Determination method of methotrexate liquid preparation

Abstract

The invention discloses a determination method of a methotrexate liquid preparation. Relates to the technical field of pharmaceutical analysis. The determination method is used for detecting methotrexate and preservatives, the determination method adopts liquid chromatography, and the conditions of the liquid chromatography are as follows: a chromatographic column is an octadecyl silane bonded silica gel column or an efficacy equivalent column; a mobile phase A is a 0.08-0.12% v / v glacial acetic acid solution, a mobile phase B is acetonitrile, and an elution procedure is gradient elution. According to the method, specific chromatographic conditions are adopted, so that methotrexate and the preservative in the methotrexate liquid preparation can be effectively separated at the same time, different detection conditions do not need to be independently developed, the development and detection cost is reduced, the detection time is greatly shortened, and the detection efficiency is improved; and interference generated by a blank solvent and an auxiliary material solution can be eliminated, and the method has the advantages of good specificity, good separation degree and good accuracy.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical analysis, and particularly to a method for determining a liquid preparation of methotrexate. Background Art

[0002] Methotrexate (MTX) is an antimetabolite drug belonging to the class of antifolate drugs and is used to treat various diseases such as rheumatoid arthritis (RA), acute lymphoblastic leukemia (ALL), and malignancies. Methotrexate inhibits dihydrofolate reductase (DHFR), thereby inhibiting the conversion of dihydrofolate to tetrahydrofolate, resulting in the inhibition of DNA, RNA, and protein synthesis, and further inhibiting cell proliferation, especially rapidly proliferating tumor cells and immune cells. This mechanism of action makes methotrexate an important chemotherapeutic drug and immunosuppressant in clinical practice.

[0003] The liquid preparation of methotrexate is an important form of pharmaceutical preparation. One of its prominent advantages is that it can be rapidly absorbed by the digestive system and is suitable for patient groups who are unable to swallow solid tablets, such as children, the elderly, or those with swallowing function affected by certain diseases. In addition, the liquid preparation can flexibly adjust the dosage according to the specific situation of the patient. Especially in some patients who require long-term treatment, it can improve treatment compliance and the flexibility of medication.

[0004] However, one problem with liquid preparations is their susceptibility to microbial contamination, especially during the long-term storage of the drug. To address this issue, preservatives are usually added to prevent the growth and reproduction of microorganisms, thereby ensuring the safety and effectiveness of the drug and avoiding adverse reactions caused by contamination with bacteria, molds, etc. Adding preservatives can not only effectively inhibit the growth of microorganisms but also ensure the stability of the drug within a certain period after opening.

[0005] Although the use of preservatives can effectively prevent drug contamination, it also brings some challenges. On the one hand, the substances decomposed by the preservatives after long-term storage may react with the active ingredients of the drug, affecting the stability and efficacy of the drug; on the other hand, some preservatives may cause certain irritation or allergic reactions to patients during long-term use. For example, certain preservatives may decompose during long-term storage or in a high-temperature environment, generating degradation products harmful to the human body. Therefore, in the quality control process of drugs, it is necessary to not only monitor the content of methotrexate but also effectively detect and control the preservatives and their degradation products.

[0006] To solve these problems, in recent years, many drug quality control standards and detection methods have been continuously developed. International pharmacopoeias (such as the Chinese Pharmacopoeia, the United States Pharmacopoeia, and the British Pharmacopoeia) have provided clear specifications for the determination of the content of active ingredients in various dosage forms of methotrexate. These specifications usually include the quantitative method of methotrexate and the impurity analysis that may be involved. However, although these pharmacopoeias provide detailed technical requirements for the determination of methotrexate content, there is no corresponding detection method for how to simultaneously detect methotrexate, preservative ingredients, and their degradation products. Summary of the Invention

[0007] The object of the present invention is to provide a method for simultaneously detecting methotrexate and preservatives in methotrexate liquid preparations.

[0008] Specifically, the technical solution according to the present invention is as follows:

[0009] A method for determining a methotrexate liquid preparation for detecting methotrexate and preservatives, the determination method using liquid chromatography, and the conditions of the liquid chromatography including:

[0010] Chromatographic column: octadecylsilane-bonded silica gel column or a column with equivalent efficiency;

[0011] Mobile phase A: 0.08 - 0.12% glacial acetic acid solution v / v, mobile phase B: acetonitrile;

[0012] The elution procedure is:

[0013] Time (min) Mobile Phase A (%) Mobile Phase B (%) 0 88-92 12-8 20 63-67 37-33 23 38-42 62-58 25 38-42 62-58 25.1 88-92 12-8 30 88-92 12-8 .

