Method for determining enantiomer in levocarnitine intermediate by gas chromatography

Abstract

The invention belongs to the technical field of medicine detection, and particularly relates to a method for determining enantiomers in a levocarnitine intermediate by gas chromatography. The method for determining the enantiomer in the levocarnitine intermediate by the gas chromatography comprises the following steps: taking a capillary column which takes dimethyl polysiloxane bonded with cyclodextrin as a stationary liquid as a chromatographic column; the method comprises the following steps: dissolving and diluting a test product (R)-4-chloro-3-hydroxy ethyl butyrate by using ethyl acetate, and deriving by adding acetic anhydride and pyridine to prepare a test product solution; and injecting the test solution into a gas chromatograph, recording a chromatogram, and calculating the content of the enantiomer (S)-4-chloro-3-hydroxybutyric acid ethyl ester in the levocarnitine intermediate (R)-4-chloro-3-hydroxybutyric acid ethyl ester according to an area normalization method. The method for determining the enantiomer in the levocarnitine intermediate by the gas chromatography provided by the invention is accurate and fast in detection, and provides a basis for quality control of levocarnitine.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical detection, and particularly relates to a method for determining enantiomers in a L-carnitine intermediate by gas chromatography. Background Art

[0002] L-Carnitine is a small molecule, water-soluble amino acid derivative widely present in body tissues. It is an essential substance for promoting energy metabolism, especially fatty acid metabolism. The main function of L-Carnitine is to promote lipid metabolism. It is clinically applicable to a series of complications caused by L-Carnitine deficiency in the body, such as cardiomyopathy, skeletal myopathy, arrhythmia, hyperlipidemia, as well as hypotension and muscle spasm during dialysis. (R)-4-chloro-3-hydroxybutyrate is a key intermediate in the synthesis of L-carnitine. During the synthesis process, the isomer (S)-4-chloro-3-hydroxybutyrate in the intermediate (R)-4-chloro-3-hydroxybutyrate will affect the quality of L-carnitine. (R)-4-chloro-3-hydroxybutyrate will be further synthesized into L-carnitine, while (S)-4-chloro-3-hydroxybutyrate will be further synthesized into D-carnitine. Therefore, controlling the content of enantiomers in the L-carnitine intermediate from the source will be more conducive to the production of L-carnitine API with qualified quality.

[0003] Zhejiang University master's thesis "Enzymatic Resolution of 4-chloro-3-hydroxybutyric acid ethyl ester (CHBE) and its kinetics (Huang Yang)" disclosed a method for the resolution of chiral compounds, established a determination method for CHBE and CABE (4-chloro-3-acetoxybutyric acid ethyl ester), and used gas chromatography to analyze their contents. The chromatographic conditions were chromatographic column HP Chiral (10% Permethylated B-cyclodextrin) 0.32mm×25mm, column box temperature 79℃, injector temperature 250℃, detector temperature 250℃, FID detector, H 2 :40mL / min, Ar: 450mL / min, N 2 : 1.3mL / min, split ratio 100:1, tail blowing: 40mL / min. This method describes the use of lipase to catalyze the reaction to generate (R)-CABE, but this reaction does not generate (S)-CABE, and the reaction steps are cumbersome and the reaction time is long, which is not suitable for the operational requirements of daily detection, and is mainly for the enzymatic separation of (R)-CHBE and (S)-CHBE. Summary of the invention

[0004] The technical problem to be solved by the present invention is to overcome the above-mentioned defects of the prior art and provide a method for determining the enantiomers in a L-carnitine intermediate by gas chromatography. The method is accurate and has fast detection, thus providing a basis for the quality control of L-carnitine.

[0005] The method for determining enantiomers in a L-carnitine intermediate by gas chromatography of the present invention comprises the following steps: (1) Setting chromatographic conditions: using a capillary column with cyclodextrin-bonded dimethylpolysiloxane as the stationary liquid as the chromatographic column; (2) Preparation of test solution: Dissolve and dilute the test sample (R)-4-chloro-3-hydroxybutyric acid ethyl ester with ethyl acetate, and then derivatize it by adding acetic anhydride and pyridine to prepare a test solution; (3) Determination: Inject the test sample solution into a gas chromatograph, record the chromatogram, and calculate the content of the enantiomer (S)-4-chloro-3-hydroxybutyrate ethyl ester in the L-carnitine intermediate (R)-4-chloro-3-hydroxybutyrate ethyl ester by the area normalization method.

