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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.

Barium ion and citric acid are analyzed in the presence of sucrose and Tween 80 on Obelisc N HILIC mixed-mode column. Both compounds are retained in ion-exchange mode and HILIC is not employed with this method. Obelisc N HPLC column has basic and acidic ion-exchange groups separated by long hydrophilic chain. Presence of that long chain allows both cation- and anion-exchange mechanisms to exist simultaneously. Obelisc N column can be used for analysis of cation and anions in HILIC/ion-exchange mode and in ion-exchange mode. Analytes can be monitored by ELSD/CAD and LC/MS.

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Pseudoephedrine is a drug used in cough and cold compositions. One of the preservatives in solutions of pseudoephedrine is citric acid. Both compounds are hydrophilic in nature, with pseudoephedrine being a basic compound and citric acid an acidic compound. Separation of these two compounds is achieved on a Primesep N column. Compounds are separated by combination of HILIC, cation-exchange and anion-exclusion mechanisms. Pseudoephedrine and citric acid are monitored by UV. This HPLC method can be adopted as generic approach for analysis of pseudoephedrine and other hydrophilic drugs and preservatives in mixtures.

Citric acid and phosphate ion are separated by HILIC on Primesep N HILIC HPLC column. Both compounds are very hydrophilic. Citric acid and phosphate ions are well separated with excellent peak shape. Method can be used for quantitation of citrate and phosphate in various formulations. Because of the lack of UV activity, separation can be monitored by LC/MS, ELSD or Corona CAD. Method can be used for other hydrophilic organic and inorganic acids.

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.

Alginate is used in various pharmaceutical preparations. Chemically, it is a linear copolymer with homopolymeric blocks of (1-4)-linked ?-D-mannuronate (M) and its C-5 epimer ?-L-guluronate (G) residues, respectively, covalently linked together in different sequences or blocks. Alginic acid can be separated from benzoate, citric acid and saccharin by mixed-mode chromatography on Primesep C HPLC column. This method can be used to quantitate alginic acid, citric acid or saccharin in complex mixtures. Various detection technique can be used (UV, ELSD, LC/MS), based on mobile phase selection.