A method for analyzing chiral impurities in L-carnitine key intermediate L-carnitine

Abstract

The present invention belongs to the field of pharmaceutical chemical technology, and specifically relates to a method for analyzing chiral impurities of L-carnitine key intermediate L-carnitine. The method first uses (S)-2-(6-methoxy-2-naphthyl) propionyl chloride as a derivatization reagent to derive L-carnitine, and then uses high performance liquid chromatography to separate and detect the derivatized L-carnitine. The present invention proposes for the first time to use HPLC to determine the chiral purity of L-carnitine, which has simple steps and mild conditions, and is conducive to industrial promotion and application; the method of the present invention has strong specificity, high sensitivity, and good durability, and can be used for the accurate determination of the chiral purity of L-carnitine.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical chemical industry, and particularly relates to a method for analyzing chiral impurities of L-carnitine key intermediate L-carnitine. Background Art

[0002] L-carnitine is an important food nutritional enhancer, with the chemical name (3R)-3-hydroxy-4-trimethylaminobutyric acid. It is widely used in many fields such as medicine, nutrition and feed. It can not only be used to treat diseases such as chronic renal failure, cardiomyopathy, coronary heart disease and organic acidemia, but also can be used as an auxiliary treatment for chronic liver disease, and has a certain protective effect on endotoxemia and liver damage. In addition, in the field of nutrition and feed, L-carnitine is also often used as an additive to improve the nutritional value of food and feed.

[0003] As food safety requirements become increasingly stringent, research on impurities in L-carnitine is becoming increasingly in-depth, especially for the chiral purity requirements of L-carnitine. USP has made clear provisions for the chiral purity of L-carnitine, requiring its impurities to be less than 0.2%. The preparation process of L-carnitine involves high temperature and strong acid, which may cause product racemization during the production process. L-carnitine nitrile is a key intermediate in the synthesis of L-carnitine, and its chiral purity is a key influencing factor in controlling the chiral impurities of L-carnitine.

[0004] At present, the commonly used method for detecting L-carnenitrile is to perform optical rotation detection on it. Optical rotation detection can reflect the chiral purity of the compound, but its disadvantages are also obvious. Optical rotation detection is related to the concentration and purity of the sample. The inorganic salts in the sample will directly affect the concentration of the configured sample and cause errors in the optical rotation measurement. The chiral impurities contained in the sample are optically active and will also affect the optical rotation measurement of the product. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention provides an analysis method for chiral impurities of L-carnitrile. L-carnitrile has a simple structure and no ultraviolet absorption, and needs to be detected by a special detector. The present invention adopts a chiral derivatization reagent to perform pre-column derivatization on L-carnitrile, and the derivatized compound has two chiral centers and ultraviolet absorption, and the formed diastereomers can be separated by a common chromatographic column, the derivatization step is simple, and the operation is convenient, the chiral derivatization reagent is cheap, and the method has high market application value.

[0006] A method for detecting chiral impurities of L-carnitrile comprises the following steps:

[0007] (1) At a certain temperature, in a buffer solution, a L-carnitrile sample is subjected to a derivatization reaction with (S)-2-(6-methoxy-2-naphthyl)propionyl chloride to obtain a derivatized product, as shown in the following reaction formula (I);

[0008]

[0009] (2) Separating and detecting the derivatized product of step (1) by high performance liquid chromatography.

[0010] Furthermore, step (1) is to mix the aqueous solution of L-carnitrile, the acetonitrile solution of (S)-2-(6-methoxy-2-naphthyl)propionyl chloride and the carbonate buffer solution (preferably with a pH of 10.2) evenly, heat in a water bath at 58 to 62° C. (preferably 60° C.) for 1.5 to 2.5 h (preferably 2 h), immediately add the acetate buffer solution (preferably with a pH of 4.2), mix evenly, filter and collect the filtrate.

[0011] Furthermore, in step (1), the mass ratio of L-carnitrile to (S)-2-(6-methoxy-2-naphthyl)propionyl chloride is 1:20; and the amount ratio of L-carnitrile, carbonate buffer solution and acetate buffer solution is 1 mg:1 mL:74 mL.