[0006] The chromatographic conditions include: the detector is an FID detector, the FID detector temperature is 270-290°C; the temperature program is: the initial column temperature is 110-130°C, maintained for 15-20 minutes, heated to 160-180°C at a rate of 5-15°C / min, and maintained for 10-15 minutes; the injection port temperature is 260-280°C; the column pressure is 90-110 kPa; and the injection volume is 0.5-2 μL.

[0007] The chromatographic column is CP-ChirasilDex CB with specifications of 25m×0.25mm and 0.25μm.

[0008] The chromatographic conditions included an injection port temperature of 270°C, a FID detector temperature of 280°C, a column pressure of 100 kPa, and an injection volume of 1 μL.

[0009] The temperature program in the chromatographic conditions was as follows: the initial column temperature was 120°C, maintained for 17 min, increased to 170°C at a rate of 10°C / min, and maintained for 12 min.

[0010] The specific steps for preparing the test solution are as follows: take the test sample (R)-4-chloro-3-hydroxybutyric acid ethyl ester and use ethyl acetate to make a solution with a concentration of 10~50mg / mL, accurately measure 4~6mL and put it into a 20mL volumetric flask, add 4~6mL of acetic anhydride and 0.4~0.6mL of pyridine, shake it ultrasonically and place it in a 95~105℃ oil bath for 25~35min, cool it, add 4~6mL of saturated sodium chloride aqueous solution, dilute it to the scale with ethyl acetate, shake it for at least 2min, let it stand to separate, and take the upper layer as the test solution.

[0011] The L-carnitine intermediate (R)-4-chloro-3-hydroxybutyric acid ethyl ester derivative [(R)-4-chloro-3-acetoxybutyric acid ethyl ester] and the enantiomer (S)-4-chloro-3-hydroxybutyric acid ethyl ester derivative [(S)-4-chloro-3-acetoxybutyric acid ethyl ester] of step (3) are eluted in sequence, and the resolution between the two is greater than 1.5.

[0012] The calculation formula of the area normalization method is: enantiomer (S)-4-chloro-3-hydroxybutyric acid ethyl ester content (%) = ((S)-4-chloro-3-acetoxybutyric acid ethyl ester peak area / (R)-4-chloro-3-acetoxybutyric acid ethyl ester and (S)-4-chloro-3-acetoxybutyric acid ethyl ester peak area sum) × 100%.

[0013] The method is applicable to the determination of the content of the enantiomer [(S)-4-chloro-3-hydroxybutyric acid ethyl ester] in the L-carnitine API intermediate [(R)-4-chloro-3-hydroxybutyric acid ethyl ester], and the quality control standard is that the enantiomer content is ≤1.5%. The detection limit of the enantiomer (S)-4-chloro-3-hydroxybutyric acid ethyl ester derivative is 0.6~1.8μg / mL, and the quantitative limit is 2~6μg / mL.

[0014] Specifically, the method for determining enantiomers in a L-carnitine intermediate by gas chromatography comprises the following steps: (1) Chromatographic conditions: Detector: FID detector; Chromatographic column: Capillary column with cyclodextrin-bonded dimethylpolysiloxane as stationary liquid, CP-ChirasilDexCB, specifications: 25m×0.25mm, 0.25μm; Temperature program: the initial column temperature is 120±10℃, maintained for 15~20min, then increased to 170±10℃ at a rate of 10±5℃ / min, and maintained for 10~15min. Preferably, the initial column temperature is 120℃, maintained for 15min, then increased to 170℃ at a rate of 10℃ / min, and maintained for 10min. Inlet temperature: 270±10℃, preferably 270℃; FID detector temperature: 280±10℃, preferably 280℃; Column pressure: 100±10 kPa, preferably 100 kPa; Injection volume: 0.5~2μL, preferably 1μL.

[0015] (2) Preparation of test solution: Take the test sample and make a solution with a concentration of 30 mg / mL in ethyl acetate. Accurately measure 5 mL and place it in a 20 mL volumetric flask. Add 5 mL of acetic anhydride and 0.5 mL of pyridine. After ultrasonic shaking, place it in a 100°C oil bath for 30 min. Cool it, add 5 mL of saturated sodium chloride aqueous solution, dilute it to the scale with ethyl acetate, shake it for 2 min, let it stand to separate the layers, and take the upper layer.