[0014] According to an embodiment of the present invention, at least one of the following advantages or beneficial effects is achieved by one of the technical solutions in the above technical solution:

[0015] The method of the present invention uses specific chromatographic conditions, such as the type of mobile phase and the gradient elution procedure, to be able to simultaneously and effectively separate methotrexate and preservatives in methotrexate liquid preparations, so that there is no need to separately develop different detection conditions, reducing the development and detection costs, greatly shortening the detection time, and improving the detection efficiency; and it can eliminate the interference generated by blank solvents and excipient solutions, having the advantages of good specificity, good resolution, and good accuracy. The method of the present invention can be used for the content detection of methotrexate and preservatives in the preparation process, finished products, and storage process of methotrexate drug preparations, as an important part of drug quality control, thereby reducing the risk of drug clinical use.

[0016] According to an embodiment of the present invention, the elution procedure is:

[0017] Time (min) Mobile Phase A (%) Mobile Phase B (%) 0 90 10 20 65 35 23 40 60 25 40 60 25.1 90 10 30 90 10

[0018] 。The elution procedure is a preferred elution procedure.

[0019] According to an embodiment of the present invention, the elution procedure is as follows:

[0020]

[0021]

[0022] According to an embodiment of the present invention, the elution procedure is as follows:

[0023] Time (min) Mobile Phase A (%) Mobile Phase B (%) 0 92 8 20 67 33 23 42 58 25 42 58 25.1 92 8 30 92 8 。

[0024] According to an embodiment of the present invention, the mobile phase A is a 0.09 - 0.11% v / v glacial acetic acid solution.

[0025] According to an embodiment of the present invention, the mobile phase A is a 0.10% v / v glacial acetic acid solution.

[0026] According to an embodiment of the present invention, the chromatographic conditions of the liquid chromatography further include: the detection wavelength is 249 - 259 nm.

[0027] According to an embodiment of the present invention, the chromatographic conditions of the liquid chromatography further include: the injection volume is 15 - 25 μL.

[0028] According to an embodiment of the present invention, the chromatographic conditions of the liquid chromatography further include: the column temperature is 25 - 35°C; and / or,

[0029] the flow rate is 0.9 - 1.1 mL / min; and / or,

[0030] The chromatographic column is Welch Ultimate XB - C18, 4.6 * 150 mm, 3 μm.

[0031] According to an embodiment of the present invention, the column temperature is 30°C, the flow rate is 1.0 mL / min, the injection volume is 20 μL, and the detection wavelength is 254 nm. A suitable column temperature can optimize the separation process and improve the analysis efficiency.

[0032] According to an embodiment of the present invention, the preservative includes at least one of methylparaben, ethylparaben, and p - hydroxybenzoic acid. In one embodiment, the method for determining the methotrexate liquid preparation of the present invention can simultaneously detect methotrexate, methylparaben, ethylparaben, and p - hydroxybenzoic acid in the methotrexate liquid preparation. Among them, methylparaben, ethylparaben, and p - hydroxybenzoic acid are preservatives added to the methotrexate liquid preparation and / or degradation products of the preservative.

[0033] According to an embodiment of the present invention, the methotrexate liquid preparation includes a methotrexate oral solution.

[0034] Additional aspects and advantages of the present invention will be given in part in the following description, become apparent in part from the following description, or be learned through the practice of the present discovery. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The above and / or additional aspects and advantages of the present invention will become apparent and be readily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0036] Figure 1 It is a chromatogram obtained by detecting the methotrexate oral solution using the method of Comparative Example 1, wherein the blue chromatogram is the test solution and the black chromatogram is the impurity localization solution.

[0037] Figure 2 It is a chromatogram obtained by detecting the methotrexate oral solution using the method of Comparative Example 2.

[0038] Figure 3 It is a chromatogram obtained by detecting the methotrexate oral solution using the method of Comparative Example 3.

[0039] Figure 4 It is a chromatogram obtained by detecting the methotrexate oral solution using the method of Comparative Example 4.

