Method for detecting L (-)-malic acid in D (+)-malic acid by reversed-phase liquid chromatography

Abstract

The invention belongs to the technical field of medicine raw material detection, and particularly discloses a method for detecting L (-)-malic acid in D (+)-malic acid through reversed-phase liquid chromatography, and the method comprises the following steps: precisely weighing a D (+)-malic acid sample to be detected, placing in a measuring flask, adding water for dissolving, diluting to a scale, and uniformly shaking to obtain a test solution; precisely weighing a proper amount of an L (-)-malic acid reference substance, precisely weighing, adding water for dissolving, and quantitatively diluting to prepare a reference substance stock solution; precisely weighing a D (+)-malic acid sample to be detected, putting the sample into a measuring flask, precisely adding 1ml of the reference substance stock solution, adding water for dissolving and diluting to a scale, and uniformly shaking; precisely measuring the test solution and the reference solution, respectively injecting into a liquid chromatograph, and recording a chromatogram to three times of the retention time of a main peak; according to the method provided by the invention, maleic acid, fumaric acid, L (-)-malic acid and D (+)-malic acid can be effectively separated, and the content of L (-)-malic acid in D (+)-malic acid can be simply, accurately and quickly detected.

Description

Technical Field

[0001] The invention belongs to the technical field of drug raw material detection, relates to the detection of an initial raw material, malic acid, and particularly designs a method for detecting L(-)-malic acid in D(+)-malic acid by using a reversed phase liquid chromatography method. Background Art

[0002] D(+)-malic acid is an important chiral compound, which is widely used in the fields of chiral drug synthesis, chiral separation reagents and chiral auxiliary agents, and has important development and application prospects.

[0003] The industrial production of D(+)-malic acid is mainly produced by enzyme engineering. Due to the limitations of process control, fumaric acid, maleic acid and L(-)-malic acid and other process impurities will be produced during the preparation of D(+)-malic acid. Among them, L(-)-malic acid is an optical isomer impurity of D(+)-malic acid. The structures of D(+)-malic acid and each impurity are as follows:

[0004]

[0005] The L(-)-malic acid contained in D(+)-malic acid will lead to a high level of optical isomer impurities in the final synthetic product, which means that the content of L(-)-malic acid must be strictly controlled. Therefore, the study on the determination method of L(-)-malic acid in D(+)-malic acid is of great significance.

[0006] Duan Xianzhe et al., Determination of the enantiomer D-malic acid in L-malic acid by normal phase high performance liquid chromatography, Analytical Instruments, Issue 2, 2016, Pages 25-29: The enantiomer D-malic acid in L-malic acid was determined by a normal phase liquid chromatography system. Fumaric acid and maleic acid were not added to the system suitability solution. According to this method, an attempt was made to detect L(-)-malic acid in D(+)-malic acid. Maleic acid and L(-)-malic acid could not be effectively separated. The maleic acid in the sample interfered with the determination of L(-)-malic acid. By optimizing the chromatographic conditions such as the mobile phase ratio, effective separation was still not possible.

[0007] Chinese patent application CN 112881579 A discloses a normal phase liquid chromatography method for detecting D-malic acid in L-malic acid. According to this method, attempts were made to detect L(-)-malic acid in D(+)-malic acid. The peaks of fumaric acid, maleic acid, L(-)-malic acid and D(+)-malic acid can be effectively separated. Further research found that the linear relationship between the concentration and peak area of ​​L(-)-malic acid under this method is poor, and the repeatability of the L(-)-malic acid detection results is poor, and the content of L(-)-malic acid cannot be accurately detected.

