Method for detecting methotrexate intermediates and their enantiomers by high performance liquid chromatography

Abstract

The present invention provides a detection method for determining R-type isomers or R-type isomer salts contained in (S)-2-(4-(methylamino)benzamide) glutaric acid or its salt by high performance liquid chromatography, wherein the chromatographic condition is a chromatographic column with amylose-tris(3,5-dimethylphenylcarbamate) as a filler, and the mobile phase is a mixed solvent of a mobile phase A normal hexane and a mobile phase B anhydrous ethanol-acetonitrile-trifluoroacetic acid system, wherein the mobile phase A and B are isocratically eluted in a volume ratio of (78:22) to (85:15). The detection method provided by the present invention can quickly and accurately detect the content of enantiomeric impurities of methotrexate intermediate (S)-2-(4-(methylamino)benzamide) glutaric acid, thereby being able to effectively control the R-type isomer impurities to be further converted into methotrexate isomer impurities F in the intermediate process, and plays a guiding role in the synthesis process of methotrexate, which is of great significance.

Description

Technical Field

[0001] The present invention relates to the field of analysis and detection, and relates to a method for detecting methotrexate intermediates and enantiomers thereof by high performance liquid chromatography, and specifically relates to a method for detecting R-isomers or R-isomer salts contained in (S)-2-(4-(methylamino)benzamido)glutaric acid or its salts. Background Art

[0002] Methotrexate (MTX) is a folate reductase inhibitor and an anti-folate anti-tumor drug. It mainly inhibits the synthesis of tumor cell DNA by inhibiting dihydrofolate reductase, thereby inhibiting the growth and reproduction of tumor cells. It selectively acts on the S phase and is a cell cycle-specific drug. Clinically, it has good therapeutic effects in acute leukemia (especially acute lymphocytic leukemia), choriocarcinoma and malignant hydatidiform mole. It has certain therapeutic effects on head and neck tumors, breast cancer, lung cancer and pelvic tumors. It can also be combined with other drugs to treat Burkitts lymphoma, advanced lymphosarcoma (stages III and IV, Peter's stage system) and advanced mycosis fungoides. The structural formula of methotrexate is:

[0003]

[0004] In the methotrexate synthesis method disclosed in the prior art, (S)-2-(4-(methylamino)benzamido) glutaric acid disodium (abbreviated as M3) is usually used as a key chiral intermediate in the synthesis process of methotrexate, which is synthesized from diethyl p-methylaminobenzoylglutamate, with a molecular formula of C 13 H 14 N2Na2O5, molecular weight 324.24, white to light yellow powder. Its structural formula is:

[0005]

[0006] The enantiomer of the intermediate is (R)-2-(4-(methylamino)benzamido) glutaric acid disodium (abbreviated as M3-A), with the molecular formula C 13 H 14 N2Na2O5, molecular weight 324.24, white crystalline particles. Its structural formula is:

[0007]

[0008] In the subsequent process steps of methotrexate, the R-isomer impurity will be further converted into the methotrexate isomer impurity (referred to as impurity F), whose structural formula is:

[0009]

[0010] Since the purification process of the final product methotrexate has no obvious removal effect on the methotrexate isomer impurity F, it is necessary to detect and control the R isomer impurity of the intermediate during the preparation process of the intermediate and control its content at a very low level to ensure that the corresponding impurity content in the final product meets the quality requirements.

[0011] After searching, there are currently no literature reports on chromatographic monitoring methods for enantiomeric impurities in the methotrexate intermediate (S)-2-(4-(methylamino)benzamido)glutaric acid. Therefore, it is necessary to develop a method that can accurately monitor the content of the isomeric impurity (R)-2-(4-(methylamino)benzamido)glutaric acid in the intermediate process to solve the above problems. Summary of the invention

[0012] The object of the present invention is to provide a method for quickly and accurately detecting the content of the enantiomeric impurity (R)-2-(4-(methylamino)benzamido)glutaric acid or its salt in the methotrexate intermediate (S)-2-(4-(methylamino)benzamido)glutaric acid, thereby effectively controlling the R-type isomer impurity to be further converted into the methotrexate isomer impurity F during the intermediate process.

[0013] To achieve the above object, the present invention provides a method for detecting R-isomer or R-isomer salt contained in (S)-2-(4-(methylamino)benzamido)pentanedioic acid or its salt by high performance liquid chromatography, wherein the high performance liquid chromatography uses a chromatographic column with amylose-tris(3,5-dimethylphenylcarbamate) as a filler, and the mobile phase is a mixed solvent of a mobile phase A of n-hexane and a mobile phase B of an anhydrous ethanol-acetonitrile-trifluoroacetic acid system, wherein the mobile phase A and the mobile phase B are isocratically eluted in a volume ratio of (78:22) to (85:15);

[0014] Further, the chromatographic column of the present invention is DALCELCHIRALPAK AD-H, specification is 250mm×4.6mm, 5μm or equivalent performance column;

[0015] Furthermore, the volume ratio of anhydrous ethanol to acetonitrile in the mobile phase B of the present invention is (85:15) to (95:5), and the volume fraction of trifluoroacetic acid is 0.05% to 1%;

[0016] Preferably, the volume ratio of anhydrous ethanol to acetonitrile in the mobile phase B is 90:10;

[0017] Preferably, the volume fraction of trifluoroacetic acid is 0.1% to 0.5%, more preferably 0.3%;

[0018] Further, the volume ratio of mobile phase A to mobile phase B is (78:22) to (80:20), preferably 80:20;