[0012] Furthermore, the following chromatographic conditions are used for detection in step (2):

[0013] Chromatographic column: C18 column;

[0014] Mobile phase A: 1% (v / v) triethylamine aqueous solution (pH adjusted to 3.4-3.6 with phosphoric acid, preferably pH 3.5)-tetrahydrofuran [(86-90):(14-10), preferably 88:12];

[0015] Mobile phase B: acetonitrile;

[0016] Gradient elution program:

[0017] Time (min) Mobile phase A (%) Mobile phase B (%) 0 100 0 25 100 0 25.1 30 70 30 30 70 30.1 100 0 40 100 0

[0018] Detector: DAD 210nm;

[0019] Flow rate: 0.9-1.1 mL / min (preferably 1.0 mL / min);

[0020] Column temperature: 30±2℃;

[0021] Injection volume: 20μl.

[0022] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0023] (1) The present invention proposes for the first time to use HPLC to determine the chiral purity of L-carnitrile, which has simple steps, mild conditions, and is conducive to industrial promotion and application;

[0024] (2) The method of the present invention has strong specificity, high sensitivity and good durability, and can be used to accurately determine the chiral purity of L-carnitrile. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is the chromatogram of the derivatized solution of sample 1 in Example 1;

[0026] Figure 2 This is the chromatogram of the derivatized solution of sample 2 in Example 1. DETAILED DESCRIPTION

[0027] The applicant will further explain the present invention in detail below in conjunction with specific examples, with the aim of enabling those skilled in the art to more clearly understand the present invention, but the following content should not be construed in any way as limiting the scope of protection claimed in the claims of this application. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used can all be obtained from commercial channels.

[0028] Table 1: Some reagent information

[0029]

[0030] Example 1: Analysis method establishment

[0031] (1) Chromatographic conditions

[0032] Chromatographic column: Zorbax Eclipse XDB-C18, 150 mm × 4.6 mm × 5 μm;

[0033] Mobile phase: Mobile phase A: 1% (v / v) triethylamine aqueous solution (pH adjusted to 3.5 with phosphoric acid)-tetrahydrofuran (88:12); Mobile phase B: acetonitrile;

[0034] Elution procedure:

[0035]

[0036]

[0037] Detector: DAD 210nm;

[0038] Flow rate: 1.0 mL / min;

[0039] Column temperature: 30°C;

[0040] Injection volume: 20μl.

[0041] (2) Solution preparation:

[0042] Derivatization test solution: Take an appropriate amount of (S)-2-(6-methoxy-2-naphthyl)propionyl chloride and dissolve it in acetonitrile to make a 5 mg / mL solution.

[0043] Carbonate buffer solution: Take 530 mg of sodium carbonate and 420 mg of sodium bicarbonate, mix, add water to dissolve and dilute to 100 mL, and measure the pH to be 10.2.

[0044] Acetate buffer: Measure 0.3 mL of glacial acetic acid, add 90 mL of water to dissolve, adjust the pH to 4.2 with 30 wt% sodium hydroxide solution, and then dilute to 100 mL with water.

[0045] Test sample 1 solution: Take about 25 mg of L-carboxylic acid nitrile reference substance and about 5 mg of D-carboxylic acid nitrile reference substance, accurately weigh, place in the same 100mL volumetric flask, add water to dissolve and dilute to the scale, shake well; then accurately measure 1.0 ml, place in a 100ml volumetric flask, add water to dilute to the scale, and shake well.

[0046] Sample 2 solution: Take about 25 mg of L-carnenitrile sample, accurately weigh it, place it in a 25 mL volumetric flask, add water to dissolve and dilute to the scale, and shake well.

[0047] Blank derivatization solution: Accurately measure 25 μl of purified water and place it in a 2 mL volumetric flask, add 25 μl of carbonate buffer solution and 100 μl of derivatization solution, mix well, heat in a 60°C water bath for 2 h, immediately add acetate buffer to dilute to the scale, shake well, filter, and take the filtrate.

[0048] Derivatization solution of sample 1: accurately measure 25 μl of sample 1 solution and place it in a 2 mL volumetric flask, add 25 μl of carbonate buffer solution and 100 μl of derivatization test solution, mix well, heat in a 60°C water bath for 2 h, immediately add acetate buffer to dilute to the mark, shake well, filter, and take the filtrate. Prepare the derivatization solution of sample 2 in the same way.

