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Research Article
Simultaneous quantitative determination of remdesivir, dexamethasone, rosuvastatin, and atorvastatin in human plasma using HPLC-UV analysis
expand article infoMiglena Smerikarova‡, Stanislav Bozhanov‡, Vania Maslarska‡
‡ Medical University-Sofia, Sofia, Bulgaria

Corresponding author: Miglena Smerikarova ( m.smerikarova@pharmfac.mu-sofia.bg )

Academic editor: Ivanka Pencheva
© 2025 Miglena Smerikarova, Stanislav Bozhanov, Vania Maslarska.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Smerikarova M, Bozhanov S, Maslarska V (2025) Simultaneous quantitative determination of remdesivir, dexamethasone, rosuvastatin, and atorvastatin in human plasma using HPLC-UV analysis. Pharmacia 72: 1-13. https://doi.org/10.3897/pharmacia.72.e165238
Open Access

Abstract

A new reversed-phase high-performance liquid chromatographic method was developed for the simultaneous determination and quantification of two clinically proven drugs in COVID-19 treatment – remdesivir and dexamethasone – and two 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors – rosuvastatin and atorvastatin – in human plasma by isocratic elution and ultraviolet detection. A mobile phase consisting of 0.1% trifluoroacetic acid in water and acetonitrile (60:40, v/v) was used in the proposed analytical procedure, and the chromatographic determination was performed on a Purospher® RP–18 column at room temperature. The developed method was validated for linearity in the range of 1–24 µg/ml, with correlation coefficients greater than 0.998. The percentage recovery of the analyzed drugs was 100.87, 99.71, 102.41, and 93.38% for remdesivir, dexamethasone, rosuvastatin, and atorvastatin, respectively. The developed, effective, and specific method is suitable for implementation in routine quality control and clinical laboratory practice.

Keywords

remdesivir, dexamethasone, statins, HPLC, plasma

Introduction

The COVID-19 virus has led to a reevaluation of traditional therapeutic strategies for significant diseases. Numerous COVID-19 patients acquire dyslipidemia, which can result in life-threatening metabolic diseases and thrombotic complications (Liu et al. 2022). Statins, which can lower the levels of serum total cholesterol and low-density lipoproteins, are among the potential treatment agents in these situations (Liu et al. 2022). Statins inhibit 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA), reducing the synthesis of isoprenoids. This is particularly important in cases where the virus penetrates cells through angiotensin-converting enzyme 2 protein and lipid rafts. Statins also reduce the number of lipid rafts by lowering endogenous cholesterol synthesis, which may limit viral penetration into host cells (Olszewska-Parasiewicz et al. 2021). They also minimize the regeneration potential of vascular endothelium and reduce the overexpression of pro-inflammatory cytokines, primarily interleukin-6. Statins, especially atorvastatin and rosuvastatin, reduce the incidence of recurrent pulmonary embolism, a severe thromboembolic condition. The increase in arachidonic acid levels – whose deficit may raise the chance of acquiring COVID-19 – is a notable indirect antiviral mechanism of statin action. Thus, statins have a variety of direct and indirect benefits that can improve the prognosis of COVID-19 patients (Surma et al. 2021).

Single administration or a combination of an antiviral drug and a corticosteroid is one of the treatment regimens recommended by the World Health Organization (WHO) for use in hospitalized patients with moderate and severe cases of COVID-19 (National Institute of Health 2023). The antiviral pharmaceutical remdesivir (RDV) (Fig. 1), which has a positive safety profile (Lin et al. 2021) and is effective in both severe and moderate cases of COVID-19 (Morris et al. 2021), has shown the best results. Clinical investigations have demonstrated statistically significant improvements with RDV administration in mild cases not requiring oxygen therapy, with considerably faster recovery times compared to placebo controls, suppression of respiratory disease progression, and decreased mortality (Beigel et al. 2020; Wong et al. 2022a). The impact of treatment with RDV and corticosteroids as combination therapy has been demonstrated (Pan et al. 2020), especially in cases of pneumonia, which may result in serious respiratory failure. Due to its anti-inflammatory and immunosuppressive effects, dexamethasone (DXM) (Fig. 1) is widely used in clinical practice and has been linked to a significant reduction in mortality (Ahmed and Hassan 2020; RECOVERY Collaborative Group et al. 2021). Therapy with RDV and DXM has been associated with significantly shortened recovery times for hospitalized patients and improved health conditions (Gandhi et al. 2020; Wong et al. 2022b). It is not recommended to stop ongoing statin therapy during COVID-19 treatment; however, there is no significant evidence from clinical trials on initiating such therapy de novo, except in specific clinical situations (Pawlos et al. 2021).

