This application note describes the optimization of a rapid LC/MS/MS method for vitamin B related compounds (4-aminobenzoic acid, nicotinamide, riboflavin, and nicotinic acid) on an Agilent ZORBAX Rapid Resolution High Definition HILIC Plus column using an Agilent 1290 Infinity LC System with an Agilent 6410A Triple Quadrupole Mass Spectrometer. The method uses isocratic elution with an acetonitrile rich mobile phase and an ammonium acetate buffer. All analytes have good peak shape with this hydrophilic interaction chromatography (HILIC) application. The method is then used to demonstrate the importance of choosing an appropriate injection solvent when performing LC analyses in HILIC mode. Various injection solvents are investigated, including water, acetonitrile, methanol, and combinations. Of the solvents explored, it was determined that pure acetonitrile yields the best retention and peak shape for all compounds, with water being the worst solvent for analytical performance and peak shape.
This document details a rapid LC/MS/MS analysis method for four Vitamin B related compounds: 4-aminobenzoic acid, nicotinamide, riboflavin, and nicotinic acid. The method utilizes an Agilent ZORBAX Rapid Resolution High Definition (RRHD) HILIC Plus column with an Agilent 1290 Infinity LC System and an Agilent 6410A Triple Quadrupole Mass Spectrometer. The analysis employs isocratic elution with an acetonitrile-rich mobile phase containing an ammonium acetate buffer at pH 3.2.
A key focus of the study is the evaluation of injection solvent effects on chromatographic performance in Hydrophilic Interaction Liquid Chromatography (HILIC) mode. The document investigates various injection solvents, including water, acetonitrile, methanol, and their combinations. Results indicate that pure acetonitrile yields the best retention and peak shape for all analytes, whereas water, being the strongest solvent in HILIC mode, severely distorts peak shape and retention times.
| Column Dimensions | 2.1 x 50 mm and 2.1 x 100 mm |
|---|---|
| Particle Size | 1.8 ym |
| Mobile Phase Composition | CH3CN/100 mM NH4HCO3, pH 3.2 (9:1) |
| Flow Rate Options | 0.4, 0.7 or 1.0 mL/min |
| MS Source | Positive ESI |
| Capillary Voltage | 4000 V |
Pure acetonitrile yields the best retention and peak shape for all compounds.
Water is the worst solvent for analytical performance and peak shape.
The compounds analyzed are 4-aminobenzoic acid, nicotinamide, riboflavin, and nicotinic acid.