A Novel Liquid Chromatographic Method for the Quantitative Determination of Degradation Products in Remdesivir Injectable Drug product
H. Ramakrishna Reddy1,2,*, S.R. Pratap3,4, N. Chandrasekhar1,2 and S.Z.M. Shamshuddin1,3
Abstract
An effectual and stability signifying technique has been validated for the quantitative verification of degradation products in Remdesivir Injectable pharmaceutical products by employing high- performance liquid chromatography with ultraviolet detector. The process was optimized by using an octyldecylsilane chemically bonded column (Kromasil KR100-5 C18; USP L1 phase) with dimensions; 250 mm length × 4.5 mm inner diameter and 5-μm particle size. The method was validated as per International Conference on Harmonization and other current regulatory guidelines for analytical method validation. The anticipated process was found to be robust, accurate, specific, linear, precise, stable and rugged in the concentration ranging from quantification level to 200% of the specification level of specified and unknown degradation impurities. The technique was effectively applied to analyze degradation products in Remdesivir Injectable drug products.
Introduction
Remdesivir is a white to an off-white crystalline powder, having molecular formula and weight C27H35N6O8P and 602.58 g/mol correspondingly (Figure 1). Remdesivir is an adenosine triphosphate analog by impending antivirus activity versus a diversity of RNA virus/s. Remdesivir is a carboxylic ester ensuing from the condensation of the carboxy group of N-[(S)-{[(2R,3S,4R,5R)-5-(4-aminopyrrolo [2,1-f] [1,2,4] triazin-7-yl)-5-cyano3,4dihydroxytetrahydrofuran-2-yl]methoxy} (phenoxy)phosphoryl]-L-alanine with the hydroxy group of 2- ethylbutan-1-ol. Upon administration, Remdesivir, being a prodrug, is metabolized into its active form GS-441524 (1). Remdesivir is an antiviral drug discovered by Gilead Sciences in the brand name Veklury to combat the West African Ebola viral pandemic in 2010. Also, it exhibits activity against the hemorrhagic fever Marburg virus (MARV), the Middle East respiratory syndrome-related coronavirus (MERS-CoV). Remdesivir is also being examined as a potential treatment to (severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]) for COVID-19 (2–8). Remdesivir was granted a Food and Drug Administration disaster utility authorization on 1 May 2020 (9). The literature survey reveals that thus far Avataneo and companions examined the analysis of Remdesivir (its metabolite GS-441524) by employing the UHPLC–MS–MS technique (10). Hence, there is a noteworthy requirement for a specific methodology for the quantitative estimation of Remdesivir and its degradation products in Remdesivir Injectable products.
Some of the articles referred in which the authors worked on complex degradation products of different antiviral drug products using liquid chromatographic techniques (11–13). This paper outlines a typical permanence demonstrating reverse phase high-performance liquid chromatography (RP-HPLC) tech- nique to quantify Remdesivir degradation products in Remdesivir Injectable drug products. The proposed technique was validated in 3.0 ± 0.05 with diluted potassium hydroxide solution. Solution was filtered using nylon-66 membrane disc filter.
Mobile phase A preparation
Buffer solution was used as mobile phase A. line with International Conference on Harmonization (ICH) and other regulatory guidelines. The methodology was noticed to be accurate, specific and precise, from quantification level (QL) to 200% of proposed specification level for the specified and unknown degradation products of Remdesivir in drug product Remdesivir for injection.
Experimental
Chemicals and standards
Remdesivir (standard with 99.8% purity), triol nitrile impurity (98.0% potency), acetonide nitrile impurity (99.0% potency), tri- O-benzyl nitrile impurity (98.0% potency); orthophosphoric acid (85% potency), potassium hydroxide (99.7% purity, grade: AR, Make: Merck), methanol (solvent with 99.8% purity, grade: HPLC, Make: Merck), acetonitrile (solvent with 99.8% purity, grade: HPLC, Make: Merck) and Water (Milli-Q Grade).
Instrumentation and chromatographic conditions
High-performance liquid chromatography having ultraviolet (UV)/ PDA detector (Waters make Empower software) was utilized to perform the study. HPLC column (Kromasil 100 5-C18, Make: Akzonobel, Part No. MO5CLA25) having length: 250 mm, ID: 4.6 mm and particle size: 5 μm were used in the study. UV detector wavelength as 242 nm and flow rate of 0.7 mL/min was selected. A column oven temperature of 50◦C and sampler cooler temperature of 5◦C was set with 10 μL injection volume. Gradient elution with total run time of 75 minutes as follows was programmed.
Buffer preparation
Buffer solution was prepared by mixing 1 mL of orthophosphoric acid (85%) in 1,000 mL of purified water and pH was adjusted to
Mobile phase B preparation
Mobile phase B was prepared by mixing acetonitrile, methanol and water in the ratio 70:20:10, respectively. Solution using Nylon-66 membrane disc filter was filtered.