[0016] (3) Determination method: Accurately measure the solvent (ethyl acetate) and the test solution, inject them into the gas chromatograph, record the chromatogram, and separate (R)-4-chloro-3-acetoxybutyric acid ethyl ester and (S)-4-chloro-3-acetoxybutyric acid ethyl ester. In the chromatogram of the test solution, determine the chromatographic peak with a relative retention time of about 1.1 in the obtained test solution, and calculate it by the area normalization method.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The method for determining enantiomers in a L-carnitine intermediate by gas chromatography of the present invention has the advantages of fast analysis speed, strong specificity, high sensitivity and good separation, and can accurately and quantitatively detect the content of (S)-4-chloro-3-hydroxybutyric acid ethyl ester in (R)-4-chloro-3-hydroxybutyric acid ethyl ester. (R)-4-chloro-3-hydroxybutyric acid ethyl ester is a key intermediate in the synthesis process of L-carnitine, and its enantiomer content has a great influence on the quality of L-carnitine. The detection method of the present invention can more accurately detect the enantiomer level in (R)-4-chloro-3-hydroxybutyric acid ethyl ester, and provides a better front-end control standard for the preparation of L-carnitine raw material medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is the GC chromatogram of the solvent in Example 1.

[0019] Figure 2 It is the GC chromatogram of the test solution in Example 1.

[0020] Figure 3 It is the GC chromatogram of the test solution in Example 2.

[0021] Figure 4 It is the GC chromatogram of the test solution in Example 3.

[0022] Figure 5 It is the GC chromatogram of the test solution in Example 4.

[0023] Figure 6 It is the GC chromatogram of the test solution in Example 5.

[0024] Figure 7 It is the GC chromatogram of the test solution in Example 6.

[0025] Figure 8 It is the GC chromatogram of the test solution in Example 7.

[0026] Fig. 9 It is the GC chromatogram of the test solution in Example 8.

[0027] Fig.10 It is the GC chromatogram of the test solution in Example 9.

[0028] Fig.11 It is the GC chromatogram of the test solution in Example 10.

[0029] Fig.12 It is the GC chromatogram of the test solution in Example 11.

[0030] Fig.13 This is the GC chromatogram of the test solution in Example 12.

[0031] Fig.14 This is the GC chromatogram of the test solution in Example 13.

[0032] Fig.15 It is the GC chromatogram of the test solution in Comparative Example 1.

[0033] Fig.16 This is a comparison chart of specificity tests under method validation. DETAILED DESCRIPTION

[0034] The present invention will be further described below in conjunction with specific embodiments.

[0035] Example 1 The method for determining the enantiomers of L-carnitine intermediates by gas chromatography comprises the following steps: (1) Chromatographic conditions: Detector: FID detector; Chromatographic column: Capillary column with cyclodextrin-bonded dimethylpolysiloxane as stationary liquid, CP-ChirasilDexCB, specifications: 25m×0.25mm, 0.25μm; Temperature program: the initial column temperature was 120°C, maintained for 15 min, then increased to 170°C at a rate of 10°C / min, and maintained for 10 min; Inlet temperature: 270°C; FID detector temperature: 280°C; Column pressure: 100 kPa; Injection volume: 1 μL.

[0036] (2) Preparation of test solution: Take the test sample and make a solution with a concentration of 30 mg / mL in ethyl acetate. Accurately measure 5 mL and place it in a 20 mL volumetric flask. Add 5 mL of acetic anhydride and 0.5 mL of pyridine. After ultrasonic shaking, place it in a 100°C oil bath for 30 min. Cool it, add 5 mL of saturated sodium chloride aqueous solution, dilute it to the scale with ethyl acetate, shake it for 2 min, let it stand to separate the layers, and take the upper layer.

[0037] (3) Determination method: Accurately measure the solvent (ethyl acetate) and the test sample (R)-4-chloro-3-hydroxybutyric acid ethyl ester solution, inject them into the gas chromatograph, record the chromatogram, and separate (R)-4-chloro-3-acetoxybutyric acid ethyl ester and (S)-4-chloro-3-acetoxybutyric acid ethyl ester. In the chromatogram of the test sample solution, determine the chromatographic peak with a relative retention time close to 1.1 in the obtained test sample solution and calculate it by the area normalization method.

[0038] The GC chromatogram of the solvent in this example is as follows Figure 1 As shown, the solvent peak does not interfere with the detection of this method.

[0039] The GC chromatogram of the test solution in this example is as follows Figure 2 As shown by Figure 2 It can be seen that (R)-4-chloro-3-acetoxyethyl butyrate and (S)-4-chloro-3-acetoxyethyl butyrate are eluted in sequence, and the separation degree between the two peaks is greater than 1.5. The peak area and other data tables are shown in Table 1. The content of enantiomer (S)-4-chloro-3-hydroxyethyl butyrate in the test solution is 0.785%.

