Citric 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

Mannitol, ibandronic and citric acids are part of the 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. 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 salt buffer to achieve better retention and elution control.

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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:	ACN/water/Buffer
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:	Ibandronic acid, Citric acid, Mannitol

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

Pseudoephedrine is a sympathomimetic medication in the phenylamine and amphetamine class of drugs. It is a stimulant. Pseudoephedrine is a part of numerous drug formulations used as cough suppressants. It is used as a precursor and starting material in the illicit production of methamphetamine and methcathinone. Pseudoephedrine is polar and basic in nature. Citric acid is an organic acid containing three carboxylic groups. It is an intermediate in the citric acid cycle. Citric acid naturally occurs in citrus fruits. It is used widely as an acidifier, preservative, flavoring and chelating compound. Citric acid is also used as a cleaning agent. It is often added to drug formulations for stability. Citric acid is polar and strongly acidic compound. We have developed a HILIC/cation-exchange anion exclusion method for the separation of these two polar compounds on the Heritage N HILIC column. The retention time is controlled by the amount of ACN and 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, cation-exchange, anion-exclusion
Column Dimenstions:	4.6 x 150 mm, 5 um, 100A
Mobile Phase:	60% ACN with 20 mM Na2HPO4 pH 7
Detection:	UV 205 nm
Sample:	0.3 mg/ml
Injection:	5 uL
Flow rate:	1 ml/min

Analytes

Class of compounds:	Aromatic base, Drug, Organic acid, Preservative, Stimulant
Nature of compounds:	Acidic, Basic, Hydrophilic, Polar
Compounds:	Citric acid, Pseudoephedrine