Sample solvent preparation
Sample solvent (diluent) was prepared by mixing methanol and water in the ratio 70:30, respectively.
Standard solution preparation
Standard solution was prepared to get a final concentration of 2.5 μg/mL of Remdesivir in the sample solvent.
Placebo solution preparation
Placebo solution was prepared by reconstituting one vial of placebo with 19 mL of water and the contents were transferred into a 200 mL volumetric flask using methanol. Then, 40 mL of water was added and diluted with methanol. The solution was filtered through a 0.45- μm PVDF filter.
Sample solution preparation
Sample solution was prepared by reconstituting one vial of a sample (equivalent to 100 mg/vial of Remdesivir) with 19 mL of water and the contents were transferred into a 200 mL volumetric flask using methanol. Then, 40 mL of water was added and diluted with methanol to get a final concentration of 500 μg/mL of Remdesivir. The solution was filtered through a 0.45-μm PVDF filter.
Validation of the method
Method validation was carried out as per USP (1225), ICH (Q2R1) and other comprehensive method validation guidelines related to cur- rent regulatory requisition (14–23). The method validation param- eters include specificity, stress study, linearity, quantification limit (QL), detection limit (DL), accuracy, precision, analytical solution stability, ruggedness, robustness, range and system suitability were appraised.
Specificity and stress study
The specificity of the technique was assessed by injecting individ- ual analytes of sample solvent, standard solution, placebo solution, sample solution, individual impurities solution, sample spiked with impurities solution.
Stress study was performed by subjecting placebo and samples to various forced degradation conditions of hydrolysis, oxidation, photolytic and thermal exposure.
Precision
System precision was reviewed from six recurrent injections of stan- dard solution. Method precision was assessed by analyzing six indi- vidual sample preparations (n = 6) spiked with impurities at 100% specification level. %RSD was calculated for retention time and peak response of standard solution, and % recovery of impurities obtained from spiked sample determinations.
Ruggedness
Ruggedness of process was assessed by executing the analysis of sample by different analyst on different date with different column and instrument. %RSD was calculated for recovery of impurities obtained from intra and inter precision analysis of 12 cumulative samples (n = 12).
Linearity
Remdesivir standard and impurities with varied concentrations of solutions ranging from the lowest QL to about 200% level of nom- inal concentration were prepared from the linearity stock standard solution and analyzed.
A linear graph was plotted between Remdesivir and impurities concentration and respective area responses. Statistical analysis for the obtained data has been assessed.
Detection limit and quantification limit
DL and QL were estimated from the slope and the residual deviation of linearity data. The appropriate concentration levels of Remdesivir and impurities solutions was prepared and analyzed.
Accuracy and range
Accuracy test was executed by spiking impurities in the test samples and Remdesivir in placebo sample at QL, 50%, 100%, 150% and 200% level of nominal concentrations.
Spiked test samples were prepared in triplicate for each level. Accuracy was calculated from “amount added” and the “amount recovered.”
The test method range was drawn from linearity, accuracy and method precision determined through the recovery of impurities and active from the sample matrix.
Robustness
Robustness of test method were performed from Remdesivir standard solution and spiked test sample with impurities at the 100% level by varying experimental conditions such as; flow rate (0.6, 0.7 and 0.8 mL/min) and column temperature (45, 50 and 55◦C). %RSD for peak area response, peak symmetry, theoretical plates and recovery were assessed for each varying experimental condition.
Solution stability
Stability of analytical solutions was evaluated for Remdesivir stan- dard solution and test sample solution spiked with impurities from different time intervals at room temperature (25 ± 2◦C). The %varia- tion in peak area response between initial and each time interval was estimated.
System suitability
System suitability was assessed for Remdesivir peak from all valida- tion parameters. %RSD for peak area response, theoretical plates and peak symmetry were evaluated.
Results
Method validation
Specificity and stress study
The chromatograms acquired for standard, test and spiked test samples exhibited no interference at Remdesivir and impurities, demonstrating that the method is specific (Figures 2–4).
From the stress study, it was found that the sample degraded more in an alkaline stressed condition and slightly in all other stressed con- ditions. Conversely, no anonymous impurities initiated and interfered with Remdesivir and its related impurities peaks, which demonstrates that the technique is stability-indicating (Supplementary Table XIV, Supplementary Figures S5–S14).
Precision
The %RSD results for area response and retention time of Remdesivir peak attained from six repeated standard determinations found 0.4% and 0.0%, which shows the method repeatability.
The %RSD result of the quantified contents of impurities from six spiked test sample determinations found 1.2% for triol nitrile, 0.9% for acetonide nitrile and 0.6% for tri-O-benzyl nitrile, which shows the method reproducibility.
