CN100368803C - A kind of separation and determination method of pregabalin chiral isomer - Google Patents

Abstract

The present invention belongs to the field of analytical chemistry, which relates to a process for separating and measuring chiral isomers (impurities) of pregabalin after derivatization by a method of high efficiency liquid chromatography. The present invention concretely comprises that N-(5-fluorine-2, 4-dinitrophenyl)-L-amino acids are used as derivatization reagents which are processed by pre-column derivatization with the pregabalin; the pregabalin and the chiral isomers thereof are separated and measured by a method of high efficiency liquid chromatography or high efficiency liquid chromatography-mass spectrometry. The pre-column derivatization of the present invention has the advantages of mild reaction condition, stable derivatization product, high reaction speed, a little side effect, etc.; the excess derivatization reagents have no influence on measuring; the use of the common chromatographic column can separate the pregabalin from the chiral isomers thereof. The present invention can separate and measure the pregabalin and the chiral isomers containing pregabalin preparations.

Description

A kind of separation and determination method of pregabalin chiral isomer

technical field

The invention belongs to the field of analytical chemistry, relates to the derivatization and determination method of compounds before the column, in particular to N ^α -(5-fluoro-2,4-dinitrophenyl)-L-alanine amide and pregabalin (C ₈ H ₁₇ NO ₂ ) pre-column derivatization, using high performance liquid chromatography to separate and determine the chiral isomers of pregabalin.

Background technique

Pre-column derivatization is a method to improve detection sensitivity. For most compounds with no UV absorption or weak UV absorption, with appropriate derivatization methods, accurate determination including content and chiral isomers can be carried out.

Pregabalin is a chiral drug for the treatment of epilepsy and neuralgia. Its chemical name is (S)-3-(aminomethyl)-5-methylhexanoic acid, and its molecular formula is C ₈ H ₁₇ NO ₂ ; Pregabalin The chiral isomer of Bahrain, whose chemical name is (R)-3-(aminomethyl)-5-methylhexanoic acid. The ultraviolet absorption of pregabalin is very weak, and it is difficult to measure it with an ultraviolet detector. In the prior art, there has been a report on the determination of pregabalin content by derivatization method "see Journal of Chromatography B 674 (1995) 143-148", but there is no use of chiral derivatization reagents to carry out derivatization reactions with pregabalin, Determination of reports of its chiral isomers.

The chiral isomer of pregabalin is the impurity of pregabalin, which is an important indicator for the quality control of pregabalin. The separation and determination of chiral isomers of pregabalin by chiral chromatographic column is not only costly, but also difficult to separate. The present invention adopts pre-column derivatization of chiral derivatization reagents to realize rapid and accurate separation and determination of pregabalin chiral isomers with a general separation chromatographic column (C ₁₈ or C ₈ chromatographic column). Finally, the derivatized products of pregabalin and its chiral isomers have a resolution of 4.5, thereby realizing the control of the chiral impurities of pregabalin.

Contents of the invention

The object of the present invention is to provide a method for the separation and determination of chiral isomers of pregabalin using liquid chromatography, characterized in that pregabalin is derivatized with a derivatization reagent before the column, wherein the derivatization reagent is N ^α -(5-fluoro-2,4-dinitrophenyl)-L-amino acid compounds. When pregabalin is derivatized, its chiral isomers are also derivatized, so that the separation and determination of pregabalin and its chiral isomers (impurities) can be realized by using a general chromatographic column. The chiral isomers of pregabalin mentioned here are regarded as important impurities of pregabalin.

The isomer of pregabalin mentioned in the present invention refers to (R)-3-(aminomethyl)-5-methylhexanoic acid.

The above-mentioned derivatization reagent is selected from the following compounds: N ^α -(5-fluoro-2,4-dinitrophenyl)-L-propanamine amide, N ^α -(5-fluoro-2,4-dinitro Phenyl)-L-phenylalanine amide, N ^α -(5-fluoro-2,4-dinitrophenyl)-L-proline amide or N ^α -(5-fluoro-2,4-dinitrobenzene base)-L-valinamide, preferably N ^α -(5-fluoro-2,4-dinitrophenyl)-L-propanamine amide.