[0008] Both of the above methods use normal phase liquid chromatography: the method uses n-hexane and isopropanol as mobile phases. Since n-hexane is highly volatile, the laboratory is filled with the smell of n-hexane, which is very unpleasant; the chromatographic peak retention time of normal phase chromatography is greatly affected by temperature, especially in summer, which often causes a significant drift in the retention time. Summary of the invention

[0009] In view of the deficiencies in the prior art, the object of the present invention is to provide a method for detecting L(-)-malic acid in D(+)-malic acid by reverse phase liquid chromatography. The method of the present invention controls the content of L(-)-malic acid in D(+)-malic acid, thereby ensuring that D(+)-malic acid is used as a chiral compound in the synthesis to ensure the controllability of the optical isomer impurities of the final product.

[0010] The present invention is achieved through the following technical solutions:

[0011] A method for detecting L(-)-malic acid in D(+)-malic acid by reverse phase liquid chromatography comprises the following steps:

[0012] (1) Prepare the test solution: accurately weigh the D(+)-malic acid sample to be tested, place it in a volumetric bottle, add water to dissolve and dilute to the scale, and shake well to obtain the test solution;

[0013] (2) Preparation of reference substance stock solution: Accurately weigh an appropriate amount of L(-)-malic acid reference substance, accurately weigh it, dissolve it in water and quantitatively dilute it to prepare the reference substance stock solution;

[0014] (3) Preparation of reference solution: Accurately weigh the D(+)-malic acid sample to be tested and place it in a volumetric flask. Accurately add 1 ml of the reference stock solution, dissolve it in water and dilute to the mark, then shake well.

[0015] (4) Determination: Accurately measure the test solution and the reference solution, inject them into the liquid chromatograph respectively, and record the chromatogram to 3 times the retention time of the main peak;

[0016] The chromatographic conditions were as follows: octadecylsilane bonded silica gel was used as the filler (250 mm × 4.6 mm, 5 μm);

[0017] A mixed solution of anhydrous copper acetate and L-leucine was used as the mobile phase;

[0018] The flow rate is 1.0 ml per minute;

[0019] The detection wavelength is 302nm;

[0020] The column temperature was 30°C;

[0021] Injection volume: 50 μl.

[0022] A further improvement of the present invention is:

[0023] The concentration of the test solution is 2 mg / ml.

[0024] Furthermore, the concentration of the reference substance stock solution is 100 μg / ml.

[0025] Furthermore, the concentration of the sample to be tested in the reference solution is 2 mg / ml.

[0026] Furthermore, the concentration of anhydrous copper acetate in the mobile phase is 1.5 mg / ml, and the concentration of L-leucine is 2 mg / ml.

[0027] The beneficial effects of the present invention are:

[0028] The method provided by the present invention can effectively separate maleic acid, fumaric acid, L(-)-malic acid and D(+)-malic acid, and can simply, accurately and quickly detect the content of L(-)-malic acid in D(+)-malic acid. The method of the present invention is based on reversed-phase liquid chromatography, is mature and universal, uses an aqueous mobile phase, avoids the use of any organic solvent, and does not have the defects of normal-phase liquid chromatography in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the chromatogram of L(-)-malic acid reference solution;

[0030] Figure 2 is the chromatogram of the test solution;

[0031] Figure 3 is the chromatogram of blank solvent water;

[0032] Figure 4 Chromatogram of the spiked solution of the test product;

[0033] Among them, the peak order is: fumaric acid, maleic acid, L(-)-malic acid, D(+)-malic acid;

[0034] Figure 5 is the standard curve of L(-)-malic acid. DETAILED DESCRIPTION

[0035] The present invention is described in detail below in conjunction with specific embodiments.

[0036] Instrument and chromatographic column information used in this invention:

[0037]

[0038] Reagent information used in the examples of the present invention:

[0039] Reagent name factory batch number Level / content Anhydrous acetic acid ketone Maclean C16314826 AR L-Leucine Maclean C16573504 99％

[0040] Reference substance information used in the examples of the present invention:

[0041]

[0042]

[0043] Example 1: Detection of L(-)-malic acid in D(+)-malic acid by reversed phase liquid chromatography

[0044] (1) Solution preparation

[0045] Test solution: Accurately weigh about 20 mg of D(+)-malic acid sample to be tested, place it in a 10 ml volumetric flask, add water to dissolve and dilute to the scale, and shake well to obtain the test solution.