[0040] Table 1 Peak table of test solution in Example 1

[0041] Example 2 The detection method and chromatographic conditions used in this embodiment are the same as those in Example 1, except that the temperature program is adjusted as follows: the initial column temperature is 110°C, maintained for 15 min, and then increased to 160°C at a rate of 10°C / min, and maintained for 10 min; the test solution is injected into the gas chromatograph and the chromatogram is recorded.

[0042] In this embodiment, the GC chromatogram of the test solution is as follows: Figure 3 As shown, the peak area and other data are shown in Table 2. Figure 3 As can be seen from Table 2, the resolution between the two peaks is greater than 1.5, the resolution is good, and the content of enantiomers in the test solution is 0.812%.

[0043] Table 2 Peak table of test solution in Example 2

[0044] Example 3 The detection method and chromatographic conditions used in this embodiment are the same as those in Example 1, except that the heating program is adjusted as follows: the initial column temperature is 130°C, maintained for 15 min, and then heated to 180°C at a rate of 10°C / min, and maintained for 10 min; the test solution is injected into the gas chromatograph and the chromatogram is recorded.

[0045] In this embodiment, the GC chromatogram of the test solution is as follows: Figure 4 As shown, the peak area and other data are shown in Table 3. Figure 4 As can be seen from Table 3, the resolution between the two peaks is greater than 1.5, the resolution is good, and the content of enantiomers in the test solution is 0.768%.

[0046] Table 3 Peak table of test solution in Example 3

[0047] Example 4 The detection method and chromatographic conditions used in this embodiment are the same as those in embodiment 1, except that the injection port temperature is adjusted to 260° C. The test sample solution is injected into the gas chromatograph and the chromatogram is recorded.

[0048] In this embodiment, the GC chromatogram of the test solution is as follows: Figure 5 As shown, the peak area and other data are shown in Table 4. Figure 5 As can be seen from Table 4, the resolution between the two peaks is greater than 1.5, the resolution is good, and the content of enantiomers in the test solution is 0.781%.

[0049] Table 4 Peak table of test solution in Example 4

[0050] Example 5 The detection method and chromatographic conditions used in this embodiment are the same as those in embodiment 1, except that the injection port temperature is adjusted to 280° C. The test sample solution is injected into the gas chromatograph and the chromatogram is recorded.

[0051] In this embodiment, the GC chromatogram of the test solution is as follows: Figure 6 As shown, the peak area and other data are shown in Table 5. Figure 6 As can be seen from Table 5, the resolution between the two peaks is greater than 1.5, the resolution is good, and the content of enantiomers in the test solution is 0.781%.

[0052] Table 5 Peak table of test solution in Example 5

[0053] Example 6 The detection method and chromatographic conditions used in this embodiment are the same as those in embodiment 1, except that the detector temperature is adjusted to 270° C. The test sample solution is injected into the gas chromatograph and the chromatogram is recorded.

[0054] In this embodiment, the GC chromatogram of the test solution is as follows: Figure 7 As shown, the peak area and other data are shown in Table 6. Figure 7 As can be seen from Table 6, the resolution between the two peaks is greater than 1.5, the resolution is good, and the content of enantiomers in the test solution is 0.786%.

[0055] Table 6 Peak table of test solution in Example 6

[0056] Example 7 The detection method and chromatographic conditions used in this embodiment are the same as those in embodiment 1, except that the detector temperature is adjusted to 290° C. The test sample solution is injected into the gas chromatograph and the chromatogram is recorded.

[0057] In this embodiment, the GC chromatogram of the test solution is as follows: Figure 8 As shown, the peak area and other data are shown in Table 7. Figure 8 As can be seen from Table 7, the resolution between the two peaks is greater than 1.5, the resolution is good, and the content of enantiomers in the test solution is 0.783%.

[0058] Table 7 Peak table of test solution in Example 7

[0059] Example 8 The detection method and chromatographic conditions used in this embodiment are the same as those in Example 1, except that the column pressure is adjusted to 90 kPa; the test solution is injected into the gas chromatograph and the chromatogram is recorded.

[0060] In this embodiment, the GC chromatogram of the test solution is as follows: Fig. 9 As shown, the peak area and other data are shown in Table 8. Fig. 9 As can be seen from Table 8, the resolution between the two peaks is greater than 1.5, the resolution is good, and the content of enantiomers in the test solution is 0.791%.