[0019] Furthermore, the flow rate of the mobile phase of the present invention is 0.3 to 1.2 mL / min, preferably 0.7 to 0.8 mL / min, and more preferably 0.7 mL / min;

[0020] Further, the column temperature of the present invention is 20-35°C, preferably 20-25°C, more preferably 25°C;

[0021] Furthermore, the injection volume of the present invention is 5 to 20 μl, preferably 5 μl;

[0022] Further, the detection wavelength of the present invention is 295±5nm, preferably 295nm;

[0023] Further, the salt of (S)-2-(4-(methylamino)benzamido)pentanedioic acid and / or the salt of the R isomer described in the present invention is a disodium salt;

[0024] Furthermore, the detection described in the present invention is qualitative and / or quantitative detection;

[0025] Furthermore, the quantitative detection described in the present invention is to calculate the content of the R isomer contained in the test solution of (S)-2-(4-(methylamino)benzamido)glutaric acid or its salt by the area normalization method;

[0026] According to some specific embodiments of the present invention, the detection method comprises the following steps:

[0027] (1) Preparation of test solution: Take an appropriate amount of methotrexate intermediate M3 test sample, dissolve it in methanol and dilute it to make a solution containing about 1.0 mg M3 per 1 ml;

[0028] (2) Preparation of system suitability solution: Dissolve the test sample M3 and the enantiomeric impurity M3-A reference substance in methanol and dilute them to make a mixed solution containing approximately 1.0 mg of M3 and 30 μg of M3-A enantiomer per 1 ml.

[0029] (3) Setting the chromatographic conditions: Using amylose-tris (3,5-dimethylphenylcarbamate) as the filler (DALCELCHIRALPAK AD-H, specification: 250mm×4.6mm, 5μm or chromatographic column with equivalent performance); isocratic elution was performed with a mixed solution of n-hexane and anhydrous ethanol-acetonitrile-trifluoroacetic acid (90:10:0.3) in a volume ratio of 80:20 as the mobile phase; detection wavelength was 295nm; column temperature was 25℃; flow rate was 0.7ml / min;

[0030] (4) Determination: Take 5 μL of the blank solution methanol, the solution of step (1) and (2), respectively, and inject them into the high performance liquid chromatograph to record the chromatogram; in the system suitability solution chromatogram, the peak order is the intermediate M3 and its enantiomer M3-A peak, among which the separation degree between the M3-A peak and the adjacent peak should be greater than 1.5, and the enantiomer content of the intermediate M3 in the test solution is calculated by the area normalization method.

[0031] The present invention has the following beneficial effects:

[0032] The present invention provides a method for detecting enantiomeric impurities of a methotrexate intermediate (S)-2-(4-(methylamino)benzamide)pentanedioic acid, which can achieve qualitative and quantitative detection of the intermediate isomeric impurities, and has the advantages of high accuracy, high sensitivity, high precision, and good specificity. The detection method provided by the present invention can quickly and accurately detect the content of the R-type enantiomer in the methotrexate intermediate (S)-2-(4-(methylamino)benzamide)pentanedioic acid, thereby effectively controlling the R-type isomeric impurities to be further converted into methotrexate isomeric impurities F in the intermediate process, and plays a guiding role in the synthesis process of methotrexate, which is of great significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Screening case 1 reversed phase HPLC chromatogram;

[0034] Figure 2 Screening case 2 reversed phase HPLC chromatogram;

[0035] Figure 3 Normal phase HPLC chromatogram of screening case three;

[0036] Figure 4 Optimization case four normal phase HPLC chromatogram;

[0037] Figure 5 Normal phase HPLC chromatogram of optimization case five;

[0038] Figure 6 Normal phase HPLC chromatogram of optimization case VI;

[0039] Figure 7 Normal phase HPLC chromatogram of optimization case seven;

[0040] Figure 8 Optimization case eight normal phase HPLC chromatogram;

[0041] Fig. 9 Optimization case nine normal phase HPLC chromatogram;

[0042] Fig.10 Optimization case ten normal phase HPLC chromatogram;

[0043] Fig.11 Optimization case 11 Normal phase HPLC chromatogram;

[0044] Fig.12 Optimization case twelve normal phase HPLC chromatogram;

[0045] Fig.13 Optimization Case 13 Normal phase HPLC chromatogram;

[0046] Fig.14 Optimization Case 14 Normal phase HPLC chromatogram;

[0047] Fig.15 Optimization case 15 Normal phase HPLC chromatogram;

[0048] Fig.16 Normal phase HPLC chromatogram of optimization case 16;

[0049] Fig.17 Optimization Case 17 Normal phase HPLC chromatogram;

[0050] Fig.18 Optimization case 18 Normal phase HPLC chromatogram;

[0051] Fig.19 Normal phase HPLC chromatogram of optimization case 19;

[0052] Fig. 20 Optimization case 20 Normal phase HPLC chromatogram;

[0053] Fig.21 Optimization Case 21 Normal phase HPLC chromatogram.

[0054] Fig. 22 Normal phase HPLC chromatogram of intermediate M3 test sample.

[0055] Fig.23 Reversed-phase high-performance liquid chromatogram of methotrexate. DETAILED DESCRIPTION

[0056] The present invention provides a method for rapidly and accurately separating and detecting enantiomeric impurities in an intermediate (S)-2-(4-(methylamino)benzamido)glutaric acid, which is used to guide the development and control of a synthetic process, thereby ensuring that the quality of a methotrexate raw material meets standard requirements and satisfies clinical drug safety.