Figure 1. 

Chemical structures of RDV, DXM, RVS, FVS, and AVS.

Observational studies have suggested the protective effects of statins in COVID-19 patients (Daniels et al. 2020; Zhang et al. 2020; Diaz-Arocutipa et al. 2021; Peymani et al. 2021; Lee et al. 2021; Wu et al. 2021; Bouillon et al. 2022; Cho et al. 2022; Kow and Hasan 2022; Santosa et al. 2022; Rivera et al. 2023), with a decreased risk for invasive mechanical ventilation, reduced risk of intensive care admission, and lower risk of death (Liu et al. 2022). On the other hand, some studies have found no association between statin use and lower COVID-19 mortality rates (Butt et al. 2020; Kuno et al. 2022). These highly heterogeneous observational findings require confirmation by ongoing randomized clinical trials (Scheen 2021).

Severe COVID-19 increases serum glucose and creatine kinase levels, but statins can also cause side effects. Clinical trials are ongoing worldwide to validate the safety and effectiveness of statin therapy for COVID-19 patients. However, caution is needed due to potential risks of statin-associated muscle symptoms, liver injury, new-onset diabetes, renal injury, and neurological and neurocognitive disorders. Monitoring daily health conditions is crucial for COVID-19 patients using statins. Patients with neurological or renal disorders should use them cautiously due to possible worsening of these conditions during COVID-19 infection. Drug interactions should be managed carefully, as most statins are metabolized by CYP450 enzymes, mainly through CYP3A4. Further analysis is needed to prescribe the appropriate intensity of statin therapy (Liu et al. 2022).

The present study aimed to develop and optimize an isocratic reversed-phase high-performance liquid chromatographic (RP-HPLC) method for the determination of RDV, DXM, and some of the most commonly used statins – rosuvastatin (RVS) and atorvastatin (AVS) (Fig. 1) – in a biological matrix (human plasma) using fluvastatin (FVS) as an internal standard. In the available literature, detailed information exists on developed liquid chromatographic methods for RDV, DXM, RVS, FVS, and AVS alone (Nakashima et al. 2001; Zarghi et al. 2005; Kumar et al. 2006; Farahani et al. 2009; Haq et al. 2018; Ibrahim et al. 2021; Kishore et al. 2021) or in combination with other drugs (Iriarte et al. 2009; Abdallah 2011; Ashutosh Kumar et al. 2015; Heda et al. 2011; Shah et al. 2011; Ashfaq et al. 2013; Lariya and Agrawal 2015; Farajzadeh et al. 2016; Sangshetti et al. 2016; Chen et al. 2017; Porwal and Talele 2017; Razzaq et al. 2017; Hamdy et al. 2022), in dosage forms (Abdallah 2011; Heda et al. 2011; Lariya and Agrawal 2015; Sangshetti et al. 2016; Chen et al. 2017; Razzaq et al. 2017; Haq et al. 2018; Ibrahim et al. 2021) and biological matrices (Nakashima et al. 2001; Zarghi et al. 2005; Kumar et al. 2006; Farahani et al. 2009; Iriarte et al. 2009; Abdallah 2011; Shah et al. 2011; Ashfaq et al. 2013; Ashutosh Kumar et al. 2015; Farajzadeh et al. 2016; Porwal and Talele 2017; Razzaq et al. 2017; Kishore et al. 2021; Hamdy et al. 2022). However, to our knowledge, no developed and validated method exists for the simultaneous quantification of these five drugs in a synthetic mixture and biological matrix. The proposed method is suitable for assaying the target pharmaceuticals in a single run and can be implemented in routine quality control and clinical laboratory practice for therapeutic drug monitoring.

Materials and methods

Materials and reagents

All chemicals, reagents, and analytical standards used for method development and validation were of HPLC grade. RDV, DXM, RVS, AVS, and FVS, each with a purity greater than 98%, were purchased from Sigma-Aldrich Co. HPLC-grade acetonitrile (ACN), methanol, and trifluoroacetic acid (TFA) were used for mobile phase and stock solution preparation. A calibration curve was built using a blank human plasma standard provided by Sigma-Aldrich Co. All additional reagents needed to develop the analytical method were suitable for HPLC analysis.

