Abstract
The main purpose of this study was to investigate chromatographic behaviors of the three types of important analytes, aliphatic, aromatic acids and nitrogen-containing heterocyclic compounds, to develop HILIC separation methods for the determination of these types of analytes. Citric, oxalic, malic, ascorbic and quinic acids are common aliphatic acids; benzoic and gallic acids belong to aromatic acids; uric acid, creatinine and glimepiride are important nitrogen-containing heterocyclic metabolites and drugs. The chromatographic behaviors of these compounds were investigated using Hydrophilic Interaction Liquid Chromatography (HILIC) with a propyl amino column by changing the mobile phase conditions such as content of acetonitrile (10 - 80%, interval of 10%), buffer concentrations (1.0, 2.0, 5.0, and 10.0 mM) and pH values (2.50 - 7.52). The basic retention patterns were explored through analyzing the retention factors. A HILIC method was developed for the determination of benzoic, ascorbic, malic, citric, and quinic acid in orange, lemon and other fruit juices. The study revealed different retention characteristics of aliphatic and aromatic acids and nitrogen-containing heterocyclic compounds on the HILIC column. The aliphatic, aromatic acids and nitrogen-containing heterocyclic compounds studied except glimepiride had a stronger retention as acetonitrile percent increased from 10 to 80%; one exception was that the retention of glimepiride decreased with the increase of acetonitrile content because of its low polarity; but had a weaker retention as the buffer concentration increased from 1.0 to 10.0 mM. The retention of the three types of compounds increased with the pH increase to some extent, then decreased as pH continually went up. The studies on the effect of pH and buffer phosphate concentration on retention provided experimental evidence that ion-exchange effect was important for the strong retention of acidic compounds on amino propyl column. The study on the effect of acetonitrile content on retention revealed that hydrophobic interaction was dominant within the low percent of acetonitrile, while hydrophilic interaction was the main retention force in the range of high content of acetonitrile. Hydrogen bonding interaction played an important role when hydrogen bonding donor and acceptor existed. The established HILIC method for the simultaneous determination of benzoic, ascorbic, malic, citric and quinic acid in Orange and Lemon fruit juices was validated. The calibration curves were found to be linear in the range of 0.50-10 ppm for benzoic acid, 5.0-50 ppm for ascorbic acid, 100-800 ppm for malic acid, 200-1200 ppm for citric acid and 100-800 ppm for quinic acid in orange and lemon fruit juices. The main constituents detected in orange juice were ascorbic, malic, and citric acid, with the concentration of 0.5722 mg / mL, 14.30 mg / mL, and 13.28 mg / mL, respectively. The main detected ingredients in lemon juice were ascorbic and citric acid with the concentration of 0.5588 mg / mL and 84.80 mg / mL, respectively. This validated HILIC method was proved to be fast, simple, accurate and reliable..