[0057] The present invention is completed based on the following findings of the inventors: Since the enantiomer M3-A impurity in the intermediate M3 will be further converted into the methotrexate isomeric impurity F in the subsequent process steps of methotrexate; and the currently disclosed methotrexate purification process has no obvious removal effect on the methotrexate isomeric impurity F, in order to ensure that the quality of the final product methotrexate meets the standard requirements, it is necessary to control the content of the isomeric impurity M3-A during the intermediate process. The inventors found through the study of impurity limits that when the content of the isomeric impurity M3-A is controlled below 3.0%, it can ensure that the content of the methotrexate isomeric impurity F in the synthesized final product methotrexate is maintained below 2.39% (the pharmacopoeia stipulates that the impurity limit is ≤3%), which meets the quality control requirements.

[0058] In order to achieve the above objectives, it is necessary to develop a method that can accurately detect the content of the isomer impurity M3-A to guide process control.

[0059] However, it was found during the analysis and detection process that it is not only necessary to separate the isomeric impurity M3-A and the main component intermediate M3 well, but also to effectively separate the isomeric impurity M3-A from the adjacent known or unknown impurity peaks, and to ensure that the peak shape of the isomeric impurity M3-A is good in order to achieve accurate quantification. To this end, the inventors fully investigated the detection conditions. First, the inventors tried to use a reverse phase system to separate the isomeric impurity M3-A, but found that the isomeric impurity M3-A could not be separated from the known impurities at all (screening case one and case two); so the inventors decided to use an AD-H chromatographic column, the reverse phase system was replaced with a normal phase, and the n-hexane-anhydrous ethanol-trifluoroacetic acid system was used as the mobile phase for preliminary screening, but the result was that the intermediate M3 peak and its enantiomer M3-A peak were not completely separated. The inventors further screened by changing the type of eluent in the mobile phase, the ratio of the eluent, the type and dosage of the acid, the flow rate, and the column temperature conditions to obtain the best detection conditions.

[0060] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0061] The raw materials and equipment used in the specific embodiments of the present invention are all known products and are obtained through commercial purchase.

[0062] In the present invention, the abbreviations and professional terms involved have the following meanings:

[0063] M3: (S)-2-(4-(methylamino)benzamido)glutarate disodium salt;

[0064] M3-A: (R)-2-(4-(methylamino)benzamido)glutarate disodium salt;

[0065] M3-B&C: (S)-4-methylaminobenzoylglutamic acid monoethyl ester mixture, in which the structure of M3-B is

[0066] The structure of M3-C is

[0067] M3-D: (S)-4-dimethylaminobenzoylglutamate;

[0068] M3-E: (S)-4-aminobenzoylglutamate;

[0069] M3-F: 4-(Methylamino)benzoic acid;

[0070] SM3: (S)-4-Methylaminobenzoylglutamate diethyl ester.

[0071] Area normalization method: Prepare the test solution according to the regulations under each item, take a certain amount of sample, and record the chromatogram. Measure the area of ​​each peak and the total chromatographic peak area on the chromatogram except the solvent peak, and calculate the percentage of each peak area to the total peak area.

[0072] Self-control method: When determining the impurity content, dilute the test solution into a solution equivalent to the impurity limit according to the impurity limit specified under each variety, use it as the control solution, inject the sample, record the chromatogram, measure the peak area of ​​each impurity on the chromatogram of the test solution, and compare it with the peak area of ​​the main component of the control solution to calculate the impurity content.

[0073] External standard method: According to the regulations under each variety, accurately weigh (measure) the reference substance and test substance, prepare them into solutions, accurately take a certain amount of each, inject the sample, record the chromatogram, measure the peak area (or peak height) of the substance to be tested in the reference substance solution and the test sample solution, and calculate the impurity content.

[0074] Example 1 Detection of the impurity content of the enantiomer of intermediate M3

[0075] Test solution: Take an appropriate amount of methotrexate intermediate M3 test sample, dissolve it in methanol and dilute it to make a solution containing about 1.0 mg per 1 ml.

[0076] System suitability solution: Take the test sample M3 and the isomeric impurity M3-A reference substance respectively, dissolve them in methanol and dilute them to make a mixed solution containing approximately 1.0 mg of the test sample and 30 μg of the enantiomer per 1 ml.

[0077] Chromatographic conditions: Using amylose-tris (3,5-dimethylphenylcarbamate) as filler (DALCELCHIRALPAK AD-H, specification: 250mm×4.6mm, 5μm or chromatographic column with equivalent performance); isocratic elution was performed with a mixed solution of n-hexane and anhydrous ethanol-acetonitrile-trifluoroacetic acid (90:10:0.3) in a volume ratio of 80:20 as the mobile phase; detection wavelength was 295nm; column temperature was 25℃; flow rate was 0.7ml / min; injection volume was 5μl.

[0078] System suitability requirements: In the system suitability solution chromatogram, the order of peaks is intermediate M3, isomeric impurity M3-A, among which the separation degree between the M3-A peak and the adjacent peaks should be greater than 1.5.

[0079] Determination method: Accurately measure the test solution and system suitability solution, inject them into the liquid chromatograph respectively, record the chromatogram, and measure the content of isomeric impurity M3-A in the test solution to be 0.45%.

[0080] Limit: In the chromatogram of the test solution, the content of the intermediate M3 enantiomer shall not exceed 3.0% calculated by area normalization.

[0081] Example 2 Study on the detection method of enantiomeric impurities of intermediate M3

[0082] In the intermediate M3 of methotrexate, in addition to its R-enantiomer impurity (M3-A), there are also multiple other known or unknown impurities. In the detection process, it is necessary to effectively separate the M3-A peak from the intermediate M3 and other adjacent impurities to achieve the determination of the isomer impurity content. The following is the effective separation of impurity peaks through the screening of chromatographic conditions.

