Introduction:
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the causative agent of the global pandemic known as Coronavirus Disease 2019 (COVID-19) [1,2]. This novel virus spread rapidly and, by the end of 2019, had reached nearly every country worldwide. The primary mode of transmission is through respiratory droplets expelled when an infected individual coughs, sneezes, or talks. Transmission can also occur via contact with contaminated surfaces.
COVID-19 presents a wide range of clinical manifestations, from mild to severe [1]. Common symptoms include fever, cough, shortness of breath, fatigue, loss of smell (anosmia), and loss of taste (ageusia). In more severe cases, the disease can progress to pneumonia and acute respiratory distress syndrome (ARDS), significantly increasing mortality risk, particularly in elderly individuals and those with underlying health conditions.
In response to the pandemic, governments and health organizations worldwide implemented numerous public health measures, including mass diagnostic testing, social distancing, mask mandates, temporary lockdowns, and large-scale vaccination campaigns [2]. Although various treatment strategies have been explored to alleviate symptoms and improve clinical outcomes [6,7], vaccines have proven particularly effective in reducing disease severity and transmission rates [3,4,5].
This multi-pronged approach underscores the complexity of managing a global health crisis such as COVID-19. Scientific research and public health strategies continue to evolve in pursuit of more effective control and prevention methods.
Once developed, the performance of any analytical method must be validated through a rigorous process. For the quantification of Remdesivir in rat plasma, method validation will assess critical parameters including stability under different storage conditions, linearity over a broad dynamic range, selectivity to minimize interference from endogenous substances, and accuracy and precision across various concentration levels. Accurate measurement of Remdesivir is essential in both preclinical animal studies and human clinical trials. Bioanalytical methods play a vital role in evaluating the safety and efficacy of therapeutic agents, thereby supporting potential regulatory approval [9].
Research is ongoing to explore the therapeutic benefits of Remdesivir, whether as a standalone treatment or in combination with other therapies. In future studies, bioanalytical techniques will be indispensable in evaluating the effectiveness of such interventions [10,11]. Numerous studies have focused on the development and validation of analytical methods for Remdesivir using platforms such as HPLC and LC-MS/MS [12,13]. These tools are crucial for both researchers and clinicians, enhancing the understanding and application of this promising antiviral agent.
Currently, efforts are underway to develop a sensitive and accurate HPLC method for quantifying Remdesivir in rat plasma. The successful validation and application of such a method will significantly impact its therapeutic potential. The primary objective of this work is to establish a robust bioanalytical technique that enables precise quantification of Remdesivir, thereby advancing knowledge of its pharmacological properties [14] and facilitating its broader clinical use.
Materials and Methods:
Chemicals and Reagents
Remdesivir and Favipiravir (used as the internal standard) were analyzed using a validated reverse-phase high-performance liquid chromatography (RP-HPLC) method. The instrumentation comprised a Waters e2695 HPLC system equipped with a photodiode array (PDA) detector and a Dikma Spursil RP-EP 18 column. A standard stock solution of Remdesivir was prepared by dissolving 60 mg in a suitable diluent to yield a concentration of 600 µg/mL, from which serial dilutions were made to generate working standards. Calibration samples were obtained by spiking rat plasma with these working solutions. Favipiravir stock solution was prepared by dissolving 20 mg in the same diluent.
Sample preparation was performed using a straightforward protein precipitation technique. Spiked plasma samples were mixed with the internal standard and extraction solvent, vortexed thoroughly, and centrifuged before being introduced into the chromatographic system. The mobile phase consisted of acetonitrile and 0.1% formic acid, which was filtered and sonicated prior to use. This systematic procedure facilitated the development of a robust, precise, and reproducible method for the quantification of Remdesivir in rat plasma, which is critical for pharmacokinetic and preclinical studies.
Preparation of Solutions
The mobile phase consisted of a 1:1 ratio of acetonitrile and 0.1% formic acid (prepared by diluting 1 mL of formic acid in 1000 mL of water). A stock solution of Remdesivir was prepared by dissolving 60 mg in 100 mL of diluent to obtain a 600 µg/mL concentration. This solution was further diluted with plasma to generate calibration standards. For the internal standard, 20 mg of Favipiravir was dissolved in 100 mL of diluent to create a stock solution, which was then diluted as required. Spiked plasma samples were prepared by adding both the internal standard and Remdesivir solutions, followed by protein precipitation for subsequent chromatographic analysis.
