Improving Peak Shape and Recovery in LC Studies of Phosphated Vitamin B2

Phosphated vitamin B2 (riboflavin) vitamers were analyzed by UHPLC and a performance comparison was made in terms of peak shape and recovery obtained on an Agilent 1290 Infinity II LC System (classic stainless steel LC) versus an Agilent 1290 Infinity II Bio LC System (biocompatible LC), and on classic versus metal-free column hardware. The results clearly demonstrate the necessity of biocompatible flow paths for accurate analysis of riboflavin diphosphates and monophosphates to prevent nonspecific interactions of these compoundswith the metal surfaces. The iron-free flow path allows the use of volatile MS/ELSD-compatible mobile phases for the direct analysis of the phosphorylated species without the need for enzymatic treatment with phosphatase.