[0046] Reference substance stock solution: Take an appropriate amount of L(-)-malic acid reference substance, weigh accurately, dissolve in water and quantitatively dilute to make a solution containing approximately 100 μg per 1 ml.

[0047] Reference substance solution: Accurately weigh 20 mg of D(+)-malic acid sample to be tested, accurately weigh, place in a 10 ml volumetric flask, accurately add 1 ml of reference substance stock solution, dissolve with water and dilute to the scale, and shake well.

[0048] Mobile phase: Weigh 1.5 g of anhydrous copper acetate and 2 g of L-leucine, dissolve in water and dilute to 1000 ml.

[0049] (2) Chromatographic conditions

[0050] Octadecylsilane bonded silica gel was used as filler (250 mm × 4.6 mm, 5 μm);

[0051] A mixed solution of anhydrous copper acetate and L-leucine was used as the mobile phase;

[0052] The flow rate is 1.0 ml per minute;

[0053] The detection wavelength is 302nm;

[0054] The column temperature was 30°C;

[0055] Injection volume: 50 μl.

[0056] (3) Determination: Accurately measure the test solution and the reference solution, inject them into the liquid chromatograph respectively, and record the chromatogram until 3 times the retention time of the main peak. The chromatogram of the reference solution is as follows Figure 1 ; Chromatogram of the test solution is as follows Figure 2 .

[0057] Example 2: Specificity verification of the detection method of the present invention

[0058] (1) Blank solvent: water.

[0059] (2) Fumaric acid reference solution: Take an appropriate amount of fumaric acid reference substance, weigh it accurately, dissolve it in water and quantitatively dilute it to make a solution containing about 200 μg per 1 ml.

[0060] (3) Maleic acid reference solution: Take an appropriate amount of maleic acid reference substance, weigh it accurately, dissolve it in water and quantitatively dilute it to make a solution containing about 100 μg per 1 ml.

[0061] (4) D(+)-malic acid localizing solution: Take an appropriate amount of D(+)-malic acid reference substance, weigh it accurately, dissolve it in water and quantitatively dilute it to make a solution containing about 1 mg per 1 ml.

[0062] (5) L(-)-malic acid localizing solution (reference substance stock solution): Take an appropriate amount of L(-)-malic acid reference substance, accurately weigh it, dissolve it in water and quantitatively dilute it to make a solution containing approximately 100 μg per 1 ml.

[0063] (6) Test solution: Take about 20 mg of the product, weigh accurately, place in a 10 ml volumetric flask, add water to dissolve and dilute to the scale, and shake well.

[0064] (7) Reference substance solution: Take about 20 mg of the product, weigh it accurately, place it in a 10 ml volumetric flask, accurately add 1 ml of the reference substance stock solution, dissolve it in water and dilute it to the mark, and shake well.

[0065] (8) Test sample spike solution: Take about 20 mg of the product, accurately weigh it, place it in a 10 ml volumetric flask, accurately add 1 ml of L(-)-malic acid reference stock solution, 1 ml of fumaric acid fixative solution and 0.1 ml of maleic acid fixative solution, dissolve it in water and dilute it to the scale, and shake well.

[0066] Accurately measure each of the above solutions, inject them into the liquid chromatograph, record the chromatogram, and examine whether the blank solvent interferes with the determination of D(+)-malic acid optical isomers; whether the fumaric acid, maleic acid and L(-)-malic acid peaks can be effectively separated, and whether the L(-)-malic acid peak and the D(+)-malic acid peak can achieve baseline separation. The chromatogram of the blank solvent water is shown in Figure 1. Figure 3 The chromatogram of the spiked solution of the test sample is as follows: Figure 4 .