Chromatographic conditions

The proposed method was developed on a SHIMADZU Corporation chromatographic system equipped with a vacuum degasser, pump, auto-injector, and UV-VIS detector. LabSolution software was used for recording and processing results. A Purospher® RP-18 (150 × 4.6 mm, 5 µm) chromatographic column, equipped with an ODS guard column, was used as the stationary phase at ambient temperature. The mobile phase consisted of water and acetonitrile (60:40, v/v) containing 0.1% TFA, filtered through a 0.45 µm membrane filter, and sonicated for 10 min in an ultrasonic bath. A flow rate of 1.0 ml/min, isocratic elution, and a run time of 30 min were used. The detector wavelength was set at 245 nm, and the injection volume was 20 µl.

Preparation of standard stock solutions and laboratory-prepared mixtures

Five stock solutions (RDV-SS, DXM-SS, RVS-SS, FVS-SS, and AVS-SS) with a concentration of 1000 µg/ml of RDV, DXM, RVS, FVS, and AVS, respectively, were prepared by dissolving 20 mg of each substance in methanol using a 20.0 ml class A volumetric flask. A 40 µg/ml mixed stock solution (RDRFA-SS) was prepared by transferring 1.00 ml aliquots of RDV-SS, DXM-SS, RVS-SS, FVS-SS, and AVS-SS into the same 25.0 ml volumetric flask and diluting with methanol. After a series of appropriate dilutions and analyses, a calibration curve was constructed using six different concentrations for each drug in the range of 1–24 µg/ml.

Preparation of synthetic mixture working solutions

Six working solutions (RDRFA-SM1, RDRFA-SM2, RDRFA-SM3, RDRFA-SM4, RDRFA-SM5, and RDRFA-SM6) with concentrations of 1, 2, 4, 8, 16, and 24 µg/ml were prepared by pipetting aliquots of 0.25, 0.5, 1.0, 2.0, 4.0, and 6.0 ml from the RDRFA-SS solution and diluting with methanol to 10.0 ml in volumetric flasks. All solutions were stored at 2–4 °C before analysis. For intraday and interday precision and accuracy analysis, three working solutions (RDRFA-QC1, RDRFA-QC2, and RDRFA-QC3) with concentrations of 1.5, 6, and 20 µg/ml were prepared by diluting 0.75, 3.0, and 10.0 ml aliquots of the RDRFA-SS solution to 20.0 ml with methanol in volumetric flasks.

Preparation of plasma working solutions

A second stock solution (RDRA-SS) with a concentration of 200 µg/ml of RDV, DXM, RVS, and AVS, respectively, and six working solutions (RDRA-P1, RDRA-P2, RDRA-P3, RDRA-P4, RDRA-P5, and RDRA-P6) with concentrations of 5, 10, 20, 40, 80, and 120 µg/ml were prepared by pipetting aliquots of 0.125, 0.25, 0.5, 1.0, 2.0, and 3.0 ml from the RDRA-SS solution and diluting with methanol to 5.0 ml in volumetric flasks. The solutions were used for plasma method validation and stored at 2–4 °C before analysis. Three working solutions (RDRA-QC1, RDRA-QC2, and RDRA-QC3) with concentrations of 7.5, 30, and 100 µg/ml were prepared by diluting 0.75, 3.0, and 10.0 ml aliquots, respectively, in the same diluent.

Preparation of internal standard solution

FVS was chosen as the internal standard (IS) for bioanalytical method development and validation. The working solution (FVS-WS) was prepared by pipetting 2.4 ml from the FVS-SS, transferring it to a 25.0 ml volumetric flask, and diluting with methanol to obtain a concentration of 96 µg/ml.

Preparation of calibration and quality control solutions for the plasma assay

All calibration and quality control solutions were prepared with human plasma standards. A portion of 400 µl plasma was mixed with 100 µl of RDRA-P1, RDRA-P2, RDRA-P3, RDRA-P4, RDRA-P5, RDRA-P6, RDRA-QC1, RDRA-QC2, or RDRA-QC3, respectively. The resulting concentrations were 1, 2, 4, 8, 16, and 24 µg/ml for the calibration standards and 1.5, 6, and 20 µg/ml for the quality control samples. During preparation, blank plasma was vortex mixed for 2 min, the indicated volumes of the stock solutions were added, and the samples were mixed carefully for another 5 min.

Sample preparation

The procedure for plasma protein precipitation was previously developed by our research team and adapted for the present method (Smerikarova et al. 2023). It was carried out as follows: a 150 µl plasma aliquot was first mixed for 5 min with 50 µl of the FVS-WS, then 600 µl of acetonitrile was added, and the sample was vortex mixed for 10 min. This was followed by 15 min of sonication in an ultrasonic bath and 15 min of shaking at 500 rpm. The supernatant was separated after 10 min of centrifugation at 13 000 rpm, and 600 µl was filtered through a 0.45 µm syringe filter. HPLC analysis was carried out with an injection volume of 20 µl.

