Etidronic acid

This method outlines the High-Performance Liquid Chromatography (HPLC) separation of ascorbic acid, citric acid, etidronic acid, and ethylenediaminetetraacetic acid (EDTA) utilizing an Amaze HA Mixed-Mode Column. Each compound's unique properties are considered for effective separation, ensuring accurate quantification. Ascorbic Acid: Also known as vitamin C, ascorbic acid is a water-soluble antioxidant involved in various physiological processes. It possesses acidic properties, making it amenable to interaction with the stationary phase of the HPLC column. Citric Acid: Widely found in citrus fruits, citric acid is a tricarboxylic acid that contributes to the sour taste of fruits. It is water-soluble and contains multiple acidic functional groups, allowing for interaction with the column's stationary phase. Etidronic Acid: A bisphosphonate used in the treatment of osteoporosis, etidronic acid is water-soluble and contains phosphonic acid functional groups. Its unique structure influences its behavior during HPLC separation. Ethylenediaminetetraacetic Acid (EDTA): A chelating agent commonly used in analytical chemistry, EDTA is water-soluble and forms complexes with metal ions. Its chelating properties present specific challenges and opportunities in chromatographic separation. Column Properties: Amaze HA Mixed-Mode Column: This column features a mixed-mode stationary phase, offering both hydrophobic and ionic interactions. The hydrophobic component enables interaction with non-polar compounds, while the ionic component facilitates interactions with charged species. This versatility allows for effective separation of polar and ionizable compounds like ascorbic acid, citric acid, etidronic acid, and EDTA. By employing this method, precise separation and quantification of ascorbic acid, citric acid, etidronic acid, and EDTA can be achieved, facilitating their analysis in pharmaceutical formulations, food products, and environmental samples.

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Conditions of Experiment

Column:	Amaze HA
Separation Modes:	Anion-exchange
Column Dimenstions:	3x100 mm, 3 um, 100 A
Mobile Phase:	50% ACN with 50 mM ammonium formate pH 3
Detection:	ELSD, 45*C
Sample:	various-0.5 mg/ml
Injection:	3 uL
Flow rate:	0.6 ml/min

Analytes

Class of compounds:	Aromatic acid, Drug, Organic acid, Preservative, Supplement, Vitamin
Nature of compounds:	Acidic, Hydrophilic, Polar, Zwitterionic
Compounds:	Etidronic acid, Citric acid, Ascorbic acid, Ethylenediaminetetraacetic acid

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.

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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

Analytes

Class of compounds:	Drug, Organic acid, Preservative, Sugar
Nature of compounds:	Acidic, Hydrophilic, Neutral, Polar
Compounds:	Citric acid, Etidronic acid, Mannitol