Separation and detection method of glycyl-L-glutamine chiral isomer

Abstract

The invention provides a derivatization separation and detection method of a glycyl-L-glutamine chiral isomer, and belongs to the field of analytical chemistry. The method comprises the steps that before the glycyl-L-glutamine chiral isomer enters a liquid chromatographic column, derivatization is conducted through a derivatization reagent, and the derivatization reagent adopts an N<alpha>-(5-fluorine-2,4-dinitrophenyl)-L-amino acid compound. The separation determination method is high efficiency liquid chromatography. The used chromatographic column is a chromatographic column which takes base octadecyl bonded silica as filler. The separation and detection method mainly comprises the steps of preparing a sample solution and a derivative solution, conducting a derivative reaction, conducting derivative reaction aftertreatment and conducting separation determination through a high performance liquid chromatographic instrument. The pre-column derivatization method has the advantages that the reaction condition is mild, derived products are stable, the reaction velocity is high, the side reactions are few, determination is not disturbed by the excess derivatization reagent, and separation between glycyl-L-glutamine and the chiral isomer of the glycyl-L-glutamine can be achieved by using an ordinary chromatographic column.

Description

technical field

[0001] The invention relates to a method for separating and detecting chiral isomers of glycyl-L-glutamine, which belongs to the field of analytical chemistry. technical background

[0002] Glycyl-L-glutamine is an important source of glutamine, and it is one of the dipeptide components in the compound amino acid (15) dipeptide (2) injection, which overcomes the problem of glutamine being unstable in solution, The formula of amino acid liquid has been enriched. Its chemical name is glycyl-L-glutamine monohydrate, and its molecular formula is as follows:

[0003]

[0004] It can be seen from the molecular formula that there are chiral isomers, and its chemical name is glycyl-D-glutamine. There is no determination of glycyl-L-glutamine isomers in the existing technology and literature. Related reports.

[0005] The chiral isomer of glycyl-L-glutamine is its impurity, which is an important control index of glycyl-L-glutamine and a key focus item in drug pr...

Examples

Embodiment 1

[0037] (1) Instruments and conditions

[0038] Chromatographic conditions:

[0039] Chromatographic column: base-passivated octadecyl-bonded silica gel column (4.6mm×250mm, 5μm)

[0040] Mobile phase: Phase A: 0.09% trifluoroacetic acid (adjust pH 2.3 with NaOH)-acetonitrile (94:6)

[0041] Phase B: Acetonitrile

[0042] Detection wavelength: 340nm; column temperature: 25°C; flow rate: 1.6ml / min; diluent: water

[0043] Gradient elution program:

[0044]

[0045] (2) Experimental steps

[0046] Derivatization test solution: take Merfey's [N α -(5-fluoro-2,4-dinitrophenyl)-L-alanamine amide] reagent amount, accurately weighed, add acetone to dissolve and dilute to make a 10mg / ml solution, shake well, and place in the refrigerator ( 2℃~8℃) for storage.

[0047] Sample solution: Take an appropriate amount of glycyl-L-glutamine and its isomers, dissolve and dilute with water to make a mixed solution containing about 30mg of glycyl-L-glutamine and 30μg of isomers per 1ml,...

Embodiment 2

[0051] (1) Instruments and conditions

[0052] Chromatographic conditions:

[0053] Chromatographic column: base-passivated octadecyl-bonded silica gel column (4.6mm×250mm, 5μm)

[0054] Mobile phase: Phase A: 0.11% trifluoroacetic acid (adjust pH 2.7 with NaOH)-acetonitrile (86:14)

[0055] Phase B: Acetonitrile

[0056] Detection wavelength: 340nm; column temperature: 35°C; flow rate: 2.0ml / min; diluent: water

[0057] Gradient elution program:

[0058]

[0059] (2) Experimental steps

[0060] Derivatization test solution: take Merfey's [N α -(5-fluoro-2,4-dinitrophenyl)-L-alanamine amide] reagent amount, accurately weighed, add acetone to dissolve and dilute to make a 10mg / ml solution, shake well, and place in the refrigerator ( 2℃~8℃) for storage.

[0061] Sample solution: take appropriate amount of glycyl-L-glutamine and glycyl-D-glutamine, dissolve and dilute with water to make about 30mg of glycyl-L-glutamine, glycyl-D-glutamine per 1ml - A mixed solution of 30...

Embodiment 3

[0065] (1) Instruments and conditions

[0066] Chromatographic conditions:

[0067] Chromatographic column: base-passivated octadecyl-bonded silica gel column (4.6mm×250mm, 5μm)

[0068] Mobile phase: Phase A: 0.1% trifluoroacetic acid (adjust pH 2.5 with NaOH)-acetonitrile (88:12)

[0069] Phase B: Acetonitrile

[0070] Detection wavelength: 335nm; column temperature: 30°C; flow rate: 1.8ml / min; diluent: water

[0071] Gradient elution program:

[0072]

[0073] (2) Experimental steps

[0074] Derivatization test solution: take Merfey's [N α -(5-fluoro-2,4-dinitrophenyl)-L-alanamine amide] reagent amount, accurately weighed, add acetone to dissolve and dilute to make a 10mg / ml solution, shake well, and place in the refrigerator ( 2℃~8℃) for storage.