[0083] Solution preparation:

[0084] Diluent: methanol.

[0085] M3-A impurity localization solution: Weigh about 1 mg of the methotrexate intermediate M3 enantiomer impurity sample into a 10 ml volumetric flask, add methanol to dissolve and dilute to the scale.

[0086] M3-B&C impurity localization solution: Weigh about 5 mg of methotrexate intermediate M3 impurity B&C, place it in a 10 ml volumetric flask, add methanol to dissolve and dilute to the scale.

[0087] M3-D impurity localization solution: Weigh about 5 mg of methotrexate intermediate M3 impurity D, place in a 10 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and filter.

[0088] M3-E impurity localization solution: Take about 5 mg of methotrexate intermediate M3 impurity E, place it in a 10 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and filter.

[0089] M3-F impurity localization solution: Take about 5 mg of methotrexate intermediate M3 impurity F, place it in a 10 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and filter.

[0090] SM3 impurity localization solution: Take about 5 mg of methotrexate intermediate M3 starting material SM3, place it in a 10 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and filter.

[0091] Mixed solution-1: Weigh about 10 mg of methotrexate intermediate M3 sample into a 10 ml volumetric flask, add an appropriate amount of methanol to dissolve, transfer 3 ml of M3-A, M3-B & C impurity location solution respectively, continue to add methanol to dilute to the scale, shake well, and filter.

[0092] Mixed solution-2: Weigh about 50 mg of methotrexate intermediate M3 sample into a 50 ml volumetric flask, add an appropriate amount of methanol to dissolve, transfer 3 ml of M3-A, M3-B&C, M3-D, M3-E, M3-F and SM3 impurity location solution respectively, continue to add methanol to dilute to the scale, shake well, and filter.

[0093] 2.1 Screening Case 1

[0094] Use DAICELZWIX(+) 0.30cm×25cm, 3μm as the chromatographic column, methanol (containing 25mmol / L formic acid and 12.5mmol / L diethylamine) as mobile phase A, water as mobile phase B, and mix the mobile phases A and B in a volume ratio of 98:2 for isocratic elution; the detection wavelength is 295nm; the column temperature is 30℃; the flow rate is 0.3ml / min. Take 5μl of the mixed solution-1 and inject it into the high performance liquid chromatograph to record the chromatogram.

[0095] Chromatogram see Figure 1 The results showed that the elution time of the isomer M3-A peak was consistent with that of the impurity peak M3-B&C (7.120 min), and the two could not be separated.

[0096] 2.2 Screening Case 2

[0097] On the basis of screening case 1, except for changing the mobile phase, other detection conditions remain unchanged; the specific mobile phase is a mixed solution of methanol and acetonitrile (containing 50mmol / L formic acid and 50mmol / L diethylamine) with a volume ratio of 1:4 as mobile phase A, and water as mobile phase B. After mixing the mobile phase A and B at a volume ratio of 80:20, isocratic elution is performed. Take 5μl of the mixed solution-1 and inject it into the high performance liquid chromatograph to record the chromatogram.

[0098] Chromatogram see Figure 2 The results showed that the peak elution time of M3 enantiomer M3-A was consistent with that of M3-B&C impurity peak (4.930 min), and the two could not be separated.

[0099] 2.3 Screening Case 3

[0100] AD-H column and n-hexane-anhydrous ethanol-trifluoroacetic acid system were used as mobile phase for preliminary screening.

[0101] Using amylose-tris (3,5-dimethylphenylcarbamate) as filler (DALCEL CHIRALPAK AD-H, specification: 250mm×4.6mm, 5μm) chromatographic column, with a mixed solution of n-hexane and anhydrous ethanol (containing 0.05% trifluoroacetic acid by volume) in a volume ratio of 70:30 as the mobile phase; detection wavelength 295nm; column temperature 30℃; flow rate 1.0ml / min. Take 5μl of the mixed solution-1 and inject it into the high performance liquid chromatograph to record the chromatogram.

[0102] Chromatogram see Figure 3 The results showed that the intermediate M3 peak (elution time 7.920 min) and its enantiomer M3-A peak (elution time 8.316 min) could not be completely separated.

[0103] 2.4 Optimization Case 4

[0104] Based on the screening case 3, except for adjusting the ethanol ratio in the mobile phase, other detection conditions remain unchanged; the adjusted mobile phase is a mixed solution of n-hexane and anhydrous ethanol (containing 0.05% trifluoroacetic acid by volume) with a volume ratio of 80:20. Take 5μl of the mixed solution-1 and inject it into the high performance liquid chromatograph to record the chromatogram.

[0105] Chromatogram see Figure 4 The results showed that the M3 peak (peak time 15.211 min) and the M3-A peak (retention time 16.320 min) failed to achieve baseline separation.

[0106] 2.5 Optimization Case 5

[0107] On the basis of the optimization case 4, except for adjusting the amount of trifluoroacetic acid (from the original volume fraction 0.05% to 0.1%), other detection conditions remain unchanged. 5 μl of the mixed solution-1 was injected into the high performance liquid chromatograph and the chromatogram was recorded.

[0108] Chromatogram see Figure 5The results showed that the M3 peak (peak time 16.949 min) and the M3-A peak (peak time 17.949 min) failed to achieve baseline separation.

