UHPLC Analysis of Riboflavin and Impurities on Titan™ C18 with MS Detection

UHPLC Analysis of Riboflavin and Impurities on Titan™ C18 with MS Detection


column Titan C18, 10 cm x 2.1 mm I.D., 1.9 µm particles (577124-U)
column temp. 35 °C
mobile phase [A] 0.1% formic acid in water; [B] 0.1% formic acid in acetonitrile (95:5)
gradient 5% to 25% in 6 min, return to 5% b in 0.1 min, and re-equilibrate for 4 min
flow rate 500 µL/min
pressure 5000 psi (345 bar)
injection 2 µL
detector Diode Array at 276 nm
detector ESI (+), MRM and TIC


Analysis Note Riboflavin (vitamin B2) is a water-soluble vitamin. It is synthesized by all plants and many microorganisms, but it is not produced by higher animals. It is a precursor of coenzymes that are required for the enzymatic oxidation of carbohydrates, so it is essential to basic metabolism. Riboflavin is used as an additive to food and feed and is also used in fortification of baby food and cereal. The current USP method for impurity analysis is non-quantitative, and uses an ion pair reagent in the mobile phase. An alternative to USP and EP methods using LC-MS/MS to identify and quantify the riboflavin and impurities is presented. This new proposed method is also compatible with UV-Vis detection.
A riboflavin certified reference material (CRM) was used in this study. MRM transitions for the determination of ions for the parent and four impurites were made. Three transitions were used for each ion. Structures for each of these major impurities were confirmed by MS. In addition to improving the specificity of the method with MS/MS detection, two additional goals were to decrease analysis time and eliminate the need for ion pairing reagents that are not compatible with MS.
Categories Analytical Chromatography, Clinical, vitamins, Vitamins and Natural products
Featured Industry Clinical
Pharmaceutical (small molecule)
Legal Information Titan is a trademark of Sigma-Aldrich Co. LLC
Other Notes Sample:
USP and EP methods use 0.1 M NaOH to solubilize riboflavin.
• Not suitable for MS and also may cause the compounds to breakdown further and NaOH leads to formation of Na adducts in MS.
• Solubility studies done in following solvent systems:
• Mixtures and neat solutions of water/acetonitrile/methanol/formic acid/acetic acid showed no limited solubility of riboflavin in these common MS solvents, even with extensive sonication.
• Further literature searches suggested Dimethyl Sulfoxide (DMSO) as a possible solvent for the riboflavin and impurities.
• Stock solution prepared in DMSO at 1 mg/mL with 5 minutes sonication yielded total solubility of the riboflavin and impurities.

Dilute with mobile phase as necessary.
suitability application for UHPLC


Related Links