The pre-column derivatization of pregabalin refers to the quantitative reaction of pregabalin and its chiral isomers with derivatization reagents to generate derivatives. The molar ratio of pregabalin and derivatization reagents is 1:1～1:500 , preferably 1:1 to 1:5.

The liquid chromatography mentioned above refers to high performance liquid chromatography or the combined application of high performance liquid chromatography and mass spectrometry.

The above-mentioned separation assay method mainly comprises the following steps:

①. Take an appropriate amount of pregabalin or a preparation containing pregabalin, dissolve it in a dilute acid solution, then adjust the pH value to 6.0-8.0 with an alkaline solution, add an appropriate amount of water, shake well, and prepare a preparation containing 0.5%-5.0% pregabalin The solution;

②, take pregabalin solution and 1mol/L carbonate solution, mix, add N ^α -(5-fluoro-2,4-dinitrophenyl)-L--amino acid compound solution, mix well;

③. React at 20-60°C for 20-120 minutes, and add 2mol/L hydrochloric acid solution after the reaction is completed;

④. The reactants are dried in the pre-set drying system of phosphorus pentoxide and potassium hydroxide until the solvent is fully evaporated, then add 4ml of dimethyl sulfoxide or N,N-dimethylformamide to dissolve the residue, and filter , and the filtrate was used as the test solution;

5. Separate and measure pregabalin with high performance liquid chromatography or high performance liquid chromatography-mass spectrometry. The previous step of the test solution of pregabalin in the above step 1. said pregabalin can contain its chiral isomer (called pregabalin) Bahrain racemate), or contains a very small amount of its chiral isomer (impurity), or does not contain its chiral isomer.

Above-mentioned step 1. said dilute acid solution or mineral acid such as dilute hydrochloric acid, dilute sulfuric acid, solutions such as dilute phosphoric acid, can also be organic acid such as formic acid, acetic acid, trifluoroacetic acid; Said alkaline solution or inorganic base solution such as Sodium hydroxide, potassium hydroxide, ammonia water, organic alkali solution such as triethylamine,

Said carbonate solution of above-mentioned step 2. can be sodium bicarbonate, potassium bicarbonate, potassium carbonate, sodium carbonate solution; Also can above-mentioned carbonate mix the composition gained after according to arbitrary ratio.

The N ^α -(5-fluoro-2,4-dinitrophenyl)-L--amino acid compound in the above step ② is selected from: N ^α -(5-fluoro-2,4-dinitrophenyl base)-L-alanamine amide, N ^α -(5-fluoro-2,4-dinitrophenyl)-L-amphetamine amide, N ^α -5-fluoro-2,4-dinitrophenyl)- L-proline amide or N ^α -(5-fluoro-2,4-dinitrophenyl)-L-valinamide.

The chromatographic column that the above-mentioned steps 5. said high-performance liquid chromatography or high-performance liquid chromatography-mass spectrometry can be the chromatographic column that octadecylsilane bonded silica gel is packing, can also be that octylsilane bonded silica gel is Packed columns.

The HPLC method mentioned in the above step ⑤ is used to determine pregabalin and its chiral isomers, the detection wavelength is 200-380nm, and the optimal detection wavelength is 340±3nm.

Said method of adopting high performance liquid chromatography of the present invention to separate and measure pregabalin and its chiral isomers, mobile phase can be triethylamine aqueous solution-acetonitrile system, can also be triethylamine aqueous solution-methanol system, three The ratio of ethylamine solution to acetonitrile or methanol is 1:2 to 2:1, the concentration of triethylamine is 0.1 to 5%, and the pH is adjusted to 2.0 to 5.0 with phosphoric acid.

The inventive method is specifically realized through the following technical solutions and steps:

Take pregabalin or pregabalin-containing preparation samples, add dilute hydrochloric acid solution and shake to dissolve, and adjust the pH value to 5.0-9.0 (preferably pH value 6.0-8.0) with dilute alkali solution, add water and shake well, take the solution and add An appropriate amount of chiral derivatization reagent and sodium bicarbonate solution is reacted at 20-60° C. (preferably 35-45° C.) for 20-120 minutes (preferably 30-60 minutes). After the reaction is completed, dilute hydrochloric acid solution is added, dried under reduced pressure, and an appropriate amount of dimethyl sulfoxide or N, N-dimethylformamide is added to prepare a solution, which is determined by high performance liquid chromatography or high performance liquid chromatography-mass spectrometry.