[0109] 2.6 Optimization Case 6

[0110] On the basis of optimization case 5, except for replacing trifluoroacetic acid with diethylamine, other detection conditions remain unchanged. Take 5 μl of mixed solution-1 and inject it into the high performance liquid chromatograph to record the chromatogram.

[0111] Chromatogram see Figure 6 The results show that after replacing trifluoroacetic acid with diethylamine, the peaks of the main peak M3 and the impurity M3-A peak appear front or tail, so diethylamine is not desirable. Therefore, the inventors consider further reducing the proportion of anhydrous ethanol on the basis of optimizing case five.

[0112] 2.7 Optimization Case 7

[0113] On the basis of optimization case 5, except for adjusting the proportion of ethanol in the mobile phase, other detection conditions remain unchanged; the adjusted mobile phase is a mixed solution of n-hexane and anhydrous ethanol (containing 0.1% trifluoroacetic acid by volume) with a volume ratio of 85:15. Take 5μl of the mixed solution-1 and inject it into the high performance liquid chromatograph to record the chromatogram.

[0114] Chromatogram see Figure 7 The results showed that the separation degree between the M3 peak (peak time 32.906 min) and the M3-A peak (peak time 35.315 min) was 1.48 and the retention time of the chromatographic peak was relatively long. Consider replacing trifluoroacetic acid with formic acid for an experiment.

[0115] 2.8 Optimization Case 8

[0116] Based on the optimization case 7, except for replacing trifluoroacetic acid with formic acid, other detection conditions remain unchanged. Take 5 μl of the mixed solution-1 and inject it into the normal phase high performance liquid chromatograph to record the chromatogram.

[0117] Chromatogram see Figure 8 The results show that after replacing trifluoroacetic acid with formic acid, the peak shapes of the main peak and the impurity peak are not good and the M3 peak and the M3-A peak cannot be separated. Consider replacing formic acid with acetic acid for an experiment.

[0118] 2.9 Optimization Case 9

[0119] On the basis of the optimization case 8, except for replacing formic acid with acetic acid, other detection conditions remain unchanged. Take 5 μl of the mixed solution-1 and inject it into the high performance liquid chromatograph to record the chromatogram.

[0120] Chromatogram see Fig. 9The results showed that after replacing formic acid with acetic acid, the peak shapes of the main peak and the impurity peak did not improve, and the separation between the M3 peak and the M3-A peak did not improve significantly. Combining the experimental results of optimization cases eight and nine, trifluoroacetic acid was tried as a modifier in the mobile phase, and acetonitrile was added to the mobile phase based on optimization case seven.

[0121] 2.10 Optimization Case 10

[0122] Based on the optimization case 7, acetonitrile was added to the mobile phase. The specific chromatographic conditions were: using amylose-tris (3,5-dimethylphenylcarbamate) as the filler (DALCELCHIRALPAK

[0123] AD-H, specification: 250mm×4.6mm, 5μm) chromatographic column; n-hexane as mobile phase A, anhydrous ethanol-acetonitrile-trifluoroacetic acid (90:10:0.1) system as mobile phase B, mobile phase A and B are mixed at a volume ratio of 85:15 for isocratic elution; detection wavelength is 295nm; column temperature is 30℃; flow rate is 1.0ml / min. Take 5μl of the mixed solution-1 and inject it into the high performance liquid chromatograph to record the chromatogram.

[0124] Chromatogram see Fig.10 The results showed that after adding acetonitrile to the mobile phase, the separation degree between the M3 peak (peak time 26.486min) and the M3-A peak (retention time 28.491min) reached 1.52, and the separation degree between the M3-A peak and the M3-B&C peak (peak time 30.770min) reached 1.67. Consider increasing the amount of trifluoroacetic acid to increase the separation degree between the M3 peak and the M3-A peak.

[0125] 2.11 Optimization Case 11

[0126] On the basis of optimization case 10, except for increasing the amount of trifluoroacetic acid in mobile phase B (adjusted from the original volume fraction 0.1% to 0.5%), other detection conditions remain unchanged; the adjusted mobile phase B is anhydrous ethanol-acetonitrile-trifluoroacetic acid (90:10:0.5) system. Take 5μl of the mixed solution-1 and inject it into the high performance liquid chromatograph to record the chromatogram.

[0127] Chromatogram see Fig.11 The results show that when the amount of trifluoroacetic acid is increased to 0.5% (volume fraction), the resolution of M3 peak (peak time 28.379min) and M3-A peak (peak time 30.837min) increases to 1.95, but the resolution of M3-A peak and M3-B&C peak (peak time 32.636min) decreases to 1.37. Consider increasing the amount of trifluoroacetic acid further.

[0128] 2.12 Optimization Case 12

[0129] On the basis of the optimization case 11, except for increasing the amount of trifluoroacetic acid in mobile phase B (adjusted from the original volume fraction of 0.5% to 1%), other detection conditions remain unchanged; the adjusted mobile phase B is anhydrous ethanol-acetonitrile-trifluoroacetic acid (90:10:1) system. Take 5μl of the mixed solution-1 and inject it into the high performance liquid chromatograph to record the chromatogram.

[0130] Chromatogram see Fig.12 The results show that when the amount of trifluoroacetic acid is increased to 1% (volume fraction), the separation degree between the main peak and the impurity peak is not significantly improved. It is considered to reduce the proportion of acetonitrile based on the optimization case eleven.

[0131] 2.13 Optimization Case 13

[0132] On the basis of the optimization case 11, except for reducing the proportion of acetonitrile in mobile phase B, other detection conditions remain unchanged; after adjustment, mobile phase B is anhydrous ethanol-acetonitrile-trifluoroacetic acid (95:5:0.5) system. Take 5μl of mixed solution-1 and inject it into the high performance liquid chromatograph to record the chromatogram.