[0040] Figure 5 It is a chromatogram obtained by detecting the methotrexate oral solution using the method of Example 1. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0041] The terms "preferred", "more preferred", etc. in the present invention refer to embodiments of the present invention that can provide certain beneficial effects in certain cases. However, in the same or other cases, other embodiments may also be preferred. In addition, the description of one or more preferred embodiments does not imply that other embodiments are not available, nor is it intended to exclude other embodiments from the scope of the present invention.

[0042] When a numerical range is disclosed herein, the above range is considered continuous and includes the minimum and maximum values of the range, as well as each value therebetween. Further, when the range refers to integers, it includes each integer between the minimum and maximum values of the range. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein.

[0043] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of the present invention.

[0044] The reagents, methods, and equipment used in the present invention are all conventional reagents, methods, and equipment in the technical field, unless otherwise specified.

[0045] I. Main Instruments and Reagents

[0046] 1. Main Instruments

[0047] (1) High-performance liquid chromatography: Waters e2695, Arc series high-performance liquid chromatography.

[0048] (2) Electronic balance: Sartorius SQP.

[0049] 2. Main Reagents

[0050] Glacial acetic acid (analytical pure), acetonitrile (chromatographic pure).

[0051] 3. Control Substance Information

[0052] Methotrexate control substance: sourced from the National Institutes for Food and Drug Control, with a content of 99.9%;

[0053] p-Hydroxybenzoic acid control substance: sourced from the National Institutes for Food and Drug Control, with a content of 100.0%;

[0054] Methylparaben control substance: sourced from the National Institutes for Food and Drug Control, with a content of 99.9%;

[0055] Ethylparaben control substance: sourced from the National Institutes for Food and Drug Control, with a content of 100.0%.

[0056] 4. Sample Information

[0057] Test sample: Methotrexate oral solution, developed by Bemei R & D (Shenzhen) Co., Ltd., specification 60 mL: 2 mg / mL.

[0058] Blank matrix liquid medicine: provided by Bemei R & D (Shenzhen) Co., Ltd., taking excipients with the same prescription ratio as the test sample, preparing a liquid medicine without adding methotrexate, methylparaben, ethylparaben, and p-hydroxybenzoic acid, and with the same concentration as the blank matrix liquid medicine. The excipients include citric acid, disodium hydrogen phosphate, sucralose, and essence.

[0059] II. Preparation of Mobile Phase A

[0060] 0.08 - 0.12% glacial acetic acid solution (v / v): Precisely measure 1 L of ultrapure water, add 0.8 - 1.2 mL of glacial acetic acid, stir well to mix evenly, filter by suction, and sonicate to obtain. For example, mobile phase A can be 0.08% glacial acetic acid solution (v / v), 0.09% glacial acetic acid solution (v / v), 0.10% glacial acetic acid solution (v / v), 0.11% glacial acetic acid solution (v / v), 0.12% glacial acetic acid solution (v / v).

[0061] Taking 0.10% glacial acetic acid solution (v / v) as an example, its preparation method includes: Precisely measure 1 L of ultrapure water, add 1 mL of glacial acetic acid, stir well to mix evenly, filter by suction, and sonicate to obtain.

[0062] III. Solution Preparation

[0063] 1. Blank solution and diluent: Take mobile phase A and mobile phase B and mix them evenly in a volume ratio of 9:1 to obtain.

[0064] 2. Stock solution of methylparaben and ethylparaben reference substances: Precisely weigh 20 mg of methylparaben reference substance and 10 mg of ethylparaben reference substance, place them in a 50 mL volumetric flask, dissolve with the diluent, and dilute to the mark with the diluent to obtain.

[0065] 3. Stock solution of p-hydroxybenzoic acid reference substance: Precisely weigh about 12 mg of p-hydroxybenzoic acid reference substance and place it in a 200 mL volumetric flask, dissolve it by sonication with the diluent, dilute to the mark with the diluent, shake well, then precisely measure 10 mL of the above solution and place it in a 50 mL volumetric flask, dilute to the mark with the diluent, and shake well to obtain.

[0066] 4. Stock solution of methotrexate reference substance: Precisely weigh about 20 mg of methotrexate reference substance and place it in a 20 mL volumetric flask, dissolve with the diluent, and dilute to the mark with the diluent, shake well to obtain.