[0061] Table 8 Peak table of test solution in Example 8

[0062] Example 9 The detection method and chromatographic conditions used in this embodiment are the same as those in Example 1, except that the column pressure is adjusted to 110 kPa; the test solution is injected into the gas chromatograph and the chromatogram is recorded.

[0063] In this embodiment, the GC chromatogram of the test solution is as follows: Fig.10 As shown, the peak area and other data are shown in Table 9. Fig.10 As can be seen from Table 9, the resolution between the two peaks is greater than 1.5, the resolution is good, and the content of enantiomers in the test solution is 0.773%.

[0064] Table 9 Peak table of test solution in Example 9

[0065] Example 10 The detection method and chromatographic conditions used in this embodiment are the same as those in Example 1, except that the preparation method of the test solution is adjusted as follows: the test sample is prepared into a solution with a concentration of 30 mg / mL with ethyl acetate, 4 mL is accurately measured and placed in a 20 mL volumetric flask, 4 mL of acetic anhydride and 0.4 mL of pyridine are added, and after ultrasonic shaking, the solution is placed in an oil bath at 100°C for 30 min, cooled, 4 mL of saturated sodium chloride aqueous solution is added, the solution is diluted to the scale with ethyl acetate, shaken for 2 min, allowed to stand to separate, and the ethyl acetate layer (upper layer) is taken; the test sample solution is injected into a gas chromatograph, and a chromatogram is recorded.

[0066] In this embodiment, the GC chromatogram of the test solution is as follows: Fig.11 As shown, the peak area and other data are shown in Table 10. Fig.11 As can be seen from Table 10, the resolution between the two peaks is greater than 1.5, the resolution is good, and the content of enantiomers in the test solution is 0.784%.

[0067] Table 10 Peak table of test solution in Example 10

[0068] Embodiment 11 The detection method and chromatographic conditions used in this embodiment are the same as those in Example 1, except that the preparation method of the test solution is adjusted as follows: prepare a solution of the test sample with ethyl acetate at a concentration of 30 mg / mL, accurately measure 6 mL, place it in a 20 mL volumetric flask, add 6 mL of acetic anhydride and 0.6 mL of pyridine, shake it ultrasonically and place it in an oil bath at 100°C for 30 min, cool it, add 6 mL of saturated sodium chloride aqueous solution, dilute it to the scale with ethyl acetate, shake it for 2 min, let it stand to separate, and take the ethyl acetate layer (upper layer); inject the test sample solution into a gas chromatograph, and record the chromatogram.

[0069] In this embodiment, the GC chromatogram of the test solution is as follows: Fig.12 As shown, the peak area and other data are shown in Table 11. Fig.12As can be seen from Table 11, the resolution between the two peaks is greater than 1.5, the resolution is good, and the content of enantiomers in the test solution is 0.783%.

[0070] Table 11 Peak table of test solution in Example 11

[0071] Example 12 The detection method and chromatographic conditions used in this embodiment are the same as those in Example 1, except that the preparation method of the test solution is adjusted as follows: prepare a solution of the test sample with ethyl acetate at a concentration of 30 mg / mL, accurately measure 5 mL, place it in a 20 mL volumetric flask, add 5 mL of acetic anhydride and 0.5 mL of pyridine, shake it ultrasonically, place it in a 95°C oil bath for 25 min, cool it, add 5 mL of saturated sodium chloride aqueous solution, dilute it to the scale with ethyl acetate, shake it for 2 min, let it stand to separate, and take the ethyl acetate layer (upper layer); inject the test sample solution into a gas chromatograph, and record the chromatogram.

[0072] In this embodiment, the GC chromatogram of the test solution is as follows: Fig.13 As shown, the peak area and other data are shown in Table 12. Fig.13 As can be seen from Table 12, the resolution between the two peaks is greater than 1.5, the resolution is good, and the content of enantiomers in the test solution is 0.785%.

[0073] Table 12 Peak table of test solution of Example 12

[0074] Embodiment 13 The detection method and chromatographic conditions used in this embodiment are the same as those in Example 1, except that the preparation method of the test solution is adjusted as follows: prepare a solution of the test sample with ethyl acetate at a concentration of 30 mg / mL, accurately measure 5 mL, place it in a 20 mL volumetric flask, add 5 mL of acetic anhydride and 0.5 mL of pyridine, shake it ultrasonically and place it in an oil bath at 105°C for 35 min, cool it, add 5 mL of saturated sodium chloride aqueous solution, dilute it to the scale with ethyl acetate, shake it for 2 min, let it stand to separate, and take the ethyl acetate layer (upper layer); inject the test sample solution into a gas chromatograph, and record the chromatogram.

