Ascorbic acid

Ascorbic acid is a vitamin and preservative. Citric acid is a preservative. Both are widely used in food industry. Both acids are polar and acidic in nature. Retention on a regular C18 column can be achieved with addition of ion-pairing reagent. We have developed a simple and robust method for separation of ascorbic and citric acid in HILIC/anion-exchange mode on Amaze TH HPLC column. Method is using volatile mobile phase which is compatible with MS spectrometry. Method can be used for analysis of ascorbic and citric acids in various sample matrices including biofluids and complex food food matrices. Amaze TH column can retain compounds by combination of HILIC, cation- and anion-exchange. Amount of ACN, buffer concentration and buffer pH will affect retention of polar and ionizable compounds.

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Conditions of Experiment

Column:	Amaze TH
Separation Modes:	HILIC, anion-exchange
Column Dimenstions:	4.6 x 150 mm, 5 um, 100A,
Mobile Phase:	75% ACN with 25 mM AmAc pH 4.2
Detection:	ELSD
Sample:	0.3 mg/ml
Injection:	3 uL
Flow rate:	1 ml/min

Analytes

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

Ascorbic, succinic and malonic acid are weak organic acids. Ascorbic acid is a vitamin found in various foods. It is one of the more important supplements used in various formulations. Food containing Vitamin C includes fruits and vegetables. It is a very important nutrient for humans and animals. Vitamin C functions as a cofactor in many enzymatic reactions in humans and animals. It mediates a variety of essential biological functions like wound healing and collagen synthesis. Methylmalonic acid is a dicarboxylic acid. Succinic acid is a dicarboxylic acid which is used in the production of some polymers and component of some alkyl resins. It is also used as a food additive and dietary supplement. It serves as an acidity regulator. We have developed an HILIC anion-exclusion approach for the analysis of these three organic acids on Heritage N HPLC column. The retention time is controlled by the amount of ACN and the amount of buffer. No ion-pairing reagent is required. Various buffers can be used depending on your detection technique

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Conditions of Experiment

Column:	Heritage N
Separation Modes:	HILIC, anion-exclusion
Column Dimenstions:	4.6 x 150 mm, 5 um, 100A
Mobile Phase:	75% ACN with 15 mM AmAc pH 7
Detection:	ELSD
Sample:	0.3 mg/ml
Injection:	5 uL
Flow rate:	1 ml/min

Analytes

Class of compounds:	Aromatic acid, Organic acid, Supplement, Vitamin
Nature of compounds:	Acidic, Hydrophilic, Polar
Compounds:	Ascorbic acid, Methylmalonic acid, Succinic acid

Ascorbic acid, or Vitamin C, is a vitamin found in plants, fruits and other food-related sources. It is one of the main dietary supplements. Vitamin C is an essential nutrient involved in repair of tissues. It is available as generic over-the-counter medication. Dehydroascorbic acid is an oxidized form of ascorbic acid. Both ascorbic and dehydroascorbic acids have similar biological activity as antiviral medications, but dehydroascorbic acid also has neuroprotective effects. Both compounds are very polar, with limited or no retention in reversed-phase chromatography. Both acids were analyzed and retained by weak reversed-phase and anion-exchange mechanisms on a core-shell mixed-mode anion-exchange column. The method and column can be used for analysis of these and other hydrophilic acids by mixed-mode chromatography. The method is compatible with LC/MS, ELSD and CAD and can be used for quantitation of ascorbic and dehydroascorbic acids in various foods, drug formulations and biofluids. Coresep SB column combines unique selectivity of mixed-mode with ultra-high efficiency of core-shell particles technology. HPLC method is reproducible and robust.

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Conditions of Experiment

Column:	Coresep SB
Separation Modes:	reversed-phase and anion-exchange
Column Dimenstions:	3.2 x 100 mm, 2.7 um, 90A
Mobile Phase:	5% ACN with 0.05% of formic acid
Detection:	UV 250 nm
Sample:	0.5 mg/ml
Injection:	1 uL
Flow rate:	0.8 ml/mim

Analytes

Class of compounds:	Aromatic acid, Organic acid, Supplement, Vitamin
Nature of compounds:	Acidic, Hydrophilic, Polar
Compounds:	Dehydroascorbic acid, Ascorbic acid