Ruggedness
The %RSD result for the quantified contents of impurities from six spiked test sample determinations obtained from intermediate precision found 0.7% for triol nitrile, 0.1% for acetonide nitrile and 0.6% for tri-O-benzyl nitrile. The cumulative %RSD computed for 12 sample determinations (attained from method precision and inter- mediate precision) found 0.9% for triol nitrile, 0.6% for acetonide nitrile and 0.7% for tri-O-benzyl nitrile, which shows the ruggedness of the method.
Linearity
The statistical data of linearity for correlation and regression between peak areas and concentration levels of Remdesivir and impurities in the range QL to 200% level found linear (r: 0.9999 and R2: 09999 for Remdesivir; r: 0.9999 and R2: 09999 for triol nitrile; r: 0.9999 and R2: 09998 for acetonide nitrile; r: 0.9999 and R2: 09999 for tri- O-benzyl nitrile). The results are tabulated in Table I and Figures 5–8 (Supplementary Tables II–IV). The “relative response factors (RRF)” for each impurity to active (Remdesivir) based on the respective slope obtained from linearity data were established (RRF as multiplication factor; 0.59 for triol nitrile, 0.53 for acetonide nitrile and 0.89 for tri-O-benzyl nitrile).
Detection limit and quantification limit
DL and QL were assessed for the derived values for Remdesivir (0.121 and 0.398 μg/mL), triol nitrile (0.124 and 0.413 μg/mL), acetonide nitrile (0.121 and 0.399 μg/mL), tri-O-benzyl nitrile (0.126 and 0.415 μg/mL). %RSD of peak area response at DL concentration acquired from six determinations found 2.2% for Remdesivir, 2.8% for triol nitrile, 1.9% for acetonide nitrile, 1.6% for tri-O-benzyl nitrile. Discrete observable peaks were observed at DL concentration for Remdesivir and each impurity, which shows the sensitivity of the process.
Accuracy and range
The overall mean % recovery found 98.6% (precision RSD = 1.1%) for Remdesivir, 97.5% (precision RSD = 1.3%) for triol nitrile, 97.5% (precision RSD = 1.1%) for acetonide nitrile and 97.2% (precision RSD = 1.4%) for tri-O-benzyl nitrile. The correlation and regression between the mean values of “amount added” along with “amount recovered” from QL to 200% level for Remdesivir and each impurity found linear, which shows the test method is linear, accurate and precise (Supplementary Tables V–XII).
Robustness
The test method found to be reliable even with deliberate variations in chromatographic conditions (flow rate and column temperature). The system suitability and recovery results were well within the criteria (Supplementary Table XIII).
Solution stability
The % variation in the peak area response from initial to 48 hours attained for standard solution and test sample spiked with impurities at the 100% level was found to be within the acceptable limit. Thus, the solutions (analytical) were considered stable up to 48 hours.
System suitability
The system suitability criteria from all validation parameters were evaluated and found RSD < 5.0% of peak area responses, peak symmetry factor < 2.0 for Remdesivir peak. Thus, it was proved that the system is suitable across the analytical test.
Discussion
Method development and optimization
Based on the Remdesivir molecule nature, a RP-HPLC mode with UV detection was chosen for the separation. Since the product formulation contains complex degradation components, gradient elution with the longer length column was selected to achieve better separation and greater sensitivity for impurities. Several experimental trials were performed with varied chromatographic conditions, buffer and mobile phase concentrations and ratios with different gradient patterns.
The method was optimized based on the solubility and resolution of active and its impurities components in the sample matrix. The method was finalized with buffer solution having 0.001% orthophos- phoric acid as mobile phase (A); acetonitrile, water and methanol in the ratio 70:10:20 (v/v) as mobile phase (B). The sample solvent contains methanol and water in the ratio of 70:30 (v/v).
The sample compartment temperature was maintained at 5◦C to avoid further degradation of impurities in the longer sequence of sample runs. The separation of peaks of active and impurities from the test sample matrix with appropriate recovery by exploiting a C18 USP L1 phase column and eventual liquid chromatographic parameters (24–26).
Conclusion
A reverse phase liquid chromatography with UV detection method was developed and validated for the quantitative determination of degradation products in drug product Remdesivir for injection. The proposed methodology found to be capable enough to detect, discrete and quantify the potential degrading impurities of Remdesivir. The selectivity and forced degradation study indicates stability indicating nature of method throughout stability life cycle of the product. The quantitation limits for all impurities achieved below 10% level of specification limit, which proved the sensitivity of method. The test method precision, linearity and accuracy was established by covering a wide range from QL to 200% specification limit, which will be helpful to apply the method even with increased specification limits for impurities based on stability trend of the product. Thus, the proposed test method could be adapted effectively to analyze and monitor degradation products in test samples of Remdesivir injectable drug products by quality control laboratories.
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