[0075] In this embodiment, the GC chromatogram of the test solution is as follows: Fig.14 As shown, the peak area and other data are shown in Table 13. Fig.14 As can be seen from Table 13, the resolution between the two peaks is greater than 1.5, the resolution is good, and the content of enantiomers in the test solution is 0.788%.

[0076] Table 13 Peak table of test solution of Example 13

[0077] Comparative Example 1 The chromatographic conditions of this comparative example are basically the same as those of Example 1, except that the reaction temperature of the oil bath during the preparation of the test solution is adjusted to 60°C, the heating program is adjusted to isocratic, and the column temperature is 120°C; the test solution is injected into the gas chromatograph and the chromatogram is recorded.

[0078] In this comparative example, the GC chromatogram of the test solution is as follows Fig.15 As shown, the peak area and other data are shown in Table 14. Fig.15 As can be seen from Table 14, when the reaction temperature of the oil bath is adjusted to 60°C, the reaction of the L-carnitine intermediate (R)-4-chloro-3-hydroxybutyric acid ethyl ester is incomplete, and the underivatized (R)-4-chloro-3-hydroxybutyric acid ethyl ester accounts for about 43%, indicating that this comparative example is not suitable for the detection of the compounds in the present invention.

[0079] Table 14 Comparative Example 1 Test Solution Peak Table

[0080] Method verification of the method for determining the enantiomers of the L-carnitine intermediate by gas chromatography of the present invention: (1) Exclusivity Blank solution: accurately measure 5 mL of ethyl acetate, place in a 20 mL volumetric flask, add 5 mL of acetic anhydride and 0.5 mL of pyridine, shake by ultrasound, place in a 100°C oil bath for 30 min, cool, add 5 mL of saturated sodium chloride aqueous solution, dilute to the scale with ethyl acetate, shake for 2 min, let stand to separate, and take the ethyl acetate layer (upper layer).

[0081] Preparation of (S)-4-chloro-3-acetoxybutyric acid ethyl ester solution: Accurately weigh about 23 mg of (S)-4-chloro-3-hydroxybutyric acid ethyl ester, place in a 20 mL volumetric flask, add 5 mL of ethyl acetate to dissolve, add 5 mL of acetic anhydride and 0.5 mL of pyridine, shake by ultrasound, place in a 100°C oil bath for 30 min, cool, add 5 mL of saturated sodium chloride aqueous solution, dilute to the scale with ethyl acetate, shake for 2 min, let stand to separate, and take the ethyl acetate layer (upper layer).

[0082] Preparation of (R)-4-chloro-3-acetoxybutyric acid ethyl ester solution: Accurately weigh about 150 mg of (R)-4-chloro-3-hydroxybutyric acid ethyl ester, place in a 20 mL volumetric flask, add 5 mL of ethyl acetate to dissolve, add 5 mL of acetic anhydride and 0.5 mL of pyridine, shake by ultrasound, place in a 100 ° C oil bath for 30 min, cool, add 5 mL of saturated sodium chloride aqueous solution, add ethyl acetate to dilute to the scale, shake for 2 min, let stand to separate, and take the ethyl acetate layer (upper layer).

[0083] Accurately measure the blank solution, (S)-4-chloro-3-acetoxybutyric acid ethyl ester solution, and (R)-4-chloro-3-acetoxybutyric acid ethyl ester solution, inject them into the gas chromatograph, and record the chromatogram. The results are as follows: Fig.16 As shown, Fig.16 Note: From top to bottom are the chromatograms of (S)-4-chloro-3-acetoxybutyric acid ethyl ester solution, (R)-4-chloro-3-acetoxybutyric acid ethyl ester solution, and blank solution. Fig.16 It can be seen that the blank solution does not interfere with the determination of this method. The retention time of (S)-4-chloro-3-acetoxybutyric acid ethyl ester is 13.134min, the retention time of (R)-4-chloro-3-acetoxybutyric acid ethyl ester is 12.265min, and the separation degree between the two peaks in the (R)-4-chloro-3-acetoxybutyric acid ethyl ester solution is 2.191, which is greater than 1.5, indicating that the method has good specificity.

