[Plasma ascorbic acid: preanalytical factors and new HPLC method]

Area of Science:

• Biochemistry
• Analytical Chemistry
• Clinical Diagnostics

Background:

• Ascorbic acid (vitamin C) and glutathione are crucial biomarkers often analyzed together.
• Existing analytical methods can be complex and require specific handling procedures.
• The need for efficient and reliable analytical techniques for these compounds in biological samples is significant.

Purpose of the Study:

• To develop and validate a fast electro-driven high-performance liquid chromatography (ED-HPLC) method for ascorbic acid analysis.
• To introduce a novel, user-friendly mixed-mode chromatographic column for this analysis.
• To establish a common sample pretreatment method for both ascorbic acid and glutathione.

Main Methods:

• Utilized a new mixed-mode chromatographic column with a functional dodecylsilane group and quaternary ammonium.
• Employed a simplified mobile phase without counter-ions for easier column handling.
• Validated a common pretreatment solution (sulfosalicylic acid, N-ethylmaleimide, EDTA) for plasma ascorbic acid and whole blood glutathione.
• Assessed the stability of deproteinized plasma samples stored at -20°C and -80°C.

Main Results:

• The developed ED-HPLC method provides fast analysis of ascorbic acid.
• The new chromatographic column simplifies handling and eliminates the need for counter-ions in the mobile phase.
• The common pretreatment solution is effective for both ascorbic acid and glutathione.
• Plasma samples, after deproteinization, remain stable for three weeks when stored at -20°C or -80°C.

Conclusions:

• A rapid and simplified ED-HPLC method for ascorbic acid analysis has been established using a novel mixed-mode column.
• The validated common pretreatment enhances efficiency for simultaneous analysis of ascorbic acid and glutathione.
• The method offers improved sample stability, facilitating its application in clinical settings.