The present invention uses N ^α -(5-fluoro-2,4-dinitrophenyl)-L-propanamine amide and pregabalin for pre-column derivatization, which not only makes the derivatized product have stronger ultraviolet absorption, but also Moreover, the chiral isomers of pregabalin can be determined conveniently and quickly; the invention has the advantages of mild reaction conditions, fast reaction speed, no interference of excess derivatization reagents and the like. Moreover, the derivatized product of the present invention is stable, has high analytical sensitivity, and can accurately determine 0.05% chiral isomers in pregabalin. The method not only overcomes the weak ultraviolet absorption of pregabalin, which is difficult to measure with an ultraviolet detector, but also solves the problem of separating and measuring chiral impurities in pregabalin and its preparations with ordinary chromatographic columns (C ₁₈ or C ₈ chromatographic columns).

Description of drawings

Figure 1 High performance liquid chromatogram of reagent blank

The high-performance liquid chromatogram of Fig. 2 pregabalin racemate (R and S configuration)

The high performance liquid chromatogram of Fig. 3 pregabalin

The high performance liquid chromatogram of Fig. 4 pregabalin capsule

Specific embodiments: the following examples are used to further understand the present invention, but are not limited to the scope of the implementation.

Example 1

Instruments and Conditions

American Agilent 1100 high performance liquid chromatography system and workstation; automatic sampling; chromatographic column adopts octadecylsilane bonded silica gel column; ultraviolet detection wavelength: 340nm; mobile phase: triethylamine solution (take 5ml of triethylamine, add water 1000ml, adjust the pH value to 3.0 with phosphoric acid solution)-acetonitrile (55:45).

Experimental procedure

Take about 160 mg of the racemate of pregabalin into a 10 ml measuring bottle, take an appropriate amount of 1 mol/L hydrochloric acid solution, shake to dissolve, neutralize with 1 mol/L sodium hydroxide solution, and dilute to the mark with water. Shake well as a sample solution; take another 10.0 mg of Nα-(5-fluoro-2,4-dinitrophenyl)-L-alanamine amide, add 1 ml of acetone, shake well, and use it as a derivatization reagent solution. Take 50 μl of sample solution, 200 μl of derivatization reagent solution and 20 μl of 1mol/L sodium bicarbonate solution, put them in a 2ml sample bottle, seal it tightly, and heat it at 40°C for 45 minutes; after the reaction, add 10 μl of 2mol/L hydrochloric acid solution, Diphosphorus oxide and potassium hydroxide are dried in a vacuum drying system until the solvent is fully evaporated. Add 4ml of dimethyl sulfoxide and shake to dissolve the residue, filter, and use the filtrate as the test solution. Get need testing solution and carry out HPLC analysis under above-mentioned conditions, the results are shown in Figure 2.

The chromatographic peak with a retention time of 3.608 minutes in Fig. 2 is an excess derivatization reagent peak; the chromatographic peaks with a retention time of 7.904 minutes and 9.397 minutes are pregabalin (S configuration) and its chiral isomer (R configuration) respectively chromatographic peaks. It indicated that pregabalin and its chiral isomers and excess derivatization reagent could achieve baseline separation.

Example 2

Get about 80mg of pregabalin to 10ml measuring bottle, prepare need testing solution according to the experimental procedure of embodiment 1 below, and carry out high performance liquid chromatography analysis under the condition of embodiment 1, the results are shown in Figure 3.

The chromatographic peak of retention time 3.666 minutes among Fig. 3 is excess derivatization reagent peak; configuration, 0.5%) of the chromatographic peaks. Figure 3 shows that pregabalin and its chiral isomers and excess derivatization reagents can achieve baseline separation, and can accurately determine the chiral isomer impurities contained in pregabalin.

Example 3

Get an appropriate amount of pregabalin capsule content (approximately equivalent to 80 mg of pregabalin) in a 10ml measuring bottle, prepare the test solution according to the experimental procedure of Example 1 below, and carry out high performance liquid chromatography analysis by the conditions of Example 1 , the results are shown in Figure 4.