[0084] (2) Detection limit and quantification limit Using the solvent (ethyl acetate) as the blank, adjust the instrument sensitivity, inject samples three times continuously, record the instrument noise level within the peak time range of the analyte, and calculate the average noise.

[0085] Detection limit: Accurately prepare the reference solution of the test object, gradually dilute it to a certain concentration and inject it, and measure it three times in a row. Calculate the ratio of its peak height to noise (signal-to-noise ratio). The sample concentration with a signal-to-noise ratio (S / N) above 3 is the detection limit concentration, and its ratio to the theoretical sample concentration is the detection limit.

[0086] Limit of quantitation: Accurately prepare the reference solution of the substance to be measured, gradually dilute it to a certain concentration and inject it, and measure it 6 times in a row. Calculate the ratio of its peak height to noise (signal-to-noise ratio). The sample concentration with a signal-to-noise ratio (S / N) above 10 is the limit of quantitation concentration, and its ratio to the theoretical sample concentration is the limit of quantitation.

[0087] Preparation of quantitative limit solution: accurately weigh about 23 mg of (S)-4-chloro-3-hydroxybutyric acid ethyl ester, place in a 20 mL volumetric flask, add 5 mL of ethyl acetate to dissolve, add 5 mL of acetic anhydride and 0.5 mL of pyridine, shake well with ultrasound, place in a 100 ° C oil bath for 30 minutes, cool, add 5 mL of saturated sodium chloride aqueous solution, dilute to scale with ethyl acetate, shake for 2 minutes, let stand to separate, take the ethyl acetate layer (upper layer) as the stock solution. Accurately measure 2 mL of the stock solution, place in a 20 mL volumetric flask, dilute to scale with ethyl acetate, and shake well; accurately measure 1 mL, place in a 50 mL volumetric flask, dilute to scale with ethyl acetate, and shake well.

[0088] Preparation of detection limit solution: Accurately measure 3 mL of quantitation limit solution, place in a 10 mL volumetric flask, dilute to the mark with ethyl acetate, and shake well.

[0089] Accurately measure the solvent (ethyl acetate) and inject it continuously for 3 times according to the chromatographic conditions of Example 1, inject the detection limit solution continuously for 3 times, and inject the quantitative limit solution continuously for 6 times. Record the chromatogram and calculate the signal-to-noise ratio. The results are shown in Tables 15, 16, and 17.

[0090] Table 15 Baseline noise detection results

[0091] Table 16 Detection limit determination results

[0092] Table 17 Quantitation limit determination results

[0093] Conclusion: Under this method, the sensitivity of (S)-4-chloro-3-hydroxybutyric acid ethyl ester derivatives meets the requirements.

[0094] (3) Linearity and range The linear relationship between the test results and the concentration of the test solution was investigated within the concentration range of the quantitative limit to 120% for (R)-4-chloro-3-acetoxybutyric acid ethyl ester and within the concentration range of the quantitative limit to 200% for (S)-4-chloro-3-acetoxybutyric acid ethyl ester solution. The test results are shown in Tables 18 and 19.

[0095] Table 18 Linear determination results of (R)-4-chloro-3-acetoxybutyric acid ethyl ester

[0096] Table 19 (S)-4-chloro-3-acetoxybutyric acid ethyl ester linear determination results

[0097] Conclusion: The linear correlation coefficients of (R)-4-chloro-3-acetoxybutyric acid ethyl ester and (S)-4-chloro-3-acetoxybutyric acid ethyl ester are both greater than 0.99, indicating a good linear relationship.

[0098] (4) Repeatability Determine the closeness of the results obtained by the same analyst under the same measurement conditions and using the same homogeneous sample after multiple sampling.

[0099] Preparation of test solution: Accurately weigh about 150 mg of (R)-4-chloro-3-hydroxybutyric acid ethyl ester, place in a 20 mL volumetric flask, add 5 mL of ethyl acetate to dissolve, add 5 mL of acetic anhydride and 0.5 mL of pyridine, shake well with ultrasound, place in a 100°C oil bath for 30 min, cool, add 5 mL of saturated sodium chloride aqueous solution, dilute to scale with ethyl acetate, shake for 2 min, let stand to separate, and take the ethyl acetate layer (upper layer). Prepare 6 copies in parallel.

[0100] Accurately measure 6 portions of the test solution and inject them into the gas chromatograph respectively. Record the chromatogram and calculate the content of the enantiomer by the area normalization method. The results are shown in Table 20.