[0133] Chromatogram see Fig.13 The results show that after reducing the proportion of acetonitrile on the basis of optimization case 11, the separation degree between M3-A peak (peak time 35.922min) and M3-B&C peak (peak time 37.393min) is reduced to 0.89. Consider increasing the proportion of acetonitrile on the basis of optimization case 11.

[0134] 2.14 Optimization Case 14

[0135] On the basis of the optimization case 11, except for increasing the proportion of acetonitrile in mobile phase B, other detection conditions remain unchanged; after adjustment, mobile phase B is anhydrous ethanol-acetonitrile (85:15:0.5) system. Take 5 μl of mixed solution-1 and inject it into the high performance liquid chromatograph to record the chromatogram.

[0136] Chromatogram see Fig.14 The results showed that after increasing the proportion of acetonitrile in mobile phase B on the basis of optimization case eleven, the separation degree between M3 peak (elution time 21.909 min) and M3-A peak (elution time 24.362 min) increased to 2.43, and the separation degree between M3-A peak and M3-B&C peak (elution time 26.535 min) increased to 1.99 with good peak shape. Consider adjusting the proportion of eluent and increasing the column temperature to investigate the effect on peak elution.

[0137] 2.15 Optimization Case 15

[0138] On the basis of the optimization case 11, the ratio of mobile phase A to B was adjusted and the column temperature was increased to 35°C, while other detection conditions remained unchanged; the adjusted mobile phase was a mixed solution of n-hexane (mobile phase A) and anhydrous ethanol-acetonitrile-trifluoroacetic acid (85:15:0.5) system (mobile phase B) with a volume ratio of 80:20. Take 5 μl of the mixed solution-1 and inject it into the high performance liquid chromatograph to record the chromatogram.

[0139] Chromatogram see Fig.15 The results showed that after adjusting the ratio of mobile phase A to B and the column temperature, the number of impurities increased, and the separation degree between the M3 peak (peak time 11.779 min) and the M3-A peak (peak time 12.822 min) decreased to 1.95. Therefore, the inventor considered using the mobile phase ratio of optimized case 15 and reducing the column temperature on the basis of optimized case 14 for an experiment.

[0140] 2.16 Optimization Case 16

[0141] On the basis of the optimization case 15, except changing the column temperature to 25°C, other detection conditions remain unchanged. Take 5 μl of the mixed solution-1 and inject it into the high performance liquid chromatograph to record the chromatogram.

[0142] Chromatogram see Fig.16 The results show that after the column temperature is lowered, the separation between the M3 peak (peak time 12.030min) and the M3-A peak (peak time 13.129min) is 1.88, and the separation between the M3-A peak and the M3-B&C peak (peak time 15.986min) is 4.28, both of which meet the requirements. Since the inventor tried to use mixed solution-2 for separation under this chromatographic condition, but found that the M3-A peak and the M3-D peak could not be separated, the flow rate was reduced on the basis of this condition for investigation.

[0143] 2.17 Optimization Case 17

[0144] On the basis of optimizing case 16, except changing the flow rate to 0.8 ml / min, other detection conditions remain unchanged. Take 5 μl of mixed solution-2 and inject it into the high performance liquid chromatograph to record the chromatogram.

[0145] Chromatogram see Fig.17 The results show that the peak of M3-A is consistent with the peak of M3-D (peak time 16.388min), which will affect the detection of M3-A. Based on this condition, the amount of acetonitrile and trifluoroacetic acid was reduced.

[0146] 2.18 Optimization Case 18

[0147] On the basis of optimization case 17, except for reducing the amount of acetonitrile and trifluoroacetic acid in mobile phase B, the other detection conditions remain unchanged; the specific mobile phase B is anhydrous ethanol-acetonitrile-trifluoroacetic acid (90:10:0.1) system. Take 5μl of mixed solution-2 and inject it into the high performance liquid chromatograph to record the chromatogram.

[0148] Chromatogram see Fig.18 The results showed that after reducing the amount of acetonitrile and trifluoroacetic acid in mobile phase B, the M3-A peak (peak time 18.922min) and the M3-D peak (peak time 19.599min) had a tendency to separate, but the ratio of acetonitrile and trifluoroacetic acid was relatively low, so the next step was to consider lowering the column temperature for investigation.

[0149] 2.19 Optimization Case 19

[0150] On the basis of optimization case eighteen, except for lowering the column temperature to 20°C, the other detection conditions remain unchanged; 5 μl of mixed solution-2 is injected into the HPLC and the chromatogram is recorded.

[0151] Chromatogram see Fig.19 The results showed that the separation degree between the M3 peak (peak time 17.271 min) and the M3-A peak (peak time 19.049 min) was 1.97. The ratio of the eluent was adjusted based on this condition.

[0152] 2.20 Optimization Case 20

[0153] On the basis of optimization case 19, except for adjusting the ratio of mobile phase A and B, the other detection conditions remain unchanged; the specific mobile phase is a mixed solution of n-hexane (mobile phase A) and anhydrous ethanol-acetonitrile-trifluoroacetic acid (90:10:0.1) system (mobile phase B) with a volume ratio of 78:22. Take 5μl of the mixed solution-2 and inject it into the normal phase high performance liquid chromatograph to record the chromatogram.

[0154] Chromatogram see Fig. 20 The results showed that the separation degree between M3 peak (peak time 14.564min) and M3-A peak (peak time 15.848min) was 1.67. Based on the optimization case 19, the amount of trifluoroacetic acid was increased, the flow rate was reduced, and the column temperature was increased for investigation.