[0049] (3) Determination method:

[0050] Accurately measure 20 μl of blank derivatization solution, derivatization solution of test sample 1, and derivatization solution of test sample 2, respectively, and inject them into the chromatograph, record the chromatogram, and identify the chromatographic peaks of the derivatives of L-carnitrile and its enantiomers by comparing the chromatogram of the derivatization solution of test sample 1. The separation degree between the derivative peaks of L-carnitrile and its enantiomers and their adjacent peaks in the chromatogram of the derivatization solution of the test sample should be no less than 1.5. If the chromatographic peaks of the enantiomeric derivatives are detected in the chromatogram of the derivatization solution of test sample 2, calculate the enantiomeric impurity content in test sample 2 according to the following formula:

[0051]

[0052] Where: W is the content of enantiomeric impurities; A D A is the peak area of ​​the enantiomeric derivative in the chromatogram of the derivatized solution of sample 2; 总 It is the sum of the peak areas of the chromatographic peaks in the chromatogram of the derivatized solution of sample 2.

[0053] (4) Measurement results

[0054] The chromatograms of the derivatized solution of sample 1 and the derivatized solution of sample 2 are shown in Figure 1 and Figure 2 .Depend on Figure 1 , 2 It can be seen that the method of the present invention can better identify L-carnitrile and its enantiomers, and the separation degree between the derivative peak of L-carnitrile and its enantiomer and its adjacent peak is greater than 1.5, thereby achieving the detection purpose of the present invention.

[0055] Example 2: Methodology Verification Experiment

[0056] (1) Chromatographic conditions

[0057] Chromatographic column: Zorbax Ecl ipse XDB-C18, 150mm×4.6mm×5μm

[0058] Mobile phase: Mobile phase A: 1% (v / v) triethylamine aqueous solution (pH adjusted to 3.5 with phosphoric acid)-tetrahydrofuran (88:12); Mobile phase B: acetonitrile

[0059] Elution procedure:

[0060] Time (min) Mobile phase A (%) Mobile phase B (%) 0 100 0 25 100 0 25.1 30 70 30 30 70 30.1 100 0 40 100 0

[0061] Detector: DAD 210nm; flow rate: 1.0mL / min; column temperature: 30℃; injection volume: 20μl.

[0062] (2) Solution preparation

[0063] Derivatization test solution: Take an appropriate amount of (S)-2-(6-methoxy-2-naphthyl)propionyl chloride and dissolve it in acetonitrile to make a 5 mg / mL solution.

[0064] Carbonate buffer solution: Take 530 mg of sodium carbonate and 420 mg of sodium bicarbonate, mix, add water to dissolve and dilute to 100 mL.

[0065] Acetate buffer: Measure 0.3 mL of glacial acetic acid, add 90 mL of water to dissolve, adjust the pH to 4.2 with 30 wt% sodium hydroxide solution, and then dilute to 100 mL with water.

[0066] Blank solution: Accurately measure 25 μl of purified water and place it in a 2 mL volumetric flask, add 25 μl of carbonate buffer solution and 100 μl of derivatization test solution, mix well, heat in a 60°C water bath for 2 h, immediately add acetate buffer to dilute to the scale, shake well, filter, and take the filtrate.

[0067] System suitability solution: Accurately weigh 25.1415 mg of L-carnitrile reference substance and 5.0139 mg of D-carnitrile reference substance, place them in the same 25 mL volumetric flask, add water to dissolve and dilute to the mark, shake well. Accurately measure 25 μl and place it in a 2 mL volumetric flask, add 25 μl of carbonate buffer solution and 100 μl of derivatization test solution, mix well, heat in a 60°C water bath for 2 h, immediately add acetate buffer to dilute to the mark, shake well, filter, and take the filtrate.

[0068] Reference substance stock solution: Accurately weigh 5.0204 mg of D-carnenitrile reference substance, place it in a 100 mL volumetric flask, add water to dissolve and dilute to the scale, and shake well.

[0069] Reference substance solution: Take 1 mL of reference substance stock solution and place it in a 25 mL volumetric flask, add water to dilute to the scale, and shake well.

[0070] Derivatization solution of reference substance: accurately measure 25 μl of reference substance solution and place it in a 2 mL volumetric flask, add 25 μl of carbonate buffer solution and 100 μl of derivatization test solution, mix well, heat in a 60°C water bath for 2 h, immediately add acetate buffer to dilute to the scale, shake well, filter, and take the filtrate.