[0067] Table 1, specificity determination results

[0068]

[0069] The results showed that the blank solvent had no interference with the determination of D(+)-malic acid optical isomers; the fumaric acid, maleic acid and L(-)-malic acid peaks could be effectively separated, the L(-)-malic acid peak and the D(+)-malic acid peak could achieve baseline separation, and the analytical method for the determination of D(+)-malic acid optical isomers had good specificity.

[0070] Example 3: Precision verification of the detection method of the present invention

[0071] Reference substance stock solution: Take an appropriate amount of L(-)-malic acid reference substance, weigh accurately, dissolve in water and quantitatively dilute to make a solution containing approximately 100 μg per 1 ml.

[0072] Reference substance solution: Take about 20 mg of this product, weigh accurately, place in a 10 ml volumetric flask, accurately add 1 ml of reference substance stock solution, dissolve with water and dilute to the scale, shake well.

[0073] The reference solution was accurately measured and injected six times according to the determination and analysis method of D(+)-malic acid optical isomers. The RSD of the retention time and peak area of ​​L(-)-malic acid in the reference solution was investigated. The results are shown in Tables 2 and 3.

[0074] Table 2, Instrument precision measurement results (JQ1336)

[0075] — Retention time (min) Peak area 1 7.637 117240 2 7.641 119619 3 7.637 118784 4 7.637 119924 5 7.629 121995 6 7.628 122644 average value 7.635 120034 RSD(%) 0.07 1.67

[0076] Table 3, Instrument precision measurement results (JQ1312)

[0077] — Retention time (min) Peak area 1 9.329 82248 2 9.468 82090 3 9.547 77098 4 9.593 83672 5 9.630 84276 6 9.663 80548 average value 9.538 81655 RSD(%) 1.29 3.17

[0078] The results showed that the instrumental precision of the analytical method for determination of optical isomers of D(+)-malic acid was good.

[0079] Example 4: Repeatability verification of the detection method of the present invention

[0080] Reference substance stock solution: Take an appropriate amount of L(-)-malic acid reference substance, weigh accurately, dissolve in water and quantitatively dilute to make a solution containing approximately 100 μg per 1 ml.

[0081] Reference substance solution: Take about 20 mg of this product, weigh accurately, place in a 10 ml volumetric flask, accurately add 1 ml of reference substance stock solution, dissolve with water and dilute to the scale, shake well.

[0082] Test solution: Take about 20 mg of the product, weigh accurately, place in a 10 ml volumetric flask, add water to dissolve and dilute to the scale, shake well (prepare 6 portions in parallel).

[0083] According to the analytical method for determination of optical isomers of D(+)-malic acid, 6 test solutions were determined. The RSD of the content of L(-)-malic acid in the 6 test solutions was investigated, and the results are shown in Table 4.

[0084] Table 4, Repeatability test results

[0085] Test solution L(-)-Malic acid content (%) 1 0.11 2 0.11 3 0.11 4 0.11 5 0.12 6 0.12 average value(%) 0.11 RSD(%) 5.23

[0086] The results showed that the analytical method for determination of optical isomers of D(+)-malic acid had good repeatability.

[0087] Example 5: Verification of the detection limit and quantification limit of the detection method of the present invention

[0088] The detection limit and quantification limit were determined by the signal-to-noise ratio method. The corresponding concentration when the signal-to-noise ratio was about 3:1 was taken as the detection limit, and the corresponding concentration when the signal-to-noise ratio was about 10:1 was taken as the quantification limit.

[0089] (1) Detection limit

[0090] Accurately measure 5 ml of the quantitative limit solution, place it in a 15 ml volumetric flask, dilute it to the mark with water, shake it well, and use it as the detection limit solution. The detection limit concentration is 0.3655 μg / ml, which is 0.02% of the test solution concentration.

[0091] (2) Limit of quantification

[0092] Reference substance stock solution: Take an appropriate amount of L(-)-malic acid reference substance, weigh accurately, dissolve in water and quantitatively dilute to make a solution containing approximately 100 μg per 1 ml.

