HPLC Analysis of Etidronic Acid, Citric Acid and Mannitol on Amaze HD Mixed-Mode Column

Application description

Mannitol, etidronic and citric acids are part of the pharmaceutical formulation to reduce bone resorption, strengthen bones and treat osteoporosis. Etidronic acid also prevents bone calcification. Citric acid is used as a preservative in the formulation. All three compounds are very polar in nature with citric and etidronic acids being acidic and mannitol being neutral. All three are not retained in RP chromatography. In HILIC, citric acid and etidronic acid can elute with a poor peak shape due to unspecified secondary interactions. We have developed a robust HILIC/anion-exclusion method for separation of these polar compounds on the Amaze HD mixed-mode cation-exchange/anion-exclusion column. This method is compatible with major detection techniques for such compounds with no UV activity (ELSD, CAD, RI and MS). The method can be used for analysis of polar acidic, basic and neutral compounds in HILIC, cation-exchange and anion-exclusion modes. The retention time is controlled by the amount of acetonitrile, buffer pH and buffer concentration. It is recommended to use a salt buffer to achieve better retention and elution control. For complex matrices like biofluids, a sample preparation is recommended.

Conditions of Experiment
Column: Amaze HD
Separation Modes: HILIC, anion-exchange
Column Dimenstions: 4.6x50 mm, 5 um, 100A
Mobile Phase: 80% ACN with 20 mM AmFm pH 3
Detection: ELSD, 50°C
Sample: 2 mg/ml
Injection: 2 uL
Flow rate: 1 ml/min
Class of compounds: Drug, Organic acid, Preservative, Sugar
Nature of compounds: Acidic, Hydrophilic, Neutral, Polar
Compounds: Citric acid, Etidronic acid, Mannitol