Separation type: Hydrophilic interaction liquid chromatography (HILIC)

Xanthine is a purine base that naturally occurs in the human body, and is a precursor for many other stimulants, medications, and biological compounds, including caffeine and uric acid. Caffeine is one of the most well-known stimulants due to its presence in popular drinks such as coffee, tea, and soda. 1,3-Dimethyluric acid is another xanthine-derived metabolite commonly found in urine after the metabolization of xanthine derivatives. Hypoxanthine is an important purine-derivative that serves as a key nitrogen source for some types of bacteria and other cells. Uric acid is the final product of purine metabolism and is commonly found in urine. Using SIELC’s newly introduced BIST™ method, these purine-derivatives and metabolites can be retained and separated on a positively-charged anion-exchange BIST™ B+ column. 

There are two keys to this retention method: 1) a multi-charged, negative buffer, such as Sulfuric acid (H2SO4), which acts as a bridge, linking the positively-charged analytes to the positively-charged column surface and 2) a mobile phase consisting of a majority of organic solvent (such as MeCN) to minimize the formation of a solvation layer around the charged analytes. Using this new and unique analysis method, Xanthine and Uric Acid can be separated, retained, and UV detected at 260 and 290 nm.

High Performance Liquid Chromatography (HPLC) Method for Analysis of Xanthines and Uric Acid

Condition

Column	BIST™ B+, 4.6×150 mm, 5µm, 100A
Mobile Phase	MeCN – 85%
Buffer	Formic Acid – 0.5%
Flow Rate	1.0 ml/min
Detection	UV 290 nm
Retention time	9.09 min

Description

Class of Compounds	Acid
Analyzing Compounds	Uric Acid