Method validation

The developed method applied for the laboratory-prepared mixture and plasma quantification was validated according to the International Council for Harmonization (ICH) guidelines for bioanalytical method validation (ICH M10) and analytical procedure development (ICH Q14), assessing parameters including selectivity, linearity, accuracy, precision, system suitability, limit of detection, and limit of quantification (ICH M10 2019; ICH Q14 2022).

Results

Optimization of chromatographic conditions and experimental parameters

Three main parameters were optimized during method development and validation – stationary phase, mobile phase, and ultraviolet (UV) detection. The choice of chromatographic column depends on the physicochemical properties of the analytes and the biological matrix. Octadecylsilane (C18) and octylsilane (C8) reversed-phase columns with different lengths (150 or 250 mm) and diameters (4.6 or 4.0 mm) were tested, and the best results for resolution and selectivity were obtained with a 150 × 4.6 mm C18 column. The influence of particle size was also investigated, and 5 µm was determined to be the most suitable for providing stable and reliable analysis conditions while avoiding the risk of clogging during the bioassay. The column chosen for the analysis was Purospher® RP-C18 (150 × 4.6 mm, 5 µm) equipped with an ODS guard column.

According to preliminary literature data, various compositions and ratios of mobile phase solvents were tested. Several adjustments were made to achieve better separation of the target drugs, including the use of buffers and different organic solvents (pure or with modifiers) in varied ratios. First, the type of organic solvent was assessed. Both methanol and ACN were tested, and ACN was selected because it provided better results, with suitable retention times (tR) and peak asymmetry, whereas methanol delayed drug elution. Different ratios of ACN were evaluated, and– as expected – increasing the ACN ratio decreased retention times. Mobile phases consisting of ACN and pure distilled water, buffer solution, or a solution of TFA were analyzed. The use of pure water provided good separation of RDV and DXM but had a significant adverse effect on statin retention times, with all showing tR > 10 min. A mobile phase modified with potassium dihydrogen phosphate buffer also failed to meet expectations, as it caused retention times to double; even RDV, which previously had a relatively short retention time, showed tR > 7 min. The effect of pH (range 3–6) and buffer concentration (0.5 mM to 20 mM) was investigated, but satisfactory results were not obtained. All drugs exhibited long retention times and peak asymmetry values greater than 2.4. The best results were obtained with the third mobile phase composition (0.1% TFA in water/ACN mixture). A series of ratios of 0.1% TFA in water:acetonitrile (70:30 to 30:70, v/v) was tested, and 60:40 (v/v) was found to be the most suitable, as it showed the best separation of the substances from matrix components and provided the shortest analytical time. The isocratic mode of elution was used at a flow rate of 1.0 ml/min and ambient temperature. No risk to the stability of the analyzed solutions and plasma samples was detected. Based on the spectra of the investigated substances (Fig. 2), a wavelength of 245 nm was chosen as the most suitable for the analysis.

Figure 2. 

UV spectra of RDV, DXM, RVS, FVS, and AVS.

Figure 3. 

Typical chromatograms obtained from A. Mobile phase; B. Blank plasma sample.

Method validation

Selectivity

Under the optimized chromatographic conditions, the proposed method showed excellent potential for determining RDV, DXM, RVS, FVS, and AVS in their laboratory-prepared synthetic mixture or biological matrix (plasma) without any interferences. Representative chromatograms (Fig. 3A, B), obtained during the analysis of mobile phase or blank plasma samples, confirmed the high degree of specificity and selectivity of the developed method. All drug substances were well separated within the analytical run of 30 min.

Linearity

The linearity of the proposed method was studied and evaluated through triplicate analysis of six calibration standards at fixed concentration levels from 1 to 24 µg/ml. All accuracy results obtained during the analysis lay within the range of 94.8–105.5% and 82.8–112.2% for the laboratory-prepared mixture and plasma samples, respectively, fully meeting the predefined acceptance criteria (Table 1).

Table 1.
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Linearity, accuracy, and precision results for the RDV, DXM, RVS, FLV, and AVS calibration curves.