[0067] 5. Reference solution: Precisely measure 5 mL of the stock solution of methylparaben and ethylparaben reference substances, 6 mL of the stock solution of p-hydroxybenzoic acid reference substance, and 4 mL of the stock solution of methotrexate reference substance, place them in the same 50 mL volumetric flask, dilute to the mark with the diluent, and shake well to obtain.

[0068] 6. Test solution: Take the methotrexate oral solution, shake well, precisely measure 2 mL, place it in a 50 mL volumetric flask, dilute to the mark with the diluent, and shake well to obtain.

[0069] 7. Blank matrix solution: Take the blank matrix liquid medicine, shake well, precisely measure 2 mL, place it in a 20 mL volumetric flask, dilute to the mark with the diluent, and shake well to obtain.

[0070] 8. Test sample impurity spike solution: Take an appropriate amount of methotrexate oral solution, and prepare a mixed solution containing about 80 μg / mL of methotrexate and the specific impurity at the limit concentration. Shake well to obtain. Among them, the specific impurity is added to the test sample impurity spike solution to avoid the interference of this specific impurity on the separation and detection of the four target components.

[0071] IV. Determination method and conditions

[0072] A determination method for a methotrexate liquid preparation, used for detecting methotrexate and preservatives, the preservatives include at least one of methylparaben, ethylparaben and p-hydroxybenzoic acid. The determination method uses liquid chromatography, and the conditions of liquid chromatography include:

[0073] Chromatographic column: octadecylsilane-bonded silica gel column or a column with equivalent efficiency;

[0074] Mobile phase A: 0.08 - 0.12% glacial acetic acid solution (v / v), mobile phase B: acetonitrile;

[0075] The column temperature is 25 - 35 °C;

[0076] The flow rate is 0.9 - 1.1 mL / min;

[0077] An ultraviolet detector is used, and the detection wavelength is 249 - 259 nm;

[0078] The injection volume is 15 - 25 μL;

[0079] The elution program is:

[0080] Time (min) Mobile Phase A (%) Mobile Phase B (%) 0 88-92 12-8 20 63-67 37-33 23 38-42 62-58 25 38-42 62-58 25.1 88-92 12-8 30 88-92 12-8 .

[0081] V. Calculation formula

[0082] 1. The calculation formula for the content of methylparaben / ethylparaben is as follows:

[0083]

[0084] In the formula: W 对 The weighed amount of the reference substance, mg;

[0085] V 对 The dilution volume of the reference substance, mL;

[0086] P 对 The content of the reference substance, %;

[0087] A 对 The peak area of methylparaben / ethylparaben in the reference substance solution;

[0088] A 供The peak area of each preservative in the test solution (sodium methylparaben multiplied by the coefficient 1.145, sodium ethylparaben multiplied by the coefficient 1.133);

[0089] V 供 The dilution volume of the test sample, mL;

[0090] V 样 The sampling volume of the test sample, mL;

[0091] The labeled amount of sodium methylparaben in the methotrexate oral solution,

[0092] L 1.1 mg / mL; the labeled amount of sodium ethylparaben in the methotrexate oral solution, 0.54 mg / mL.

[0093] 2. The calculation formula for the content of methotrexate is as follows:

[0094]

[0095] Where: W 对 The weighed amount of the reference substance, mg;

[0096] V 对 The dilution volume of the reference substance, mL;

[0097] P 对 The content of the reference substance, %;

[0098] A 对 The peak area of the methotrexate peak in the reference substance solution;

[0099] A 供 The peak area of the methotrexate peak in the test solution;

[0100] D 供 The dilution factor of the test sample, mL;

[0101] L The labeled amount in the methotrexate oral solution, 2 mg / mL.

[0102] 3. The content formula for p-hydroxybenzoic acid is as follows:

[0103]

[0104] Where: W 对 The weighed amount of the reference substance, mg;

[0105] V 对 The dilution volume of the reference substance, mL;

[0106] P 对 The content of the reference substance, %;

[0107] A 对Peak area of p-hydroxybenzoic acid in the reference solution;

[0108] f 值 Average value of the correction factor;

[0109] A 供 Peak area of p-hydroxybenzoic acid in the test solution;

[0110] V 供 Dilution volume of the test sample, mL;

[0111] V 样 Sampling volume of the test sample, mL;

[0112] L Labeled amount in methotrexate oral solution, 2 mg / mL.

