Succinic acid

There are several groups of compounds which are used as active and inactive ingredients in various over-the-counter drug compositions. These compounds are different in nature and can be organic and inorganic. Most of the active ingredients are either hydrophobic basic, hydrophilic basic or hydrophilic neutral compounds. Basic active ingredients of cough and cold remedies used as salts of organic and inorganic acids. The most commonly used hydrophobic active ingredients are doxylamine, pyrilamine, chlorpheniramine, brompheniramine, and dextromethorphan. Doxylamine is an antihistamine drug, used to relieve symptoms of allergy, hay fever, and the common cold. Doxylamine is used in combination with other active ingredients to provide effective nighttime relief from cough and cold symptoms. Pyrilamine is an antihistamine drug, which targetsH1 receptors as an inverse agonist. Brompheniramine is an antihistamine drug of propylamine nature. Chlorpheniramine is an antihistamine used to treat symptoms such as allergic rhinitis. Dextromethorphan is a medication mostly used as a cough suppressant. It is a morphine type of medication with sedative, dissociative, and stimulant properties. Succinic acid is a hydrophilic diacid that is used as a counterion. Maleic acid is hydrophilic diacid which is used as a counterion. Both compounds have very limited retention even in RP-AQ columns. Due to the strongly hydrophobic and basic properties of these compounds, they demonstrate a poor peak shape in traditional reversed-phase chromatography due to the overloading of residual silanol groups on the surface when these basic drugs are analyzed. In addition to this, hydrophilic counterions are not retained in RP because there is no mechanism of retention for these acidic hydrophilic compounds. Both problems were eliminated by using Heritage MA reversed-phase anion-exchange column. Column and method demonstrate high efficiency and good peak shape for acidic and basic analytes. It can be used to develop methods for various cough, cold, and congestion compositions without the use of ion-pairing reagents. Compounds are well retained by a combination of one or more mechanisms. The column is compatible with 100% ACN and 100% water as well as with most buffers with pH 1.5-7. Various additives to the mobile phase can be used based on the nature of samples and detection techniques.

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Conditions of Experiment

Column:	Heritage MA
Separation Modes:	reversed-phase, cation-exclusion, anion-exchange
Column Dimenstions:	4.6x150 mm, 3 um, 100A
Mobile Phase:	5% ACN with 30 mM AmFm pH 3
Detection:	UV 275
Sample:	various
Injection:	3 uL
Flow rate:	1 ml/min

Analytes

Class of compounds:	Amines, Aromatic base, Drug, Organic acid, Pyridine
Nature of compounds:	Acidic, Basic, Hydrophilic, Hydrophobic, Polar
Compounds:	Doxylamine, Pyrilamine, Succinic acid, Chlorpheniramine, Brompheniramine, Maleic acid, Dextromethorphan

Mixed-mode stationary phases were designed to retain and separate compounds with drastic difference in properties using one column and method. Carboxylic acids, amino acids and sugars are poorly retained in reversed-phase chromatography. Derivatization procedures, ion-pairing reagents and HILIC columns can be used tp analyze these complex mixtures. The use of ion-pairing reagent makes MS detection problematic due to volatility issues of IP and signal suppression in MS. HILIC mixed-mode columns, like Amaze TH, have polar ionizable groups on the surface which help significantly increase retention time of polar ionizable analytes like amines, amino acids, carboxylic acids and sugars. We have developed a method for separation and retention of succinic acid, phenylalanine, sucrose, glycine, aspartic acid and raffinose. Isocratic conditions provide separation within 15 minutes with good peak shape and retention control. Retention time on Amaze TH column is controlled by amount of ACN, buffer concentration, buffer ph and buffer nature. Method can be used for other amines, amino acids, carboxylic acids and sugars where fast MS-compatible separation is required.

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Conditions of Experiment

Column:	Amaze TH
Separation Modes:	HILIC, cation-exchange, anion-exchange
Column Dimenstions:	4.6 x 100 mm 5 um, 100A
Mobile Phase:	80% ACN with 10 mM AmAc pH 4.8
Detection:	ELSD
Sample:	0.3 mg/ml
Injection:	3 uL
Flow rate:	1 ml/min

Analytes

Class of compounds:	Amino acid, Organic acid, Sugar, Supplement
Nature of compounds:	Acidic, Hydrophilic, Neutral, Polar, Zwitterionic
Compounds:	Succinic acid, Phenylalanine, Sucrose, Glycine, Aspartic acid, Raffinose

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