Method Development
Optimization of Chromatographic Conditions:
Multiple parameters were systematically investigated to optimize the chromatographic conditions for the detection of Remdesivir in rat plasma. The analysis was performed at a fixed detection wavelength of 225 nm, with an injection volume of 10 µL and a total run time of approximately 8 minutes.
Several columns were screened, and the DIKMA SPURSIL-EP RP18 (150 × 4.6 mm, 3 µm) column was selected for its superior separation performance. Various mobile phase compositions were evaluated, including combinations of ortho-phosphoric acid, methanol, acetonitrile, and water. The optimal separation of Remdesivir from endogenous plasma components was achieved using a mobile phase of 0.1% formic acid and acetonitrile in a 65:35 (v/v) ratio. A flow rate of 1.0 mL/min was maintained to ensure sharp, well-resolved peaks. These method development steps led to the establishment of a reliable and robust RP-HPLC method suitable for the accurate quantification of Remdesivir in rat plasma, thereby supporting pharmacokinetic profiling and preclinical research
Results and Discussion:
Method Validation:
System suitability:
| S. No. | Remdesivir | ISTD | ||
| Peak Area | RT (min) | Peak Area | RT (min) | |
| MQC-1 | 140069 | 5.288 | 306580 | 6.379 |
| MQC-2 | 140182 | 5.412 | 303097 | 6.53 |
| MQC-3 | 140073 | 5.412 | 307225 | 6.53 |
| MQC-4 | 140082 | 5.414 | 305873 | 6.534 |
| MQC-5 | 140052 | 5.416 | 302292 | 6.538 |
| MQC-6 | 140890 | 5.424 | 302394 | 6.543 |
| Mean | 5.393 | 6.5092 | ||
| SD | 0.0523 | 0.0639 | ||
| %CV | 0.973 | 0.981 | ||
Table no-1: System suitabaility
Fig No-1:Typical Chromatogram of Plasma Spiked Standard and ISTD
Sensitivity:
Sensitivity of the method was assessed by obtaining the responses of 6 replicates of LLOQ and % CV was calculated. At least 67% of the sample should be within 80-120% of nominal, and precision should be less than 20%CV. The% CV obtained for Remdesivir was 3.79%, satisfying the acceptance criterion. The sensitivity results were represented in Table 2
Sensitivity results
| S. No. | Remdesivir | |
| Cal Conc. (µg/mL) | % of Nominal Conc. | |
| LLOQ-1 | 0.029 | 96.66 |
| LLOQ-2 | 0.031 | 103.33 |
| LLOQ-3 | 0.031 | 103.33 |
| LLOQ-4 | 0.032 | 106.66 |
| LLOQ-5 | 0.03 | 100 |
| LLOQ-6 | 0.032 | 106.66 |
| Mean | 0.0308 | |
| SD | 0.0012 | |
| %CV | 3.689 | |
Table no-2: Sensitivity of Remdesivir
Linearity:
Eight spiked calibration solutions were prepared in the range of 0.06-0.9 µg/mL for RDR along with internal standard. The ratio of the analyte peak area to the ISTD peak area was used to quantify samples. Plot the response graph using peak area ratios versus plasma concentrations. The results were summarized in Table 3, calibration curve was represented in Figure 2
Linearity results
| Remdesivir | ||
| Conc. (µg/mL) | Area | Area response ratio |
| 0 | 0 | 0 |
| 0.06 | 11971 | 0.0304 |
| 0.15 | 21146 | 0.0561 |
| 0.3 | 45208 | 0.1225 |
| 0.6 | 91579 | 0.2341 |
| 0.75 | 115802 | 0.2854 |
| 0.9 | 137432 | 0.4589 |
Table no-3: Linearity results of Remdesivir
Fig no-2: Calibration curve of Remdesivir
Precision and Accuracy:
Precision and Accuracy of the assay should be evaluated both within and between runs. They are established by analyzing three distinct QC samples at low, mid, and high levels that fall within a predetermined range of the calibration curve. Each concentration should be tested with at least 5 replicates. For Intra-day batch the %CV values in the range of 0.66-7.06 % for RDR and for inter- batch variation the %CV values in the range of 2.4-6.26 % for RDR. The % mean accuracy for all analytes at HQC and LQC levels were within the acceptance limits of 85-115%. The data was presented in Table 4.