[0113] VI. Specific test content

[0114] Example 1

[0115] A method for the determination of a methotrexate liquid preparation for detecting methotrexate and preservatives, the preservatives including methyl paraben, ethyl paraben and p-hydroxybenzoic acid, and the determination method adopts liquid chromatography, and the conditions of the liquid chromatography include:

[0116] The chromatographic column is Welch Ultimate XB-C18, 4.6 * 150 mm, 3 μm;

[0117] Mobile phase A: 0.10% glacial acetic acid solution (v / v), mobile phase B: acetonitrile;

[0118] The column temperature of the chromatographic column is 30 °C;

[0119] The flow rate is 1.0 mL / min;

[0120] The injection volume is 20 μL;

[0121] An ultraviolet detector is adopted, and the detection wavelength is 254 nm.

[0122] The elution program is:

[0123] Time (min) Mobile Phase A (%) Mobile Phase B (%) 0 90 10 20 65 35 23 40 60 25 40 60 25.1 90 10 30 90 10 .

[0124] Example 2

[0125] Compared with Example 1, the flow rate was adjusted to 0.9 mL / min. Other chromatographic conditions remain unchanged.

[0126] Example 3

[0127] Compared with Example 1, the flow rate was adjusted to 1.1 mL / min, and other chromatographic conditions remain unchanged.

[0128] Example 4

[0129] Compared with Example 1, the column temperature was adjusted to 25 °C, and other chromatographic conditions remained unchanged.

[0130] Example 5

[0131] Compared with Example 1, the column temperature was adjusted to 35 °C, and other chromatographic conditions remained unchanged.

[0132] Example 6

[0133] Compared with Example 1, the glacial acetic acid content in mobile phase A was adjusted to 0.09%, and other chromatographic conditions remained unchanged.

[0134] Example 7

[0135] Compared with Example 1, the glacial acetic acid content in mobile phase A was adjusted to 0.11%, and other chromatographic conditions remained unchanged.

[0136] Example 8

[0137] Compared with Example 1, the glacial acetic acid content in mobile phase A was adjusted to 0.12%, and other chromatographic conditions remained unchanged.

[0138] Example 9

[0139] Compared with Example 1, the glacial acetic acid content in mobile phase A was adjusted to 0.08%, and other chromatographic conditions remained unchanged.

[0140] Example 10

[0141] Compared with Example 1, the elution program was adjusted to:

[0142] Time (min) Mobile Phase A (%) Mobile Phase B (%) 0 88 12 20 67 33 23 38 62 25 38 62 25.1 88 12 30 88 12

[0143] Other chromatographic conditions remained unchanged.

[0144] Example 11

[0145] Compared with Example 1, the elution program was adjusted to:

[0146] Time (min) Mobile Phase A (%) Mobile Phase B (%) 0 92 8 20 63 37 23 42 58 25 42 58 25.1 92 8 30 92 8

[0147] Other chromatographic conditions remained unchanged.

[0148] Control Example 1

[0149] For Control Example 1, the related substances / content determination method included in "Methotrexate" in the 2020 edition of Chp was used to determine the test solution 1 and the impurity localization solution, and the chromatographic conditions used were as follows:

[0150]

[0151]

[0152] The chromatogram is shown in Figure 1 。

[0153] Comparative Example 2

[0154] For Comparative Example 2, based on Comparative Example 1, the conditions were optimized and the elution program was adjusted. The chromatographic conditions are as follows:

[0155]

[0156] The chromatogram is shown in Figure 2 。

[0157] Comparative Example 3

[0158] For Comparative Example 3, the content determination method included in "Methylparaben" in the 2020 edition of Chp was used to determine the reference preparation solution, system suitability solution, and blank solution. The chromatographic conditions used are as follows:

[0159]

[0160]

[0161] The chromatogram is shown in Figure 3 。

[0162] Comparative Example 4

[0163] For Comparative Example 4, based on Comparative Example 3, the mobile phase and elution program were adjusted. The chromatographic conditions used are as follows:

[0164]

[0165] The chromatogram is shown in Figure 4 。

[0166] The test results of Examples 1 - 7 and Comparative Examples 1 - 4 are shown in Table 1.

