Analysis of Remdesivir and other Antiviral Drugs Using LC-MS/MS

Introduction

Remdesivir is a broad-spectrum antiviral drug, typically administered through injections. The drug has been tested in 2020 as a treatment for COVID-19, through the WHO Solidarity Trial. Here we present a fast and robust HPLC-MS/MS method that can be used for Remdesivir and three structural antiviral analogues. Indinavir, Nelfinavir, and Saquinavir are protease inhibitors, typically used in the treatment of the human immunodeficiency virus (HIV).

Several different stationary phases were screened through the development process, with the aim to find and provide the best selectivity for the target analytes. The best overall results were achieved with an RP-Amide column, but also C18, AQ-C18 or Phenyl-Hexyl phases can offer appropriate retention and peak shape for Remdesivir. With a short 30x2.1 mm Ascentis® Express RP-Amide stationary phase used in gradient mode it is possible to have all compounds well retained and fully resolved under two minutes. Calibration data for Remdesivir (1-250 ng/mL) display adequate linearity (R2 = 0.9995) with a Limit of detection (LOD) and Limit of quantitation (LOQ) determined as  0.7 and 1.3 ng/ml (95% confidence interval), respectively.

 

Remdesivir


 

Figure 1. Remdesivir

C27H35N6O8P (CAS No. 1809249-37-3)

 

 

Indinavir

Figure 3. Indinavir

C36H47N5O4 (CAS No. 150378-17-9)

 

Nelfinavir
Figure 2. Nelfinavir

C32H45N3O4S (CAS No. 159989-64-7)

 

 

Saquinavir

 

Figure 4. Saquinavir

C38H50N6O5 (CAS No. 127779-20-8)

 

 

 

Experimental Conditions
Column Ascentis® Express RP-Amide (2.0 µm) 2.1 x 30 mm Injection: 2 µL
Detection ESI(+), MRM (603.2/402.2 m/z; 603.2/200 m/z) Flow Rate: 0.8 mL/min
Mobile phase A 0.01 % formic acid/water Temperature: 40 °C
Mobile phase B acetonitrile
Gradient 0 min (97 % A); 0.2 min (97 %); 1.7 min (66 %); 1.75 min (0 %); 3.0 min (0 %); stop (2 min equilibration)    
Sample diluent water / acetonitrile (50/50, v/v)
Reference solution 1-250 ng/ml Remdesivir (8 calibrators)    

 

Time (min) Mobile phase A (%) Mobile phase B (%)
0.00-0.20 97 3
1.70 66 34
1.75-3.00 0 100
3.05-5.00 97 3

 

No. Compound Retention Time (min) RRT Tailing Factor
1 Indinavir 1.14 0.75 1.44
2 Saquinavir 1.33 0.87 1.38
3 Nelfinavir 1.37 0.90 1.38
4 Remdesivir 1.53 1.00 1.44

 

Remdesivir Calibration Data (1-250 ng/mL)

Indinavir

Figure 5. Chromatogram showing the separation of Indinavir, Saquinavir, Nelfinavir, and Remdesivir

An example chromatogram showning the baseline separation of Remdesivir and three related antiviral drugs

 

Indinavir

Figure 7. Remdesivir calibration curve data
(1-15 ng/mL)

Illustration of the calibration curve linearity at the lower Remdesivir concentration range examined in the current study

 

Indinavir

Figure 6. Chromatogram showing the chromatographic response from an injection of 1 ng/mL Remdesivir standard solution

Example LC-MS chromatogram showning the injection of 1 ng/mL standard solution

 

Indinavir


Figure 8. Remdesivir calibration curve data
(1-250 ng/mL)

Illustration of the full calibration range examined in the current study

 

Compound LOD / ng/mL LOQ / ng/mL
Remdesivir 0.7 1.3

 

     
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