[0155] 2.21 Optimization Case 21

[0156] On the basis of optimization case 19, except for increasing the amount of trifluoroacetic acid in mobile phase B (adjusted from the original volume fraction of 0.1% to 0.3%), increasing the column temperature to 25°C and reducing the flow rate to 0.7ml / min, the other detection conditions remain unchanged; the specific mobile phase is a mixed solution of n-hexane (mobile phase A) and anhydrous ethanol-acetonitrile-trifluoroacetic acid (90:10:0.3) system (mobile phase B) with a volume ratio of 80:20. Take 5μl of the mixed solution-2 and inject it into the high performance liquid chromatograph to record the chromatogram.

[0157] Chromatogram see Fig.21 The results showed that the separation degree between M3 peak (peak time 21.779 min) and M3-A peak (peak time 23.953 min) was 2.18; the separation degree between M3-A peak and M3-D peak (peak time 25.175 min) was 1.21, and other impurities did not interfere with the detection of M3-A.

[0158] Since the content of impurity M3-D in the actual detection process of the methotrexate intermediate M3 test sample is relatively low, the M3-D peak and the M3-A peak can be effectively separated, as shown in the chromatogram. Fig. 22 The results showed that the separation degree between M3-A (peak time 22.213min) and M3-D (peak time 23.529min) was 1.5; the concentration of impurity M3-D in mixed solution-2 was relatively high. Although the separation degree between M3-A and M3-D was measured to be 1.21 under the chromatographic conditions of "Optimization Case 21", the chromatographic conditions were able to meet the detection requirements of impurity M3-A in the intermediate M3 test sample.

[0159] Example 3 Methodology Verification

[0160] The method specificity, accuracy and sensitivity were further investigated according to the chromatographic conditions of optimized case 21.

[0161] 3.1 Exclusivity

[0162] The impurity localization solutions prepared in Example 2, the mixed solution-2 and the blank solution methanol were respectively injected into the high performance liquid chromatograph and the chromatograms were recorded. The results are shown in Tables 1 and Fig.21 .

[0163] Table 1 Peak time of each component in each impurity localization solution and mixed solution-2

[0164] name M3-F M3-E M3 M3-A M3-D M3-B&C SM3 Positioning solution 12.164min 18.806min 21.496min 23.777min 25.022min 28.997min 33.864min Mixed solution 12.073min 19.098min 21.475min 23.659min 24.914min 28.820min 33.724min

[0165] The results showed that the impurities were clearly located, the blank solvent did not interfere with the detection of each impurity, and the location of the single impurity was consistent with the retention time of the impurity in the mixed solution; the peaks of impurity B and impurity C completely overlapped but did not interfere with the detection of impurity M3-A. In addition, the separation degree between the isomer M3-A and the main peak M3 in mixed solution-2 was 2.21; the separation degree between M3-A and the adjacent peak (M3-D) was 1.21. Considering that the content of M3-D was low during the actual detection of the sample, the separation degree between the M3-A peak and the M3-D peak could meet the requirements (see Fig. 22 ).

[0166] 3.2 Linearity

[0167] M3 stock solution-1: Take about 30 mg of M3 test sample, weigh accurately, dissolve in a 100 ml volumetric flask with methanol and dilute to the scale, shake well.

[0168] M3-A stock solution: Take about 30 mg of M3-A reference substance, accurately weigh it, put it in a 100 ml volumetric flask, dissolve it with methanol and dilute it to the scale, and shake it well.

[0169] Accurately measure appropriate amounts of M3 and M3-A stock solutions, respectively, and dilute them with methanol into solutions of a series of concentrations as linear solutions of each component; accurately measure 2 μl of each linear solution of M3 and M3-A, perform detection according to the above-mentioned chromatographic conditions, record the chromatogram, perform linear regression on the peak area Y with concentration X (mg / ml), and calculate the regression equation and correlation coefficient.

[0170] Table 2 Linear results of intermediate M3 and isomer M3-A

[0171]

[0172]

[0173] The results in Table 2 show that the peak areas of M3 in the range of 0.0902 μg / ml to 60.1600 μg / ml and M3-A in the range of 0.0899 μg / ml to 59.9600 μg / ml have a good linear relationship with the measured concentrations.

[0174] 3.3 Accuracy

[0175] M3 stock solution-2: Take about 1.0000g of M3 test sample, weigh accurately, place in a 100ml volumetric flask, dissolve and dilute to the scale with methanol, and shake well.

[0176] Accurately measure 5 ml of M3 stock solution-2 and place them in different 50 ml volumetric bottles. Then accurately measure 1 ml, 2.5 ml, 5 ml, and 7.5 ml of M3-A stock solution in Example "3.2 Linearity" and place them in the above 50 ml volumetric bottles, dilute to the scale with methanol, and shake well. Prepare 3 portions of each concentration of recovery solution in parallel, for a total of 12 portions. Accurately measure 5.0 μl for injection and record the chromatogram. The results are shown in the table below:

[0177] Table 3 Isomer M3-A recovery results

[0178]

[0179]

[0180] The results in Table 3 show that the average values ​​of the impurity recoveries of the isomer M3-A are all within the range of 95.4% to 97.3% (90% to 108% as specified in the pharmacopoeia), and the RSD of the average values ​​of the 12 recoveries is 0.08%, indicating that the detection method of the present invention has good accuracy.

