Improvements in the Chromatographic Performance of Antiviral Compounds Analyzed with MaxPeak HPS Technology

Antiviral pharmaceutical compounds are currently under investigation for their role in treating mild, moderate, and severe critical illnesses such as COVID-19.¹ It is important that routine chromatographic methods developed to analyze these pharmaceutical compounds distinguish low level-impurities and degradation products well before they reach alert/action levels. To accomplish this, chromatographic parameters such as retention, peak shape, and sensitivity are critically important.

The MaxPeak High Performance Surfaces (HPS) is a new, novel hybrid organic-inorganic surface technology that reduces sample loss due to non-specific binding of the analyte to metal-ions present within the chromatographic instrument and column.^2,3 In this application note, a panel of antiviral compounds were separated by reversed-phase HPLC. The chromatographic performance of an XBridge Premier Column with MaxPeak HPS Technology, was compared to a conventional stainless-steel column.

Benefits

Antiviral therapeutic compounds separated with the XBridge Premier Column, exhibit higher peak height response, improved signal-to-noise (S/N), and narrower chromatographic peak widths without the need for strong ion-pairing agents, chelators, or lengthy passivation protocols.

Pharmaceutical laboratories are routinely tasked with developing fast, sensitive, and robust chromatographic methods. The analytical method of choice to monitor quality and stability is generally High-Pressure Liquid Chromatography (HPLC) due to the high degree of both quantitative and qualitative precision.

The HPLC column surface and instrument componentry are comprised of stainless-steel, a material selected for its availability and mechanical strength. Conversely, stainless-steel is vulnerable to corrosion when exposed to harsh liquids, such as the acidic and/or halide containing mobile phases routinely employed in chromatography. Metal ions from corrosion can bind to the column stationary phase and interact with analytes through complexation, oxidation, and epimerization reactions which can result sometimes poor, unacceptable peak shape, and potentially the complete loss of the target analyte particularly at low concentrations.²

Mobile phase additives, such as ion-pairing agents (e.g. trifluoroacetic acid (TFA)) or chelators (e.g. medronic acid) are routinely employed to mitigate analyte-metal binding within the instrument and column. Use of these agents provides some measure of success, can result in instrument contamination, irreversible modifications in column chemistry, and ion suppression in LC-MS applications.² Passivation, performing a series of analyte injections to “condition” the instrument and column with the analyte, is also commonly utilized, although this technique may not lend overall confidence in daily precision.³

Columns constructed of alternative metals, such as titanium and nickel-cobalt alloys, have also been employed for some applications due to their corrosion resistance, but these metals can still deteriorate under some conditions. Additionally, columns made of Polyetheretherketone (PEEK), instead of transition metals, are an option, although solvent compatibility and pressure restraints exist.²

In this application note, we compare the chromatographic performance of an XBridge Premier BEH C₁₈ Column to the performance of a conventional XBridge BEH C₁₈ Stainless-Steel Column for the separation of twelve representative antiviral pharmaceutical compounds. These compounds are rapidly being developed and investigated for their role in the treatment of a variety of critical illnesses.⁴

A mixture of antiviral compounds soluble in aqueous solvent (tenofovir, favipiravir, emtricitabine, ribavirin, abacavir, and oselatamavir) was prepared by dissolving approximately 0.5 mg of each compound in 10 mL of water. A mixture of antiviral compounds soluble in organic solvent (mastinib, darunavir, remdesivir, atazanavir, ritonavir, and lopinavir) was prepared by dissolving approximately 0.5 mg of each compound in 10 mL of methanol (Figure 1). Each mixture was filtered through a respective, solvent compatible, 0.2 µm syringe filter (Waters P/N WAT200806 or Waters P/N WAT200822) prior to injection. The LC instrument was flushed sequentially with 100% IPA, water, and mobile phase. New columns with no previous injections, were employed from stock inventory. To minimize potential bias due to passivation of the instrument by the analyte, injections were performed consecutively by alternating columns with the column manager switching valve for a total of 5 injections of each antiviral mixture on each column.

The chromatographic method separated all 12 antiviral compounds. Those soluble in water were retained for less than 9 minutes, while compounds soluble in methanol, were retained for greater than 9 mins (Figure 2). 

The XBridge Premier Column yielded a greater height response for each antiviral compound in the panel resulting in higher S/N values. Additionally, by minimizing analyte interaction with metal-ions via the hybrid surface technology, peak shape improved (i.e. greater symmetry, lower tailing) for several compounds in the panel (Figure 3). Peak width at 5σ, a stringent measure of homogeneity and analyte-column interaction, is severely affected by peak asymmetry.⁶ For all 12 compounds, a significant decrease in peak width at 5σ was observed when using the MaxPeak HPS Technology, which demonstrated higher column performance (Table 1).  

The work here has shown that when analyzing low-level, antiviral compounds by reversed phase, the LC column with MaxPeak High Performance Surface Technology (XBridge Premier Column) exhibits higher performance than its conventional counterpart. Increases in peak height response resulted in a higher signal-to-noise ratio (S/N), and improved peak shape (w_5σ) was observed with the XBridge Premier Column without the need for ion-pairing/chelator mobile phase additives or column passivation injections.

1. Antiviral Therapies, NIH COVID Treatment Guidelines, www.nih.gov, accessed 9/20/21.
2. Mathew DeLano, Thomas H. Walter, Matthew A. Lauber, Martin Gilar, Moon Chul Jung, Jennifer M. Nguyen, Cheryl Boissel, Amit V. Patel, Andrew Bates-Harrison, Kevin D. Wyndham. Analytical Chemistry, Vol 93 (14), 2021.
3. Plumb R. and Wilson I., “Metal-Analyte Interactions – An Unwanted Distraction”, The Column. Vol 17 (8), 2021.
4. Shamaila Kausar, Fahad Said Khan, Muhammad Ishaq Mujeeb Ur Rehman, Muhammad Akram, Muhammad Riaz, Ghulam Rasool, Abdul Hamid Khan, Iqra Saleem, Saba Shamim, Arif Malik.” A review: Mechanism of Action of Antiviral Drugs”, International Journal of Immunopathology and Pharmacology, Volume 35: 1–12, 2021.
5. 2D Structure Database, www.ChemSpider.com, accessed 9/20/21. 
6. Joseph C. Arsenault and Patrick McDonald. “Beginners Guide to Liquid Chromatography”, Waters Corporation Primer. 715001531, 2007.