| Acquisition Batch ID Date | HQC | MQC 1 | LQC | LL QC |
| Nominal Concentration (ng/ml) | ||||
| 0.8 | 0.62 | 0.32 | 0.06 | |
| Calculated Concentration (ng/ml) | ||||
| P & A Batch-I | 0.85 | 0.61 | 0.31 | 0.064 |
| 0.81 | 0.64 | 0.35 | 0.062 | |
| 0.82 | 0.63 | 0.36 | 0.062 | |
| 0.86 | 0.65 | 0.37 | 0.063 | |
| 0.84 | 0.67 | 0.34 | 0.062 | |
| 0.87 | 0.66 | 0.38 | 0.065 | |
| N | 6 | 6 | 6 | 6 |
| Mean | 0.84166667 | 0.6433333 | 0.35166667 | 0.063 |
| SD | 0.02316607 | 0.0216025 | 0.02483277 | 0.00126 |
| % CV | 2.75240402 | 3.3578967 | 7.06145233 | 2.0078 |
| % Mean Accuracy | 105.21% | 103.76% | 109.90% | 105.00% |
Table no-4: Precision and Accuracy of Remdesivir
Stability:
Short term Stock solution stability:
Stock solutions of analytes and ISTD were freshly prepared and allowed to stand at 250C of for 6 hours to assess short term stability. For comparison, the prepared stock solutions should be stored in refrigerator below 10°C. The stability can be assessed by comparing mean responses of stability solutions with comparison solutions. The results were shown in Table 5.
| Replicate No | Remdesivir | |
| HQC | LQC | |
| Nominal Concentration | ||
| 0.8 ng/ml | 0.32 ng/ml | |
| Calculated Concentration (ng/mL) | ||
| 1 | 0.83 | 0.33 |
| 2 | 0.85 | 0.36 |
| 3 | 0.81 | 0.37 |
| 4 | 0.83 | 0.35 |
| 5 | 0.85 | 0.34 |
| 6 | 0.81 | 0.33 |
| Mean | 0.83 | 0.34667 |
| SD | 0.01789 | 0.01633 |
| %CV | 2.15525 | 4.71056 |
| %Stability | 103.75% | 108.33% |
Table no-5: Short term Stock solution stability
Long term Stock solution stability:
Stock solutions of analytes and ISTD were freshly prepared and these solutions should be stored at below 100C for 9 days to assess long term stability. For comparison, the prepared stock solutions should be stored in refrigerator below 10°C. The stability can be assessed by comparing mean responses of stability solutions with comparison solutions. The results were shown in Table 6.
| Replicate No | Remdesivir | |
| HQC | LQC | |
| Nominal Concentration | ||
| 0.8 ng/ml | 0.32 ng/ml | |
| Calculated Concentration (ng/mL) | ||
| 1 | 0.81 | 0.31 |
| 2 | 0.84 | 0.35 |
| 3 | 0.82 | 0.36 |
| 4 | 0.83 | 0.32 |
| 5 | 0.88 | 0.34 |
| 6 | 0.87 | 0.33 |
| Mean | 0.84167 | 0.335 |
| SD | 0.02787 | 0.01871 |
| %CV | 3.31114 | 5.58456 |
| % Stability | 105.21% | 104.69% |
Table no-6: Long term Stock solution stability
The freeze-thaw (FT) stability was determined by freezing of freshly prepared HQC and LQC standards at -20°C and thawed to room temperature over 4 freezing cycles. Between cycles, QC samples should be kept frozen for at least 12 hours. The obtained responses of QC stability samples should be compared with the freshly prepared CC samples. The stability data was compiled in Table 7
| Replicate No | Remdesivir | |
| HQC | LQC | |
| Nominal Concentration | ||
| 0.8 ng/ml | 0.32 ng/ml | |
| Calculated Concentration (ng/mL) | ||
| 1 | 0.85 | 0.36 |
| 2 | 0.81 | 0.35 |
| 3 | 0.82 | 0.32 |
| 4 | 0.806 | 0.32 |
| 5 | 0.806 | 0.33 |
| 6 | 0.805 | 0.34 |
| Mean | 0.81617 | 0.33667 |
| SD | 0.01749 | 0.01633 |
| %CV | 2.14248 | 4.85047 |
| % Stability | 102.02% | 105.21% |
Table no-7: Freeze-Thaw stability
The dried extract stability was studied by storing the replicates in the dried form at a < 10°C for 38 hours. The obtained responses of QC samples should be compared with the freshly prepared CC samples. The results were shown in Table 8.