Laboratory-prepared mixture RDV DXM RVS FVS AVS
Concentration (µg/ml) Mean ± SD CV% d% Mean ± SD CV% d% Mean ± SD CV% d% Mean ± SD CV% d% Mean ± SD CV% d%
1 1.033 ± 0.001 0.11 3.28 1.054 ± 0.001 0.08 5.36 1.047 ± 0.006 0.55 4.68 1.053 ± 0.001 0.12 5.32 1.022 ± 0.000 0.02 2.23
2 1.935 ± 0.001 0.07 -3.25 1.941± 0.004 0.22 -2.95 1.927 ± 0.005 0.27 -3.66 1.979 ± 0.000 0.02 -1.05 1.923 ± 0.005 0.23 -3.83
4 3.794 ± 0.002 0.05 -5.15 3.890 ± 0.007 0.17 -4.79 3.807 ± 0.001 0.03 -4.83 3.893 ± 0.007 0.19 -2.66 3.807 ± 0.012 0.30 -4.82
8 8.189± 0.031 0.38 2.36 8.149 ± 0.030 0.37 1.86 8.171 ± 0.027 0.33 2.14 8.213 ± 0.039 0.48 2.66 8.192 ± 0.023 0.28 2.40
16 16.222 ± 0.103 0.64 1.39 16.189 ± 0.105 0.65 1.18 16.207 ± 0.116 0.71 1.29 15.745 ± 0.104 0.66 -1.59 16.245 ± 0.112 0.69 1.53
24 23.828 ± 0.032 0.14 -0.66 23.859 ± 0.047 0.20 -0.59 23.841 ± 0.023 0.09 -0.66 24.116 ± 0.076 0.31 0.48 23.810 ± 0.001 0.00 -0.79
Linear equation y = 58458x - 12427 y = 72925x - 16660 y = 47576x - 9610.6 y = 45022x - 1746.1 y = 48502x - 10532
R2 0.9996 0.9997 0.9997 0.9997 0.9996
Plasma RDV DXM RVS AVS
Concentration (µg/ml) Mean ± SD CV% d% Mean ± SD CV% d% Mean ± SD CV% d% Mean ± SD CV% d%
1 0.899 ± 0.007 0.83 -10.09 0.905 ± 0.013 1.47 -9.46 0.966 ± 0.017 1.71 -3.43 0.825 ± 0.009 1.11 -17.47
2 2.008 ± 0.032 1.61 0.42 2.106 ± 0.026 1.22 5.32 2.072 ± 0.036 1.73 3.62 1.795 ± 0.037 2.09 -10.23
4 3.769 ± 0.036 0.96 -5.76 3.790 ± 0.021 0.56 -5.25 3.816 ± 0.021 0.56 -4.60 3.663 ± 0.013 0.36 -8.43
8 8.001 ± 0.012 0.15 0.01 7.995 ± 0.024 0.30 -0.06 8.003 ± 0.007 0.08 0.04 7.994 ± 0.031 0.39 -0.08
16 17.162 ± 0.072 0.42 7.26 16.927 ± 0.136 0.80 5.79 16.882 ± 0.026 0.15 5.51 17.545 ± 0.169 0.96 9.66
24 27.006 ± 0.069 0.26 9.57 26.592 ± 0.057 0.21 10.80 26.296 ± 0.053 0.20 9.57 26.406 ± 0.111 0.42 10.03
Linear equation y = 0.0686x - 0.0345 y = 0.0913x - 0.0383 y = 0.0525x - 0.0191 y=0.0522x-0.0275
R2 0.9985 0.9986 0.9990 0.9993

SD: standard deviation; CV: coefficient of variation; R2: correlation coefficient.

Two calibration curves were constructed using linear regression: one based on the coordinates of peak area versus solution concentration for the mobile phase calibration curve (Fig. 4A) and another based on the area ratio of each analyte’s chromatographic peak to the IS peak versus concentration (µg/ml) for the plasma calibration curve (Fig. 4B). High correlation coefficients (R2) indicated good linearity of the developed method, and the summarized results for the linear equation and R2 values are shown in Table 1. The method’s reproducibility was also assessed. The standard deviation (SD) for each concentration level was calculated, with results ranging from 0.001 to 0.169 for both assays.

Figure 4. 

Calibration curves obtained from A. Laboratory-prepared synthetic mixture; B. Human plasma.

The minimum requirement of six concentration levels (1, 2, 4, 8, 16, and 24 µg/ml) was met. For the laboratory-prepared mixture, all working solutions were prepared by appropriate dilution of the mixed stock solution with methanol. For the plasma bioanalysis, the working standard solutions were added to blank plasma samples, followed by processing and purification of an appropriate aliquot to make it suitable for analysis. Fig. 5A, B show representative chromatograms obtained for the laboratory-prepared mixture and plasma samples.