[0071] Test solution: Take about 25 mg of L-carnenitrile sample, accurately weigh it, place it in a 25 mL volumetric flask, add water to dissolve and dilute to the scale, and shake well.

[0072] Derivatization solution of test sample: accurately measure 25 μl of the test sample solution and place it in a 2 mL volumetric flask, add 25 μl of carbonate buffer solution and 100 μl of derivatization test solution, mix well, heat in a 60°C water bath for 2 h, immediately add acetate buffer to dilute to the scale, shake well, filter, and take the filtrate.

[0073] 1. System applicability and specificity

[0074] Take blank solution, system suitability solution, reference solution, reference derivatization solution, and test sample derivatization solution, and measure according to the above chromatographic conditions. In the chromatogram, the blank solution has no interference at the peak of the derivative of L-carnitrile and its enantiomer, the separation degree of the derivative peak of L-carnitrile and its enantiomer and its adjacent peak is greater than 1.5, and the number of theoretical plates is greater than 5000; the peak purity of the derivative peak of L-carnitrile and its enantiomer is greater than 990; the reference derivatization solution is injected 5 times continuously, and the RSD of the main peak area is 0.91%. It shows that the method specificity and system applicability of the present invention are good.

[0075] 2. Limit of Quantitation and Limit of Detection

[0076] Take 2 ml of the reference solution and place it in a 10 ml volumetric flask, add water to dilute to the mark, shake well, and use it as the quantitative limit solution; treat it according to the above derivatization method, take the obtained sample for injection, and measure the main peak signal-to-noise ratio to be 23.7, which is greater than 10. Take another 3 ml of the quantitative limit solution and place it in a 10 ml volumetric flask, add water to dilute to the mark, shake well, and use it as the detection limit solution; treat it according to the above derivatization method, take the obtained sample for injection, and measure the main peak signal-to-noise ratio to be 6.28, which is greater than 3. The enantiomer limit is 0.2% (USP), and the concentration of the quantitative limit solution is 20% of the limit, which meets the daily detection needs.

[0077] 3. Linear

[0078] Accurately pipette 0.2ml, 0.5ml, 0.8ml, 1.0ml, and 1.2ml of the reference stock solution into a 25ml volumetric flask, dilute to the mark with water, shake well, and prepare a series of standard solutions with concentrations of 0.4016μg / ml, 1.0041μg / ml, 1.6065μg / ml, 2.0082μg / ml, and 2.4098μg / ml, respectively. Treat according to the above derivatization method, take the obtained sample for injection, and record the chromatogram and peak area. The peak area Y is used to perform linear regression calculation on the mass concentration X (μg / ml), and the linear regression equation is: Y = 4019.4X + 3.9529, r = 0.9999. The results show that the peak area of ​​the derivative has a good linear relationship with the concentration in the concentration range of 0.4016 to 2.4098μg / ml.

[0079] 4. Repeatability

[0080] Take about 25 mg of L-carnenitrile sample, weigh accurately, place in a 25 mL volumetric flask, add water to dissolve and dilute to scale, shake well; prepare 6 copies in parallel. Treat according to the above derivatization method, take the obtained sample for injection, record the chromatogram, and calculate the content of the enantiomer by area normalization. The RSD of the 6 results is 1.67%, indicating that the method has good repeatability.

[0081] 5. Accuracy

[0082] Take about 25 mg of L-carnitrile sample, accurately weigh it, put it in a 25 mL volumetric flask, add 0.2 ml of reference stock solution accurately measured, then add water to dissolve and dilute to the scale, shake well, as the 20% limit spike solution, and prepare 3 parallel copies. Take about 25 mg of L-carnitrile sample, accurately weigh it, put it in a 25 mL volumetric flask, add 1.0 ml of reference stock solution accurately measured, then add water to dissolve and dilute to the scale, shake well, as the 100% limit spike solution, and prepare 3 parallel copies. Take about 25 mg of L-carnitrile sample, accurately weigh it, put it in a 25 mL volumetric flask, add 1.2 ml of reference stock solution accurately measured, then add water to dissolve and dilute to the scale, shake well, as the 120% limit spike solution, and prepare 3 parallel copies. Take 9 portions of the prepared solution, treat them according to the above derivatization method, take the obtained sample for injection, record the chromatogram and peak area, calculate the content of enantiomeric impurities by area normalization method, and then calculate the recovery rate according to the following formula. The measured recovery rate is 94.1% to 102.7%, and the RSD of 9 results is 2.9%. This shows that the accuracy of this method is good.