[0101] Table 20 Repeatability test results

[0102] Conclusion: The RSD of the enantiomer content in the 6 test solutions was 1.0%, which was less than 10%, indicating that the method was accurate and reliable.

Claims

1. A method for determining enantiomers in a L-carnitine intermediate by gas chromatography, characterized in that: The following steps are involved: (1) Setting chromatographic conditions: using a capillary column with cyclodextrin-bonded dimethylpolysiloxane as the stationary liquid as the chromatographic column; (2) Preparation of test solution: Dissolve and dilute the test sample (R)-4-chloro-3-hydroxybutyric acid ethyl ester with ethyl acetate, and then derivatize it by adding acetic anhydride and pyridine to prepare a test solution; (3) Determination: Inject the test sample solution into a gas chromatograph, record the chromatogram, and calculate the content of the enantiomer (S)-4-chloro-3-hydroxybutyrate ethyl ester in the L-carnitine intermediate (R)-4-chloro-3-hydroxybutyrate ethyl ester by the area normalization method.

2. The method for determining enantiomers in a L-carnitine intermediate by gas chromatography according to claim 1, characterized in that: The chromatographic conditions include: the detector is an FID detector, the FID detector temperature is 270-290°C; the temperature program is: the initial column temperature is 110-130°C, maintained for 15-20 minutes, heated to 160-180°C at a rate of 5-15°C / min, and maintained for 10-15 minutes; the injection port temperature is 260-280°C; the column pressure is 90-110 kPa; and the injection volume is 0.5-2 μL.

3. The method for determining enantiomers in a L-carnitine intermediate by gas chromatography according to claim 1, characterized in that: The chromatographic column is CP-ChirasilDex CB with specifications of 25m×0.25mm and 0.25μm.

4. The method for determining enantiomers in a L-carnitine intermediate by gas chromatography according to claim 2, characterized in that: The chromatographic conditions included an injection port temperature of 270°C, a FID detector temperature of 280°C, a column pressure of 100 kPa, and an injection volume of 1 μL.

5. The method for determining enantiomers in a L-carnitine intermediate by gas chromatography according to claim 4, characterized in that: The temperature program in the chromatographic conditions was as follows: the initial column temperature was 120°C, maintained for 17 min, increased to 170°C at a rate of 10°C / min, and maintained for 12 min.

6. The method for determining enantiomers in a L-carnitine intermediate by gas chromatography according to claim 1, characterized in that: The specific steps for preparing the test solution are as follows: take the test sample (R)-4-chloro-3-hydroxybutyric acid ethyl ester and use ethyl acetate to make a solution with a concentration of 10~50mg / mL, accurately measure 4~6mL and put it into a 20mL volumetric flask, add 4~6mL of acetic anhydride and 0.4~0.6mL of pyridine, shake it ultrasonically and place it in a 95~105℃ oil bath for 25~35min, cool it, add 4~6mL of saturated sodium chloride aqueous solution, dilute it to the scale with ethyl acetate, shake it for at least 2min, let it stand to separate, and take the upper layer as the test solution.

7. The method for determining enantiomers in a L-carnitine intermediate by gas chromatography according to claim 1, characterized in that: The L-carnitine intermediate (R)-4-chloro-3-hydroxybutyric acid ethyl ester derivative [(R)-4-chloro-3-acetoxybutyric acid ethyl ester] and the enantiomer (S)-4-chloro-3-hydroxybutyric acid ethyl ester derivative [(S)-4-chloro-3-acetoxybutyric acid ethyl ester] of step (3) are eluted in sequence, and the resolution between the two is greater than 1.

5.

8. The method for determining enantiomers in a L-carnitine intermediate by gas chromatography according to claim 1, characterized in that: The calculation formula of the area normalization method is: enantiomer (S)-4-chloro-3-hydroxybutyric acid ethyl ester content (%) = ((S)-4-chloro-3-acetoxybutyric acid ethyl ester peak area / (R)-4-chloro-3-acetoxybutyric acid ethyl ester and (S)-4-chloro-3-acetoxybutyric acid ethyl ester peak area sum) × 100%.

9. The method for determining enantiomers in a L-carnitine intermediate by gas chromatography according to claim 8, characterized in that: The method is applicable to the determination of the content of the enantiomer [(S)-4-chloro-3-hydroxybutyric acid ethyl ester] in the L-carnitine raw material intermediate [(R)-4-chloro-3-hydroxybutyric acid ethyl ester], and the quality control standard is that the enantiomer content is ≤1.5%.

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