Figure 5. 

Typical chromatograms obtained from A. Laboratory-prepared mixture; B. Plasma sample. Legend: The retention time of RDV, DXM, RVS, FVS, and AVS was 3.7, 4.6, 7.0, 20.3, and 23.8 min, respectively.

Accuracy and precision

According to ICH M10 (ICH M10 2019), the accuracy, reliability, and reproducibility parameters of a developed method should be assessed by analyzing quality control (QC) samples at a minimum of three concentration levels within the calibration curve range. In this study, four QC samples were analyzed: the lower limit of quantification (1 µg/ml), low-concentration QC (1.5 µg/ml), medium-concentration QC (6 µg/ml), and high-concentration QC (20 µg/ml). Three separately prepared samples of each concentration were analyzed in triplicate for intraday precision. For interday validation, the same analyses were performed on three consecutive days. Detailed accuracy, reliability, and reproducibility data are summarized in Table 2.

Table 2.
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Accuracy, reliability, and reproducibility in a run and in time (n = 3).

Compound in laboratory-prepared mixture Spiked concentration (µg/ml) Intraday Interday
Mean ± SD CV% d% Mean ± SD CV% d%
RDV 1 1.095 ± 0.003 0.31 9.45 1.098 ± 0.003 0.30 9.81
1.5 1.597 ± 0.004 0.22 6.44 1.641 ± 0.011 0.69 9.43
6 5.977 ± 0.033 0.54 -0.38 6.144 ± 0.055 0.90 2.40
20 19.731 ± 0.037 0.19 -1.35 20.091 ± 0.058 0.29 0.46
DXM 1 1.117 ± 0.006 0.55 11.71 1.123 ± 0.004 0.39 12.32
1.5 1.619 ± 0.010 0.60 7.91 1.627 ± 0.010 0.62 8.50
6 5.972 ± 0.039 0.65 -0.46 6.053 ± 0.071 1.17 0.89
20 19.725 ± 0.032 0.16 -1.38 20.145 ± 0.090 0.44 0.72
RVS 1 1.083 ± 0.001 0.05 8.29 1.084 ± 0.007 0.64 8.39
1.5 1.591 ± 0.008 0.47 6.07 1.566 ± 0.013 0.85 4.37
6 5.915 ± 0.034 0.57 -1.41 5.991 ± 0.055 0.92 -0.16
20 19.493 ± 0.039 0.20 -2.54 19.714 ± 0.086 0.43 -1.43
FVS 1 0.915 ± 0.016 1.74 -8.53 0.912 ± 0.010 1.12 -8.81
1.5 1.487 ± 0.020 1.31 -0.84 1.426 ± 0.012 0.82 -4.96
6 5.657 ± 0.020 0.35 -5.71 5.431 ± 0.063 1.15 -9.49
20 19.718 ± 0.006 0.03 -1.41 17.977 ± 0.015 0.08 -10.12
AVS 1 1.041 ± 0.006 0.61 4.07 1.070 ± 0.011 1.04 6.96
1.5 1.582 ± 0.016 1.01 5.47 1.508 ± 0.015 1.03 0.53
6 5.859 ± 0.030 0.50 -2.36 5.657 ± 0.012 0.21 -5.72
20 19.156 ± 0.024 0.12 -4.22 18.619 ± 0.072 0.39 -6.91
Compound in plasma Spiked concentration (µg/ml) Intraday Interday
Mean ± SD CV% d% Mean ± SD CV% d%
RDV 1 0.955 ± 0.021 2.15 -4.53 0.924 ± 0.014 1.49 -7.57
1.5 1.467 ± 0.017 1.19 -2.18 1.485 ± 0.003 0.17 -0.98
6 6.001 ± 0.104 1.73 0.01 6.076 ± 0.123 2.03 1.27
20 22.037 ± 0.234 1.06 10.18 21.964 ± 0.160 0.73 9.82
DXM 1 0.906 ± 0.003 0.34 -9.43 0.895 ± 0.031 3.51 -10.50
1.5 1.512 ± 0.003 0.17 0.80 1.596 ± 0.058 3.64 6.40
6 6.016 ± 0.107 1.78 0.26 6.700 ± 0.144 2.15 11.66
20 21.439 ± 0.318 1.48 7.20 21.802 ± 0.287 1.31 9.01
RVS 1 0.988 ± 0.018 1.78 -1.23 0.967 ± 0.025 2.59 -3.30
1.5 1.550 ± 0.015 0.99 3.36 1.561 ± 0.052 3.31 4.04
6 6.077 ± 0.090 1.47 1.29 6.738 ± 0.154 2.29 12.30
20 21.239 ± 0.299 1.41 6.20 20.113 ± 0.344 1.71 0.56
AVS 1 0.872 ± 0.015 1.66 -12.83 0.869 ± 0.020 2.33 -13.10
1.5 1.380 ± 0.024 1.76 -8.00 1.543 ± 0.054 3.52 2.89
6 5.447 ± 0.080 1.47 -9.21 6.270 ± 0.209 3.33 4.49
20 20.716 ± 0.282 1.36 3.58 19.922 ± 0.332 1.67 -0.39