[0167] Table 1 Test Results of Methotrexate Oral Solution

[0168]

[0169] From Table 1, Figure 1 and Figure 2It can be seen that in the test solution of Comparative Example 1, the p-hydroxybenzoic acid peak did not show a peak response, and neither methylparaben nor ethylparaben was eluted and detected; in Comparative Example 2, methotrexate, methylparaben and ethylparaben could be detected, but the p-hydroxybenzoic acid peak did not show a detection response. Under the mobile phase systems of Comparative Example 1 and Comparative Example 2, at a wavelength of 302 nm, p-hydroxybenzoic acid could not meet the response requirements. Due to the large polarity difference, it was difficult to achieve the coordinated satisfaction of the retention values, resolution and response of the four target components whether in the selection of the mobile phase system or the adjustment of the elution program.

[0170] See Figure 3 , for the reference preparation of Comparative Example 3, the four target components of methotrexate, p-hydroxybenzoic acid, methylparaben and ethylparaben could be detected and responded, but there were interference peaks in both the methotrexate peak and the p-hydroxybenzoic acid peak, and they could not be completely separated. See Figure 4 , for the control solution of Comparative Example 4, methotrexate, p-hydroxybenzoic acid, methylparaben and ethylparaben could all be detected and responded, and the resolution was good. However, it was found that the purity of the p-hydroxybenzoic acid peak was greater than the purity threshold, indicating that the p-hydroxybenzoic acid peak was impure and p-hydroxybenzoic acid was not completely eluted.

[0171] From Table 1 and Figure 5 it can be seen that Example 1 adopted the specific chromatographic conditions of the present invention, and could effectively separate methotrexate and preservatives (p-hydroxybenzoic acid, methylparaben and ethylparaben) in the methotrexate oral solution at the same time. Compared with Example 1, in Examples 2-5, the flow rate was adjusted to 0.9-1.1 mL / min and the column temperature was adjusted to 25-35 °C, and it could also effectively separate methotrexate and preservatives in the methotrexate oral solution at the same time, and the detected content changes of each substance were not significant.

[0172] Compared with Example 1, the glacial acetic acid content in Examples 6-9 was adjusted to 0.08-0.12%. As the glacial acetic acid content increased, the detected contents of methotrexate, p-hydroxybenzoic acid, methylparaben and ethylparaben were not significantly different, and it could also effectively separate methotrexate and preservatives in the methotrexate oral solution at the same time. Examples 1, 10 and 11 were three different gradient elution programs, and could also effectively separate methotrexate and preservatives in the methotrexate oral solution, and the detected contents of methotrexate and preservatives were basically the same, indicating that methotrexate and preservatives could be detected under these three different gradient elution programs, and it did not affect the content detection results. This shows that the determination method of the present application can effectively separate methotrexate and preservatives in the methotrexate liquid preparation, and accurately determine the contents of methotrexate and preservatives.

[0173] This shows that the determination method of this application can simultaneously determine methotrexate and preservatives (p-hydroxybenzoic acid, methylparaben, and ethylparaben) in methotrexate oral solution, accurately detect the contents of methotrexate and preservatives, eliminate the interference caused by blank solvent and excipient solution, and can be used for the content detection of methotrexate and preservatives in the preparation process, finished products, and storage process of methotrexate pharmaceutical preparations.

[0174] VII. Method Validation

[0175] 1. System Suitability Test

[0176] Using the method of Example 1, the reference solution was detected, and the injection was carried out continuously for 5 times, and the chromatogram was recorded. The test results are shown in Table 2 below.

[0177] Table 2 Detection Results of System Suitability Test

[0178]

[0179] It can be seen from Table 2 that the method of the present invention can effectively separate methotrexate, methylparaben, ethylparaben, and p-hydroxybenzoic acid simultaneously, and the RED values of the retention times and peak areas of the above components are 0.1 - 0.4%, all not greater than 1.0%, indicating that the method of the present invention has good precision.

[0180] 2. Specificity Test

[0181] Using the method of Example 1, the blank solution, blank matrix solution, reference solution, test solution, and spiked test solution were detected. The test results are shown in Table 3 and Figure 5 .

[0182] Table 3 Detection Results of Specificity Test

[0183]

[0184]

[0185] Note: "NA" means not applicable.

[0186] It can be seen from Table 3 combined with Figure 5 that the blank solution and blank matrix solution have no interference on the detection. The target peaks of the test solution and the spiked test solution of test impurities are consistent with the retention time of the reference solution, and the minimum resolution between the target peak and the adjacent peak is greater than 1.5, indicating that the method of the present invention can effectively separate methotrexate, methylparaben, ethylparaben, and p-hydroxybenzoic acid simultaneously, and can eliminate the interference caused by blank solvent and excipient solution, indicating that the method of the present invention has good specificity.