The chromatographic peak with a retention time of 3.633 minutes in Figure 4 is the peak of excess derivatization reagent; the chromatographic peak with a retention time of 8.116 minutes is the chromatographic peak of pregabalin. Figure 4 shows that pregabalin and excess derivatization reagent can achieve baseline separation, if the sample contains pregabalin chiral isomer impurity, it can be accurately determined by this method.

Example 4

Prepare reagent blank solution according to the experimental procedure of Example 1, and carry out high performance liquid chromatography analysis under the conditions of Example 1, the results are shown in Figure 1.

The chromatographic peak with a retention time of 3.684 minutes in Figure 1 is the peak of the derivatization reagent; there is no chromatographic peak in the range of retention time 5 to 10 minutes, indicating that the derivatization reagent does not interfere with the determination.

Claims (11)

1. A method for the chiral isomers of pregabalin separated and measured by liquid chromatography, characterized in that: pregabalin is derivatized with a derivatizing reagent before entering the column, and said derivatizing reagent is selected from N ^α -(5-fluoro-2,4-dinitrophenyl)-L-propanamine amide, N ^α -(5-fluoro-2,4-dinitrophenyl)-L-amphetamine amide, N ^α One of -(5-fluoro-2,4-dinitrophenyl)-L-proline amide and N ^α -(5-fluoro-2,4-dinitrophenyl)-L-valinamide species, its separation and determination process includes: a) Before entering the column, pregabalin and the derivatization reagent are quantitatively reacted to generate derivatives; b) The derivatives are separated and determined by high performance liquid chromatography, or a combination of high performance liquid chromatography and mass spectrometry. 2. The method according to claim 1, characterized in that the molar ratio of pregabalin to the derivatization reagent is 1:1 to 1:500. 3. method as claimed in claim 1, said separation assay method comprises the following steps: ①. Take an appropriate amount of pregabalin or a preparation containing pregabalin, dissolve it in a dilute acid solution, then adjust the pH value to 6.0-8.0 with an alkaline solution, add an appropriate amount of water, shake well, and prepare a preparation containing 0.5%-5.0% pregabalin The solution; ②, take pregabalin solution and carbonate solution, mix, add derivatization reagent solution, mix; ③. React at 20-60°C for 20-120 minutes, and add hydrochloric acid solution after the reaction is completed; ④. The reactants are dried in the pre-set drying system of phosphorus pentoxide and potassium hydroxide until the solvent is fully evaporated, then add dimethyl sulfoxide or N,N-dimethylformamide to dissolve the residue, filter, The filtrate is used as the test solution; 5. Separate and measure the need testing solution in the previous step with high performance liquid chromatography or high performance liquid chromatography-mass spectrometry. 4. The method as claimed in claim 3, wherein: step 1. said dilute acid solution or a kind of inorganic acid selected from dilute hydrochloric acid, dilute sulfuric acid, dilute phosphoric acid solution, or selected from formic acid, acetic acid, three An organic acid in fluoroacetic acid; said alkali solution is an inorganic alkali solution selected from sodium hydroxide, potassium hydroxide, ammonia, or an organic alkali solution triethylamine. 5. The method as claimed in claim 3, wherein, step 2. said carbonate solution is selected from: sodium bicarbonate, potassium bicarbonate, potassium carbonate or sodium carbonate solution or their composition obtained after mixing according to any ratio . 6. the method for claim 3, wherein, the chromatographic column that step 5. said high performance liquid chromatography or high performance liquid chromatography-mass spectrometry uses can be the chromatographic column that is filler with octadecylsilane bonded silica gel column, or a chromatographic column packed with octylsilane-bonded silica gel. 7. The method according to claim 3, wherein the high performance liquid chromatography in step ⑤ has a detection wavelength of 200-380nm. 8. The method according to claim 7, wherein, step 5. said high performance liquid chromatography, its detection wavelength is 340 ± 3nm. 9. The method according to any one of claims 1 to 8, wherein the mobile phase is a triethylamine aqueous solution-acetonitrile system or a triethylamine aqueous solution-methanol system. 10. The assay method according to claim 9, characterized in that the concentration of triethylamine in the triethylamine solution is 0.1 to 5%, and the pH is adjusted to 2.0 to 5.0 with phosphoric acid. 11. assay method as claimed in claim 9 is characterized in that the ratio of triethylamine solution and acetonitrile or methyl alcohol is 1: 2～2: 1 in mobile phase.

Images