SD: standard deviation; CV: coefficient of variation.

Reproducibility within a run was assessed by the coefficient of variation (CV%), which was less than 1.74% for the bulk drug and 2.15% for the bioanalytical method at all concentration levels studied. Accuracy within a run was assessed by the percentage deviation of the average concentration compared to the nominal value (d%), ranging from −12.83 to 11.71% for both methods. Interday analysis also confirmed the method’s accuracy and reproducibility. As shown in Table 2, interday CV% values were less than 3.65%, and d% values ranged from −13.10 to 12.32%.

In conclusion, the accuracy and precision of the method fully met the predefined acceptance criteria.

System suitability

The proposed analytical method was validated for system suitability parameters according to the requirements established by the ICH Q14 guidelines (ICH Q14 2022). Retention time (tR), number of theoretical plates (N), capacity factor (κ’), selectivity (α), resolution (RS), and tailing factor (Tf), associated with the specifics of the developed chromatographic procedure, were analyzed. Fast and effective separation of the analytes was achieved under optimized chromatographic conditions. A six-fold analysis of the 6 µg/ml solution was performed in the mobile phase and plasma, and the obtained values were κ ≥ 2.04, α ≥ 1.14, N ≥ 2050, RS ≥ 2.06, and Tf ≤ 1.34. Table 3 provides the detailed mean values.

Table 3.
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Results of the system suitability tests.

Parameter (Acceptance criteria) tR N (NLT 2000) κ’ (NLT 2.0) α (NLT 1.0) RS (NLT 2.0) Tf (NMT 2.0)
Compound in:
Laboratory-prepared mixture
RDV 3.779 3294 2.043 2.712 2.411 1.257
DXM 4.676 4602 2.405 1.489 3.329 1.212
RVS 7.085 5204 2.644 1.882 7.207 1.114
FVS 20.323 8698 9.452 3.575 20.937 1.001
AVS 23.820 8189 11.250 1.190 3.633 0.978
Plasma
RDV 3.705 2053 2.095 1.140 2.065 1.167
DXM 4.617 2173 2.680 1.372 2.178 1.332
RVS 7.033 2417 4.606 1.719 4.750 1.171
FVS (IS) 20.343 4881 15.216 3.304 15.321 1.133
AVS 23.813 5680 17.981 1.182 2.851 1.054

tR: retention time; N: theoretical plates; κ’: capacity factor; α: selectivity; RS: resolution; Tf: tailing factor; NLT: no less than; NMT: no more than.

Recovery of RDV, DXM, RVS, and AVS from plasma

By spiking blank plasma samples with the laboratory-prepared mixture solution at three fixed concentration levels and using protein precipitation for plasma protein removal, the recovery of the developed method was assessed. Across the predetermined range, three separately prepared samples were analyzed and evaluated in triplicate. Table 4 summarizes the percentage recovery for the analytes, which ranged from 85.68 to 111.28%.

Table 4.
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Recovery of RDV, DXM, RVS, and AVS from spiked human plasma (n = 3).

Compound Spiked concentration (µg/ml) Concentration found (µg/ml) Recovery (%) (mean ± SD) CV %
RDV 1 0.95 95.45 ± 2.03 2.12
1.5 1.47 97.82 ± 1.15 1.17
6 6.00 100.01 ± 1.73 1.73
20 22.04 110.18 ± 1.17 1.06
DXM 1 0.91 90.57 ± 0.32 0.35
1.5 1.51 100.81 ± 0.19 0.18
6 6.02 100.27 ± 1.79 1.78
20 21.44 107.20 ± 1.59 1.48
RVS 1 0.99 98.78 ± 1.72 1.74
1.5 1.55 103.36 ± 1.03 1.00
6 6.08 101.29 ± 1.50 1.48
20 21.24 106.19 ± 1.50 1.41
AVS 1 0.87 87.14 ± 1.44 1.66
1.5 1.38 92.00 ± 1.59 1.73
6 5.45 90.79 ± 1.33 1.46
20 20.72 103.58 ± 1.41 1.36

SD: standard deviation; CV: coefficient of variation.