[0083]

[0084] Wherein: M is the sample weight (mg); W' is the content of enantiomeric impurities after spiking; W is the content of enantiomeric impurities before spiking; V is the volume of reference stock solution added (ml); C is the concentration of reference stock solution added (mg / ml).

[0085] 6. Stability

[0086] The test sample derivatization solution was prepared according to the solution preparation item of this embodiment, and the prepared test sample derivatization solution was measured at 0, 3, 6, 12, 18, 24, and 48 hours, respectively. The maximum deviation of the measured enantiomer peak area compared with the enantiomer peak area at 0 hour was 0.57%; and the maximum deviation of the enantiomer content measured at each time point compared with that at 0 hour was 0.02%, indicating that the test sample solution had good stability within 48 hours.

[0087] 7. Durability

[0088] The durability of the derivatization method was investigated at a derivatization temperature of 58-62°C and a derivatization time of 1.5-2.5h. The results obtained under each condition had a maximum deviation of 0.02% compared with the original conditions, indicating that the derivatization method had good durability. The chromatographic conditions were investigated again, such as a column temperature of 30±2°C, a flow rate of 0.9-1.1ml / min, a pH value of 3.4-3.6 for a 1% triethylamine aqueous solution in the mobile phase, and a volume ratio of 1% triethylamine aqueous solution to tetrahydrofuran in mobile phase A of (86-90):(14-10). The separation of the derivative peaks of L-carnitrile and its enantiomers and their adjacent peaks in the obtained chromatogram was greater than 1.5, and the theoretical plate numbers were greater than 5000; the maximum deviation of the enantiomer content measured compared with the original conditions was 0.01%. This indicates that the method has good durability.

Claims

1. A method for detecting chiral impurities in L-carnitrile, characterized in that: The following steps are involved: (1) at a certain temperature, in a buffer solution, a L-carnitrile sample is subjected to a derivatization reaction with (S)-2-(6-methoxy-2-naphthyl)propionyl chloride to obtain a derivatized product; (2) Separating and detecting the derivatized product of step (1) by high performance liquid chromatography.

2. The detection method according to claim 1, characterized in that: Step (1) specifically comprises: mixing the L-carnitrile aqueous solution, the (S)-2-(6-methoxy-2-naphthyl)propionyl chloride acetonitrile solution and the carbonate buffer solution uniformly, heating in a water bath at 58 to 62° C. for 1.5 to 2.5 hours, immediately adding the acetate buffer solution, mixing uniformly, filtering and collecting the filtrate.

3. The detection method according to claim 2, characterized in that: In step (1), the pH of the carbonate buffer solution is 10.2, and the pH of the acetate buffer solution is 4.

2.

4. The detection method according to claim 2, characterized in that: The water bath heating temperature in step (1) is 60° C., and the water bath heating time is 2 h.

5. The detection method according to claim 2, characterized in that: In step (1), the mass ratio of L-carnitrile to (S)-2-(6-methoxy-2-naphthyl)propionyl chloride is 1:

20.

6. The detection method according to claim 1, characterized in that: The chromatographic conditions of step (2) are: Chromatographic column: C18 column; Mobile phase A: adjust the pH of a 1% by volume triethylamine aqueous solution to 3.4-3.6 with phosphoric acid, and mix the resulting solution with tetrahydrofuran in a volume ratio of 86-90:14-10; Mobile phase B: acetonitrile; Gradient elution program: Detector: DAD 210nm; Flow rate: 0.9~1.1mL / min; Column temperature: 30±2℃.

7. The detection method according to claim 6, characterized in that: Mobile phase A: A 1% by volume aqueous solution of triethylamine was adjusted to pH 3.5 with phosphoric acid, and the resulting solution was evenly mixed with tetrahydrofuran in a volume ratio of 88:12; the flow rate of the mobile phase was 1.0 mL / min.

8. The detection method according to claim 1, characterized in that: The chromatographic conditions of step (2) also include the injection volume, which is 20 μl.

9. The detection method according to any one of claims 1 to 8, characterized in that: The method is used for qualitative and / or quantitative analysis of L-carnenitrile and chiral impurities thereof.

10. The detection method according to claim 9, characterized in that: The quantitative analysis adopted the area normalization method.

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