,3-dichlorophenyl)-5-(methoxycarbonyl)-2,6-dimethyl-1,4-dihydropyridine-3-carbonyl)oxonium was identified as the molecular ion with m/z 356. The fragmentation pathway of DP1 is shown in Fig. 8.
Mass fragmentation pathway of DP1
Citation: Acta Chromatographica 36, 2; 10.1556/1326.2022.01106
4.5 DP2 (m/z 370)
The LC/MS/MS spectrum demonstrates that the molecular ion at m/z 370 comes from DP2 (Fig. 6C). The product ion at m/z 352, which has the chemical name (4-(2,3-dichlorophenyl)-5-(ehtoxycarbonyl)-2,6-dimethyl-1,4-dihydropyridin-3-yl) (oxo)methylium, was created when water was lost and resulted in the formation of C17H16Cl2NO3. The resultant ion at m/z 324 is known chemically as (5-carboxy-4-(2,3-dichlorophenyl)-2,6-dimethyl-1,4-dihydropyridin-3-yl) (oxo)methylium and was created when C2H6O was lost. The chemical name for the product ion at m/z 224, which forms as a result of the loss of C6H4Cl2, is 3-carboxy-5-(ethoxycarbonyl)-2,6-dimethyl-1,4-dihydropyridin-4-ylium. Three product ions in total were seen during the m/z 370 fragmentation assay. The chemical formula of C17H18ClNO4 was visible in the ESI-QTOF spectrum with a 5.00 ppm inaccuracy. The (4-(2,3-dichlorophenyl)-5-(ethoxycarbonyl)-2,6-dimethyl-1,4-dihydropyridine-3-carbonyl)oxonium) molecular ion with m/z 370 was identified. The fragmentation pathway of DP2 is shown in Fig. 9.
Mass fragmentation pathway of DP 2
Citation: Acta Chromatographica 36, 2; 10.1556/1326.2022.01106
Felodipine is an antihypertensive drug approved by FDA. There are not many reports on the drug's stability or potential degradation products as of now [5, 7]. The goal of this study was to create a novel, accurate, repeatable, robust, stable, and linear method for measuring FEL and its impurities using RP UPLC, which is useful for routine use in quality control laboratories. It also aimed to create a method that is compatible with LC/MS, which is useful for both qualitative and quantitative studies using a mass spectrometer. The United States Pharmacopeia specification is met for all additional criteria, including column efficiency in terms of theoretical plates and tailing factor for the main peak [23]. According to the recovery studies, the attained recovery percentage for this approach ranges from 98.4 to 101.8% on average.
The pure drug was subjected to heat, humid, ultrasonic, peroxide, acid, and base stresses. The stressed sample's UPLC examination revealed that under the effect of acid, peroxide, ultrasonic, humidity, and temperature conditions, no degradation had taken place. However, stressed samples with base present degradation products that were seen in UPLC as distinct peaks in addition to felodipine. To determine the products generated, another UPLC/MS/MS analysis was performed on the collected degraded samples.
The two molecular ions fragments formed under the base stress condition which are as follows: 1.(4-(2,3-dichlorophenyl)-5-(methoxycarbonyl)-2,6-dimethyl-1,4-dihydropyridine-3-carbonyl)oxonium was identified as the molecular ion with m/z 356 and 2.(4-(2,3-dichlorophenyl)-5-(ethoxycarbonyl)-2,6-dimethyl-1,4-dihydropyridine-3-carbonyl)oxonium) molecular ion with m/z 370. From these stress studies, it was observed that the drug was stable under these conditions and more susceptible towards base hydrolysis degradation condition. In a study by Kancherla et al. in 2015 also showed almost similar results under stressed conditions for drug Ezetemibe (EZB) [24].
Peak purity for the peaks acquired from various degradation tests was observed utilizing tools from the empower programme, and it was declared that the FEL peak was pure and homogenous from all examined stress samples. Mass balance values were determined from the stressed samples, and it was found that all of the results are greater than 99.6% (Table 3), which is consistent with the findings of earlier studies [24]. Thus, it was determined that the devised technique was stable and specific to Imp 1 and its degradation products.
The results of the afore mentioned studies led to the conclusion that the procedure was accurate, precise, hardy, and robust. Further study is required for determining the degraded products' toxicity by quantifying the samples.
5 Conclusion
A novel technique that can explain the stress degradation behavior of FEL under a variety of conditions, including hydrolysis, oxidation, photolysis, and heat treatment, has been developed and validated using a UPLC system with a mobile phase that is compatible with LC-MS. The FEL in API and stability samples can be determined using this method in both quantitative and qualitative assessments. Using the LC/MS/MS technology, the structural characterization and mass accuracy studies of the FEL led to the creation of DP 1 and DP 2. This technique aids in the creation of reference standards for the principal compound's recognized degradation products as well as in the ongoing detection of DPs in samples. This approach might be useful in determining and characterizing research of medicines belonging to the same class.
Conflicts of interest
The authors confirm that this article has no conflicts of interest.
Acknowledgements
The authors are thankful to the USP India Private Limited, Hyderabad and Department of Engineering Chemistry, Koneru Lakshmaiah Education Foundation, Vaddeswaram, Guntur for arranging required samples, facilities and immense support to publish this research work.
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