| Replicate No | Remdesivir | |
| HQC | LQC | |
| Nominal Concentration | ||
| 0.8 ng/ml | 0.32 ng/ml | |
| Calculated Concentration (ng/mL) | ||
| 1 | 0.803 | 0.36 |
| 2 | 0.82 | 0.35 |
| 3 | 0.81 | 0.35 |
| 4 | 0.82 | 0.32 |
| 5 | 0.83 | 0.31 |
| 6 | 0.82 | 0.34 |
| Mean | 0.81717 | 0.33833 |
| SD | 0.00939 | 0.01941 |
| %CV | 1.14906 | 5.73633 |
| % Stability | 102.15% | 105.73% |
Table no-8: Dry extract stability
Long Term Stability in matrix:
The long-term stability was determined by analyzing the freshly prepared LQC and HQC spiked standard which are stored at -200C for 15 days. The obtained responses of QC samples should be compared with the freshly prepared CC samples. The data was shown in Table 9.
| Replicate No | Remdesivir | |
| HQC | LQC | |
| Nominal Concentration | ||
| 0.8 ng/ml | 0.32 ng/ml | |
| Calculated Concentration (ng/mL) | ||
| 1 | 0.812 | 0.36 |
| 2 | 0.817 | 0.34 |
| 3 | 0.821 | 0.38 |
| 4 | 0.826 | 0.37 |
| 5 | 0.809 | 0.34 |
| 6 | 0.819 | 0.35 |
| Mean | 0.81728 | 0.35667 |
| SD | 0.00614 | 0.01633 |
| %CV | 0.75109 | 4.57849 |
| % Stability | 102.16% | 111.46% |
Table no-9: Long Term Stability in matrix
The Bench top stability was assessed by analyzing six replicates of LQC and HQC samples which are allowed to stand at ambient temperature for 6 hours. The obtained responses of QC stability samples should be compared with the freshly prepared CC samples. The
stability results were shown in Table 10.
| Replicate No | Remdesivir | |
| HQC | LQC | |
| Nominal Concentration | ||
| 0.8 ng/ml | 0.32 ng/ml | |
| Calculated Concentration (ng/mL) | ||
| 1 | 0.806 | 0.31 |
| 2 | 0.812 | 0.35 |
| 3 | 0.815 | 0.36 |
| 4 | 0.795 | 0.32 |
| 5 | 0.797 | 0.33 |
| 6 | 0.813 | 0.34 |
| Mean | 0.80633 | 0.335 |
| SD | 0.00857 | 0.01871 |
| %CV | 1.06299 | 5.58456 |
| % Stability | 100.79% | 104.69% |
Table no-10: Bench Top Stability
DISCUSSION:
The present investigation described a validated bio-analytical method for determination of Remdesivir in rat plasma using RP-HPLC. The proposed method was found to be accurate, sensitive, selective and precise. Excellent recoveries obtained with simple protein precipitation used for sample extraction. The method is in compliance with the recommendations of US FDA guidelines. Based on the statistical analysis of results, the suggested approach was useful for routine commercial analysis and bioequivalence studies of Remdesivir samples in drug product or in drug samples.
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