Limit of detection (LOD) and limit of quantification (LOQ)

Two common approaches – experimental and computational – are used for LOD and LOQ determination. In this study, the experimental technique was applied. LOD was determined after a series of dilutions at a signal-to-noise ratio of 2:1, and LOQ was determined at a signal-to-noise ratio of 10:1. For the laboratory-prepared mixture assay, the LOD and LOQ were 0.15 and 1.00 µg/ml, respectively. Similar results were found during the plasma assay, where LOD was 0.2 µg/ml and LOQ was 1.0 µg/ml. These results confirmed the feasibility of the developed methods for application in clinical practice and for detecting even trace amounts of the drugs in biological matrices.

Discussion

Some of the major benefits of simultaneous drug determination include efficiency, applicability, cost-effectiveness, and reduced analysis time. The ability to use the method for either individual or combined drug analysis is also noteworthy. Although challenges may arise during chromatographic optimization – such as solvent–substance interactions with the mobile phase and the influence of pH, temperature, and flow rate – the most important step remains a detailed investigation of the elution patterns of various pharmacological compounds. In many cases, peak shape and peak area are significantly affected by changes in the percentage of organic solvent in the mobile phase, which in turn impacts the sensitivity of the analytical technique. During the development of the proposed method, this was particularly evident for the three analyzed statins (RVS, FVS, and AVS). The peaks of RDV and DXM were largely unaffected, but difficulties arose in separating them from biological matrix components. Sharp and well-separated peaks were obtained using a mobile phase consisting of 0.1% TFA in water and acetonitrile (60:40, v/v) at a flow rate of 1.0 ml/min. Quantification was carried out with UV detection at 245 nm based on peak area. The retention times were 3.77, 4.67, 7.09, 20.30, and 23.79 min for RDV, DXM, RVS, FVS, and AVS, respectively. Temperature was also a crucial parameter for elution because it influenced peak tailing and accelerated elution, which could lead to undefined changes in retention times. The quantification method was validated according to ICH M10 guidelines, and all predefined criteria were met. The selected chromatographic conditions provided both a short analysis time and reliable quantification of RDV, DXM, RVS, FVS, and AVS, with performance comparable to previously reported methods (Nakashima et al. 2001; Zarghi et al. 2005; Kumar et al. 2006; Farahani et al. 2009; Iriarte et al. 2009; Abdallah 2011; Shah et al. 2011; Ashfaq et al. 2013; Ashutosh Kumar et al. 2015; Farajzadeh et al. 2016; Porwal and Talele 2017; Razzaq et al. 2017; Kishore et al. 2021; Hamdy et al. 2022).

Conclusion

A simple, precise, and sensitive liquid chromatographic method was developed and validated for the simultaneous determination of RDV, DXM, and selected HMG-CoA reductase inhibitors (RVS, FVS, and AVS) in bulk form, laboratory-prepared mixtures, and biological matrices (human plasma). The proposed HPLC-UV method is the first to be developed for the combined application of these therapeutic drugs in COVID-19 treatment alongside statin therapy. The applied isocratic elution mode, ambient temperature, and low percentage of organic solvents highlighted the chromatographic simplicity of the method. The ability to process a large number of samples rapidly, combined with the high precision of the analytical procedure, makes it suitable for routine quality control and clinical laboratory practice.

Acknowledgements

This work was supported by the Bulgarian Ministry of Education and Science under the National Program for Research “Young Scientists and Postdoctoral Students-2” 2022/2023.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statements

The authors declared that no clinical trials were used in the present study.

The authors declared that no experiments on humans or human tissues were performed for the present study.

The authors declared that no informed consent was obtained from the humans, donors or donors’ representatives participating in the study.

The authors declared that no experiments on animals were performed for the present study.

The authors declared that no commercially available immortalised human and animal cell lines were used in the present study.

Use of AI

No use of AI was reported.

Funding

No funding was reported.

Author contributions

All authors have contributed equally.

Author ORCIDs

Miglena Smerikarova https://orcid.org/0000-0002-5106-1036

Stanislav Bozhanov https://orcid.org/0000-0002-6543-9087

Vania Maslarska https://orcid.org/0000-0002-5057-4403

Data availability

All of the data that support the findings of this study are available in the main text.

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