Separation type: Bridge Ion Separation Technology, or BIST™ by SIELC Technologies

High Performance Liquid Chromatography (HPLC) Method for Analysis of  Maleic  acid, Ascorbic acid, Nicotinic acid, Fumaric acid and Oxalic acid

The maleate ion from Maleic acid is a popular ingredient as the maleate salt in several different drugs, including Methergine, Pyrilamine, and Carfenazine, among others. Nicotinic acid, also known as Niacin or Vitamin B3, is an essential nutrient for the human body and is sometimes taken as a treatment for high cholesterol. Aconitic acid is a key intermediary in the citric acid cycle, and is also used a flavoring agent and in the production of rubbers and plastics. Fumaric acid is a popular preservative and food additive with a fruit-like taste. Using SIELC’s newly introduced BIST™ method, a mixture of these organic acids can be separated on a negatively-charged, cation-exchange BIST™ A+ column, contrary to conventional chromatographic wisdom. There are two keys to this retention method: 1) a multi-charged, positive buffer, such as N,N,N’,N’-Tetramethyl-1,3-propanediamine (TMDAP), which acts as a bridge, linking the negatively-charged anion analytes to the negatively-charged column surface and 2) a mobile phase consisting mostly of organic solvent (such as MeCN) to minimize the formation of a solvation layer around the charged analytes. Other positively-charged buffers that can generate BIST™ include Calcium acetate and Magnesium acetate. Using this new and unique analysis method, these organic acids can be separated, retained, and detected through ELSD. This method is also compatible with Mass Spectrometry (LC-MS) and CAD.

Condition

Description

Separation type: Bridge Ion Separation Technology, or BIST™ by SIELC Technologies

The maleate ion from Maleic acid is a popular ingredient as the maleate salt in several different drugs, including Methergine, Pyrilamine, and Carfenazine, among others. Nicotinic acid, also known as Niacin or Vitamin B3, is an essential nutrient for the human body and is sometimes taken as a treatment for high cholesterol. Aconitic acid is a key intermediary in the citric acid cycle, and is also used a flavoring agent and in the production of rubbers and plastics. Fumaric acid is a popular preservative and food additive with a fruit-like taste. Using SIELC’s newly introduced BIST™ method, a mixture of these organic acids can be separated on a negatively-charged, cation-exchange BIST™ A+ column, contrary to conventional chromatographic wisdom. There are two keys to this retention method: 1) a multi-charged, positive buffer, such as N,N,N’,N’-Tetramethyl-1,3-propanediamine (TMDAP), which acts as a bridge, linking the negatively-charged anion analytes to the negatively-charged column surface and 2) a mobile phase consisting mostly of organic solvent (such as MeCN) to minimize the formation of a solvation layer around the charged analytes.

Condition

Description

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Acids with different strengths can be retained and elution order controlled by the use of a mixed-mode column, which has an ion-exchange component in addition to hydrophobic retention.  In this example, the Newcrom BH mixed-mode column was used to separate fumaric, malonic and sorbic acids in HPLC.  Sorbic acid was mostly retained by hydrophobicity, while fumaric and malonic acids were also retained by the adjustment strength of the mobile phase

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The citric acid cycle (tricarboxylic acid cycle, Krebs cycle) is a key process in the metabolic pathway by which all aerobic organisms generate energy. Compounds generated during TCA are hydrophilic, acidic compounds. Some of the hydrophilic acids are very strong while others are relatively weaker. All compounds have very low hydrophobicity and do not retain by the reversed-phase mechanism on the C18 or C8 HPLC columns. Citric acid and related products were separated in reversed-phase, anion-exchange mode on the Primesep D mixed-mode column. All hydrophilic, acidic compounds are well separated and demonstrate good shape. This robust method can be used for analysis of components of the TCA cycle and other highly hydrophilic compounds. This method is fully compatible with LC/MS and prep chromatography.

This is an alternative approach for the separation of the TCA cycle intermediates that was developed using novel hydrogen-bonding HPLC column. The organic nature of the mobile phase helps obtain a highly sensitive LC/MS compatible method.

The majority of drugs in the pharmaceutical industry are administered in salt form. The presence of two counter-ions very often necessitates the use of two methods. The nature of these counterparts in drugs can be an inorganic cation and organic acid, inorganic anion and organic base, and organic cation and organic anion. Furthermore, the properties of the molecules will result in a differing stoichiometry. The task of simultaneous quantitation of counter-ions can be achieved by using mixed-mode columns. The general approach for analysis is based on properties of corresponding counter-ions. Hydrophobic basic drugs, like dextromethorphan, verapamil, trimipramine, and corresponding acidic counter-ions (chloride, chlorate, bromide, bromate, perchlorate, maleate, fumarate,tartrate, succinate, phosphate, citrate, benzosulfonate, toleuensulfonate) can be separated and quantitated in the same run on reversed-phase anion-exchange column. Basic hydrophobic drugs are retained by the reversed-phase mechanism, and counter-ions are retained by the reversed-phase and anion-exchange mechanism. Some polar counter-ions are retained only by the anion-exchange mechanism. Retention time and selectivity of HPLC separation of drugs and counter-ions can be achieved by changing the amount of acetonitrile and the amount of ions in the mobile phase. The detection technique depends on the properties of the counter-ions. In case of low or no UV activity, ELSD can be employed if the counter-ion forms a non-volatile salt with the mobile phase additive (ammonium formate). This HPLC method can be used for simultaneous quantitation of other basic drugs and counter-ions. The presence of two mechanisms of retention allows control over retention times of drug and counter-ion independently, and even allows a change of order of elution when necessary.

Hydrophilic acids are separated on Obelisc N mixed-mode HILIC column. Seven carboxylic acids are separated based on their polarity and pKa values. Changes in ionization states of acids and stationary phase can be used to control elution order of organic and inorganic acids.

Primesep D separates organic acids such as fumaric, benzoic, phthalic, naphthoic, and maleic acids by a mixture of anion exchange and reversed phase. Retention times and elution order can be changed by adjusting the percentage of acetonitrile in the mobile. This can not be done by traditional ion-exchange and ion-exclusion chromatography. The HPLC separation uses a mobile phase of water, acetonitrile (MeCN, ACN) and trifluoroacetic acid (TFA) and UV detection at 250 nm.

Primesep B combines a hydrophobic, reversed-phase mechanism with ion exchange to separate the diacids, fumaric, benzoic, phthalic, naphthoic, and maleic acids. Changing the acetonitrile content of the mobile phase reverses the peak order for naphthoic and maleic acids. Primesep B combines reversed-phase and anion-exchange mechanism with a mobile phase of water, acetonitrile (MeCN, ACN) and trifluoracetic acid (TFA) and UV detection at 250 nm.

Primesep 200 retains and separates the organic diacids (malic, succinic, fumaric, and maleic) by a combination hydrophobic, reversed-phase interactions and ion exclusion. The separation uses a mobile phase of water, acetonitrile (MeCN, ACN) and trifluoracetic acid (TFA) with UV detection at 210 nm.

Primesep 100 separates a mixture of dicarboxylic acids in ion-exclusion mode with a mobile phase of water, acetonitrile (MeCN, ACN), and sulfuric acid (H2SO4) with UV detection at 210 nm. Baseline resolution of fumaric, maleic, malic, and succinic acids is obtained in less than 8 minutes. The separation combines ion-exclusion and reversed-phase mechanisms in one method.