[0093] Accurately measure 1 ml of the reference stock solution, place it in a 100 ml volumetric flask, dilute it to the mark with water, shake well, and use it as the quantitative limit solution. The quantitative limit concentration is 1.0965 μg / ml, which is 0.05% of the test solution concentration.

[0094] The quantitative limit solution was taken and injected 6 times to examine the RSD of the L(-)-malic acid peak area. The results are shown in Table 5.

[0095] Table 5, Quantitation Limit Solution Determination Results

[0096]

[0097]

[0098] The results showed that the quantification limit solution of L(-)-malic acid had good precision.

[0099] Example 6: Linearity and range of the detection method of the present invention

[0100] Test solution: Take about 20 mg of the product, weigh accurately, place in a 10 ml volumetric flask, add water to dissolve and dilute to the scale, and shake well.

[0101] Reference substance stock solution: Take an appropriate amount of L(-)-malic acid reference substance, weigh accurately, dissolve in water and quantitatively dilute to make a solution containing approximately 100 μg per 1 ml.

[0102] According to Table 6, prepare linear 1 to linear 6 solutions of L(-)-malic acid.

[0103] Table 6, Linear solution preparation table

[0104] — Take this product (mg) Take reference stock solution (ml) Dissolve in water and dilute to (ml) 1 20 0.2 10 2 20 0.5 10 3 20 0.8 10 4 20 1.0 10 5 20 1.5 10 6 20 2.0 10

[0105] Take the quantitative limit solution and linear 1 to linear 6 solutions and inject them into the liquid chromatograph respectively. Use the concentration as the horizontal axis and the converted L(-)-malic acid peak area as the vertical axis to draw the standard curve and calculate the linear regression equation. The results are shown in Table 7. The standard curve of L(-)-malic acid is as follows Figure 5 .

[0106] Table 7, Linearity determination results

[0107]

[0108]

[0109] The results showed that L(-)-malic acid had a good linear relationship in the concentration range of 1.0965μg / ml to 21.9295μg / ml, and the linear regression equation was: y=9868.4179x-1690.1636, R 2 =0.9991.

[0110] Example 7: Verification of the accuracy of the detection method of the present invention

[0111] Reference substance stock solution: Take an appropriate amount of L(-)-malic acid reference substance, weigh accurately, dissolve in water and quantitatively dilute to make a solution containing approximately 100 μg per 1 ml.

[0112] Reference substance solution: Take about 20 mg of this product, weigh accurately, place in a 10 ml volumetric flask, accurately add 1 ml of reference substance stock solution, dissolve with water and dilute to the scale, shake well.

[0113] Test solution: Take about 20 mg of the product, weigh accurately, place in a 10 ml volumetric flask, add water to dissolve and dilute to the scale, and shake well.

[0114] 20% test sample spiked solution: Take about 20 mg of the product, accurately weigh it, put it in a 10 ml volumetric flask, accurately add 0.2 ml of the reference sample stock solution, dissolve it in water and dilute it to the scale, shake well (prepare 3 copies in parallel).

[0115] 80% test sample spiked solution: Take about 20 mg of the product, accurately weigh it, put it in a 10 ml volumetric flask, accurately add 0.8 ml of the reference sample stock solution, dissolve it in water and dilute it to the scale, shake well (prepare 3 copies in parallel).

[0116] 100% test sample spiked solution: Take about 20 mg of the product, accurately weigh it, put it in a 10 ml volumetric flask, accurately add 1 ml of the reference sample stock solution, add water to dissolve and dilute to the scale, shake well (prepare 3 copies in parallel).

[0117] 120% test sample spiked solution: Take about 20 mg of the product, accurately weigh it, put it in a 10 ml volumetric flask, accurately add 1.2 ml of the reference sample stock solution, dissolve it in water and dilute it to the scale, shake well (prepare 3 copies in parallel).