[0181] 3.4 Limit of quantification and limit of detection

[0182] Accurately measure 7.5 ml of the M3-A stock solution of Example "3.2 Linearity" into a 50 ml volumetric flask, dilute to the scale with methanol, and shake well; dilute step by step to make the M3-A peak height 10 to 30 times the baseline noise, which is the quantitative limit, and this is used as the quantitative limit solution; make the M3-A peak height 3 to 10 times the baseline noise, which is the detection limit, and this is used as the detection limit solution.

[0183] The quantitative limit and detection limit solutions were tested according to the above chromatographic conditions and the chromatograms were recorded. The results are shown in the following table:

[0184] Table 4 Results of quantification limit and detection limit of isomer M3-A

[0185] name Limit of quantitation (ug / ml) Limit of quantification (%) Detection limit (ug / ml) Detection limit (%) M3-A 0.0899 0.0090 0.0300 0.0030

[0186] The results in Table 4 show that the sensitivity of the detection method of the present invention meets the detection requirements.

[0187] 3.5 Precision

[0188] Accurately weigh about 20 mg of the methotrexate intermediate M3 test sample, place it in a 20 ml volumetric flask, dissolve it in methanol and dilute it to the mark, shake it well. Prepare 12 portions in parallel, of which 6 portions are used as repeatability solutions and the other 6 portions are used as intermediate precision solutions.

[0189] Take 5 μl of each accurately, test according to the above chromatographic conditions, and record the chromatogram. The results are shown in the table below:

[0190] Table 5 Precision results of isomer M3-A

[0191]

[0192] The results in Table 5 show that the detection method of the present invention has good precision.

[0193] Under the above-mentioned chromatographic conditions, in order to evaluate the calculation method of the content of the M3 enantiomer impurity M3-A, the area normalization method, the self-control method and the external standard method were used for calculation and comparison. The content of M3-A calculated by the area normalization method was 0.45%, the content of M3-A calculated by the self-control method was 0.47%, and the content of M3-A calculated by the external standard method was 0.45%. The three calculation results were basically consistent. Therefore, the present invention adopts the area normalization method to calculate the content of the isomer M3-A.

[0194] Example 4 Investigation of the content limit of the isomeric impurity M3-A in the intermediate M3

[0195] The intermediate M3 is used as a reactant to synthesize the final product methotrexate. Among them, the content of the isomeric impurity M3-A in the intermediate M3 is 3.02%, which is converted into impurity F during the synthesis process. By measuring the content of impurity F in the final product methotrexate sample, the content limit of the isomeric impurity M3-A in the intermediate M3 is determined.

[0196] The synthesis method is as follows: 10g of 2,4-diamino-6-bromomethylpteridine and 20g of intermediate M3 are added to 120g of hydrochloric acid solution (6mol / L), and stirred at 60-65°C for 2h under nitrogen protection, filtered, and washed with ethanol to obtain a crude product. The above crude product is added to 50mL of water, and 10% sodium hydroxide solution is added dropwise until it is dissolved, activated carbon is added for decolorization and filtration, and the filtrate is added dropwise with dilute hydrochloric acid to adjust the pH to 4, filtered, washed with ethanol, and vacuum dried to obtain methotrexate.

[0197] The content of impurity F in the methotrexate obtained by the above preparation method was detected by the European Pharmacopoeia method and was 2.39% ( Fig.23 ).

[0198] According to the European Pharmacopoeia, the limit of impurity F shall not exceed 3%. It can be seen that when the content of the intermediate M3 enantiomer impurity M3-A is controlled below 3.0%, it can ensure that the content of impurity F in the final product methotrexate meets the standard requirements.

Claims

1. A method for detecting the R-isomer or R-isomer salt contained in (S)-2-(4-(methylamino)benzamido)glutaric acid or its salt by high performance liquid chromatography, characterized in that: The high performance liquid chromatography method uses a chromatographic column with amylose-tris (3,5-dimethylphenylcarbamate) as a filler, and the mobile phase is a mixed solvent of mobile phase A n-hexane and mobile phase B anhydrous ethanol-acetonitrile-trifluoroacetic acid system, wherein the volume ratio of mobile phase A to B is (78:22) ~ (85:15) for isocratic elution, the chromatographic column with amylose-tris (3,5-dimethylphenylcarbamate) as filler is DALCEL AD-H, specifications are 250mm×4.6mm, 5μm, the volume ratio of anhydrous ethanol to acetonitrile in the mobile phase B is 90:10, the volume fraction of trifluoroacetic acid is 0.1% to 0.5%, the mobile phase flow rate is 0.7 to 0.8mL / min, the column temperature is 20 to 25°C, the sample volume is 5μl; the detection wavelength is 295nm.

2. The detection method according to claim 1, characterized in that: The volume fraction of trifluoroacetic acid is 0.3%.

3. The detection method according to claim 1, characterized in that: The volume ratio of the mobile phase A to the mobile phase B is (78:22) to (80:20).

4. The detection method according to claim 1, characterized in that The volume ratio of the mobile phase A to the mobile phase B is 80:

20.

5. The detection method according to claim 1, characterized in that: The mobile phase flow rate was 0.7 mL / min.

6. The detection method according to claim 1, characterized in that: The column temperature was 25°C.

7. The detection method according to any one of claims 1 to 6, characterized in that: The salt of (S)-2-(4-(methylamino)benzamido)pentanedioic acid and / or the salt of the R-isomer is a disodium salt.

8. The detection method according to claim 7, characterized in that: The detection is qualitative and / or quantitative detection; the quantitative detection is to calculate the content of the R isomer contained in the test solution of (S)-2-(4-(methylamino)benzamido)glutaric acid or its salt by area normalization method.

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