[0075] Sample solution: take appropriate amount of glycyl-L-glutamine and glycyl-D-glutamine, dissolve and dilute with water to make about 30mg of glycyl-L-glutamine, glycyl-D-glutamine per 1ml - A mixed solution of 3...

technical field

[0001] The invention relates to a method for separating and detecting chiral isomers of glycyl-L-glutamine, which belongs to the field of analytical chemistry. technical background

[0002] Glycyl-L-glutamine is an important source of glutamine, and it is one of the dipeptide components in the compound amino acid (15) dipeptide (2) injection, which overcomes the problem of glutamine being unstable in solution, The formula of amino acid liquid has been enriched. Its chemical name is glycyl-L-glutamine monohydrate, and its molecular formula is as follows:

[0003]

[0004] It can be seen from the molecular formula that there are chiral isomers, and its chemical name is glycyl-D-glutamine. There is no determination of glycyl-L-glutamine isomers in the existing technology and literature. Related reports.

[0005] The chiral isomer of glycyl-L-glutamine is its impurity, which is an important control index of glycyl-L-glutamine and a key focus item in drug pr...

Examples

Embodiment 1

[0037] (1) Instruments and conditions

[0038] Chromatographic conditions:

[0039] Chromatographic column: base-passivated octadecyl-bonded silica gel column (4.6mm×250mm, 5μm)

[0040] Mobile phase: Phase A: 0.09% trifluoroacetic acid (adjust pH 2.3 with NaOH)-acetonitrile (94:6)

[0041] Phase B: Acetonitrile

[0042] Detection wavelength: 340nm; column temperature: 25°C; flow rate: 1.6ml / min; diluent: water

[0043] Gradient elution program:

[0044]

[0045] (2) Experimental steps

[0046] Derivatization test solution: take Merfey's [N α -(5-fluoro-2,4-dinitrophenyl)-L-alanamine amide] reagent amount, accurately weighed, add acetone to dissolve and dilute to make a 10mg / ml solution, shake well, and place in the refrigerator ( 2℃~8℃) for storage.

[0047] Sample solution: Take an appropriate amount of glycyl-L-glutamine and its isomers, dissolve and dilute with water to make a mixed solution containing about 30mg of glycyl-L-glutamine and 30μg of isomers per 1ml,...

Embodiment 2

[0051] (1) Instruments and conditions

[0052] Chromatographic conditions:

[0053] Chromatographic column: base-passivated octadecyl-bonded silica gel column (4.6mm×250mm, 5μm)

[0054] Mobile phase: Phase A: 0.11% trifluoroacetic acid (adjust pH 2.7 with NaOH)-acetonitrile (86:14)

[0055] Phase B: Acetonitrile

[0056] Detection wavelength: 340nm; column temperature: 35°C; flow rate: 2.0ml / min; diluent: water

[0057] Gradient elution program:

[0058]

[0059] (2) Experimental steps

[0060] Derivatization test solution: take Merfey's [N α -(5-fluoro-2,4-dinitrophenyl)-L-alanamine amide] reagent amount, accurately weighed, add acetone to dissolve and dilute to make a 10mg / ml solution, shake well, and place in the refrigerator ( 2℃~8℃) for storage.

[0061] Sample solution: take appropriate amount of glycyl-L-glutamine and glycyl-D-glutamine, dissolve and dilute with water to make about 30mg of glycyl-L-glutamine, glycyl-D-glutamine per 1ml - A mixed solution of 30...

Embodiment 3

[0065] (1) Instruments and conditions

[0066] Chromatographic conditions:

[0067] Chromatographic column: base-passivated octadecyl-bonded silica gel column (4.6mm×250mm, 5μm)

[0068] Mobile phase: Phase A: 0.1% trifluoroacetic acid (adjust pH 2.5 with NaOH)-acetonitrile (88:12)

[0069] Phase B: Acetonitrile

[0070] Detection wavelength: 335nm; column temperature: 30°C; flow rate: 1.8ml / min; diluent: water

[0071] Gradient elution program:

[0072]

[0073] (2) Experimental steps

[0074] Derivatization test solution: take Merfey's [N α -(5-fluoro-2,4-dinitrophenyl)-L-alanamine amide] reagent amount, accurately weighed, add acetone to dissolve and dilute to make a 10mg / ml solution, shake well, and place in the refrigerator ( 2℃~8℃) for storage.

[0075] Sample solution: take appropriate amount of glycyl-L-glutamine and glycyl-D-glutamine, dissolve and dilute with water to make about 30mg of glycyl-L-glutamine, glycyl-D-glutamine per 1ml - A mixed solution of 3...

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