[0118] Accurately measure each of the above solutions and inject them into the liquid chromatograph respectively. Record the chromatogram and calculate the recovery rate and RSD of L(-)-malic acid in the spiked solution of the test sample. The results are shown in Table 8.

[0119] Table 8, Accuracy test results

[0120]

[0121]

[0122] The results showed that the analytical method for determination of optical isomers of D(+)-malic acid had good accuracy.

[0123] Example 8: Durability verification of the detection method of the present invention

[0124] The preparation method of reference solution and test solution is the same as that under repeatability.

[0125] According to the D(+)-malic acid optical isomer analysis method, the RSD of the L(-)-malic acid content in the test solution was determined under different wavelengths (302nm±2nm), different column temperatures (30±2℃) and different chromatographic column conditions. The results are shown in Table 9.

[0126] Table 9, Durability Test Results

[0127]

[0128] The results showed that the analytical method for determination of optical isomers of D(+)-malic acid had good durability.

[0129] Example 9: Verification of solution stability of the detection method of the present invention

[0130] The preparation method of reference solution and test solution is the same as that under repeatability.

[0131] According to the D(+)-malic acid optical isomer analysis method, the RSD of the L(-)-malic acid peak area in the reference solution and the test solution was examined within a certain period of time. The results are shown in Tables 10 and 11.

[0132] Table 10, stability test results of reference solution

[0133]

[0134]

[0135] Table 11, Test solution stability test results

[0136] Time (h) L(-)-Malic acid peak area 0 20802 10 21670 19 20872 43 20715 48 21808 average value 21173 RSD(%) 2.46

[0137] The results showed that the reference solution and the test solution had good stability within 48 hours.

[0138] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent transformation or modification made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting L(-)-malic acid in D(+)-malic acid by reverse phase liquid chromatography, characterized in that: The following steps are involved: (1) Prepare the test solution: accurately weigh the D(+)-malic acid sample to be tested, place it in a volumetric bottle, add water to dissolve and dilute to the scale, and shake well to obtain the test solution; (2) Preparation of reference substance stock solution: Accurately weigh an appropriate amount of L(-)-malic acid reference substance, accurately weigh it, dissolve it in water and quantitatively dilute it to prepare the reference substance stock solution; (3) Preparation of reference solution: Accurately weigh the D(+)-malic acid sample to be tested and place it in a volumetric flask. Accurately add 1 ml of the reference stock solution, dissolve it in water and dilute to the mark, then shake well. (4) Determination: Accurately measure the test solution and the reference solution, inject them into the liquid chromatograph respectively, and record the chromatogram to 3 times the retention time of the main peak; The chromatographic conditions were as follows: octadecylsilane bonded silica gel was used as the filler (250 mm × 4.6 mm, 5 μm); A mixed solution of anhydrous copper acetate and L-leucine was used as the mobile phase; The flow rate is 1.0 ml per minute; The detection wavelength is 302nm; The column temperature was 30°C; Injection volume: 50 μl.

2. The method for detecting L(-)-malic acid in D(+)-malic acid by reverse phase liquid chromatography according to claim 1, characterized in that: The concentration of the test solution is 2 mg / ml.

3. The method for detecting L(-)-malic acid in D(+)-malic acid by reverse phase liquid chromatography according to claim 1, characterized in that: The concentration of the reference substance stock solution is 100 μg / ml.

4. The method for detecting L(-)-malic acid in D(+)-malic acid by reverse phase liquid chromatography according to claim 1, characterized in that: The concentration of the sample to be tested in the reference solution is 2 mg / ml.

5. The method for detecting L(-)-malic acid in D(+)-malic acid by reverse phase liquid chromatography according to claim 1, characterized in that: The concentration of anhydrous copper acetate in the mobile phase is 1.5 mg / ml, and the concentration of L-leucine is 2 mg / ml.

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