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Antiviral Analysis: Remdesivir and its Metabolites

By
  • Jamie York
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Remdesivir is a broad-spectrum antiviral drug that has shown promise in the treatment of Ebola, SARS, Marburg, MERS, and SARS-CoV-2 (COVID-19).1 It is marketed under the trade name of VEKLURY and advertised as “the first and only antiviral treatment FDA approved for COVID-19.”2 In the body, Remdesivir is metabolized to GS-704277, GS-441524, and GS-443902, with GS-441524 being the major metabolite.3 GS-441524 is less effective than the activated form of Remdesivir (triphosphate GS-443902) for the treatment of Ebola, Hepatitis C, and respiratory syncytial virus, but showed good efficacy in vitro against SARS-CoV and MERS-CoV.3

blog-antiviral-analysis-remdesivir-and-its-metabolites-03.png

Figure 1. Metabolic pathway proposed for Remdesivir.3

Looking at these structures, the first thing I notice is the presence of phosphate groups. Compounds containing phosphate groups can potentially chelate and interact with metal components in your LC system. The best way to overcome this obstacle is to passivate the system before use. The use of medronic acid is popular technique for system passivation. Restek offers a ready-made passivation solution for your convenience (product: LC Passivation Solution and instructions: 480-01-001).

After passivating the system, a method was developed to analyze Remdesivir, GS-704277 (alanine metabolite), Remdesivir nucleoside monophosphate, and GS-441524 in plasma in 4.5 min. To prepare the sample, 50 µL of spiked plasma was added to a centrifuge tube. 200 µL of cold acetonitrile was added to the sample and vortexed. The sample was centrifuged for 10 min. at 4200 rpm and an aliquot of the supernatant was transferred to a vial. The extract was then evaporated to dryness under a gentle stream of nitrogen. The sample was reconstituted with 100 µL of water containing 0.1% acetic acid prior to vortexing.

Column: Raptor Biphenyl (cat.# 9309A52)
Dimensions: 50 mm x 2.1 mm ID
Particle Size: 2.7 µm
Guard Column: Raptor Biphenyl EXP guard column cartridge 2.7 µm (cat.# 9309A0252)
Temp.: 30 °C
Conc.: Metabolites 500 ppb; Remdesivir 50 ppb
Inj. Vol.: 2 µL
Mobile Phase  
A: Water, 20 mM ammonium acetate, 0.1% acetic acid
B: Methanol, 20 mM ammonium acetate, 0.1% acetic acid
Time (min)
Flow (mL/min)
%B
0.00
0.5
0
1.50
0.5
55
2.00
0.5
100
3.00
0.5
100
3.01
0.5
0
4.50
0.5
0
Ion Mode ESI (+)
Detector MS/MS
 
 

Peaks

Retention Time (min)

Precursor Ion

Product Ion 1

Product Ion 2

Product Ion 3

1. Remdesivir nucleoside monophosphate

0.8

372.1

202.1

147.2

229.1

2. GS-704277

1.1

443.1

202.2

124.1

229.2

3. GS-441524

2.1

292.0

202.1

163.1

147.1

4. Remdesivir

2.9

603.2

200.1

229.1

402.2

 

blog-antiviral-analysis-remdesivir-and-its-metabolites-02.png

This method of analysis is enabled by the use of a Raptor Biphenyl 50 x 2.1 mm, 2.7 µm column. Good selectivity and excellent peaks shapes were observed for Remdesivir and three of its metabolites in human plasma in an analysis time of only 4.5 min. Is your lab currently running or interested in this analysis? Let us know about it in the comments below!

  1. https://www.gilead.com/purpose/advancing-global-health/covid-19/about-veklury
  2. https://www.veklury.com/?gclid=EAIaIQobChMI6LXDvKni7gIVPweICR1FEwI7EAAYAiAAEgJTGfD_BwE&gclsrc=aw.ds
  3. YingjunLi, LiuCao, Ge Li, Feng Cong, Yunfeng Li, Jing Sun, Yinzhu Luo, Guijiang Chen, Guanguan Li, Ping Wang, Fan Xing, Yanxi Ji, Jincun Zhao, Yu Zhang, Deyin Guo, Xumu Zhang. Remdesivir Metabolite GS-441524 Effectively Inhibits SARS-CoV-2 Infection in Mice Models. bioRxiv 2020.10.26.353300; doi: https://doi.org/10.1101/2020.10.26.353300 
    https://www.biorxiv.org/content/10.1101/2020.10.26.353300v1.full