[0187] 3. Precision Test

[0188] Take blank solution, reference solution, and test solution (prepared in parallel for 6 portions), and determine the contents of methotrexate, methylparaben, ethylparaben, and p-hydroxybenzoic acid in the test sample according to the method of Example 1. The test results are shown in Table 4.

[0189] Table 4 Test Results of Precision Test

[0190]

[0191]

[0192] As can be seen from Table 4, the maximum RSD of the contents of the four target components, namely methotrexate, methylparaben, ethylparaben, and p-hydroxybenzoic acid, in the test solution is 0.3%, which is less than 2.0%. This indicates that the method of the present invention can effectively separate methotrexate, methylparaben, ethylparaben, and p-hydroxybenzoic acid simultaneously and has good precision.

[0193] 4. Accuracy Test

[0194] Take the reference stock solution containing methotrexate, methylparaben, ethylparaben, and p-hydroxybenzoic acid, and blank matrix medicinal liquid, and prepare accuracy solutions at the limit concentration levels of 50% - 200% according to the concentrations shown in Table 5. Prepare 3 portions in parallel for each accuracy level, inject samples respectively, and perform detection according to the method of Example 1. The test results are shown in Table 6.

[0195] Table 5 Concentrations of Samples Used in Accuracy Test

[0196]

[0197]

[0198] Table 6 Test Results of Accuracy Test

[0199]

[0200]

[0201] As can be seen from Tables 5 - 6, by using the method of Example 1, at the limit accuracy levels of 50% - 200%, the average recoveries of methotrexate, methylparaben, and ethylparaben are between 97.4% and 101.4%, and the maximum RSD of the recoveries is 1.5%, all within the standard requirements; the recovery of p-hydroxybenzoic acid is between 100.0% and 104.4%, and the RSD of the recovery is 2.4%, also within the standard requirements; indicating that the method of the present invention has good accuracy.

[0202] 5. Stability Test 1

[0203] The stability of the reference substance solution and the test solution used in the method of Example 1 was tested, and the test results are shown in Table 7.

[0204] Table 7 Detection Results of the Stability Test of the Reference Substance Solution and the Test Solution

[0205]

[0206] Note: The change rate of the content at 2d and 0h is the content at 2d / the content at 0h.

[0207] As can be seen from Table 7, by using the method of the present invention, it was found that the change rate of the reference substance solution and the test solution within 48 hours was 1.00 - 1.01, within the range of 0.98 - 1.02, indicating that the reference substance solution and the test solution remained stable for at least 48 hours.

[0208] 6. Stability Test 2

[0209] The stability of the test substance was tested by using the method of Example 1, and the test results are shown in Table 8. The test method is as follows: The test substance was divided into two batches, named finished product batch number 1 and finished product batch number 2, and the finished product batch number 1 and finished product batch number 2 were respectively subjected to stability tests under the conditions of 0 month, long-term (25°C ± 2°C; RH60% ± 5) for 6 months, and accelerated (40°C ± 2°C; RH65% ± 5) for 6 months.

[0210] Table 8 Detection Results of the Sample Stability Test

[0211]

[0212] Two parallel batches of the test substance were detected for methotrexate, p-hydroxybenzoic acid, methylparaben, and ethylparaben under different inspection conditions and different detection times. From the results in Table 8, it can be seen that the content of the target components of the test substance basically did not change after 6 months of long-term storage. At the same time, compared with 0 month of long-term, and the results trends of the two parallel batches were consistent and reasonable, which further verified the stability of the test substance.

[0213] In summary, the system suitability, specificity, precision, accuracy, etc. of the method of the present invention all meet the requirements, and it can effectively separate and accurately quantify methotrexate, methylparaben, ethylparaben, and p-hydroxybenzoic acid in methotrexate oral solution at the same time. The method has good specificity, excellent resolution, and good accuracy. In addition, the stability test for the short-term and long-term detection of the test substance shows that the test substance has high stability. The method of the present invention can be used for the daily detection of the content of methotrexate and preservatives (p-hydroxybenzoic acid, methylparaben, ethylparaben) in methotrexate oral solution, the detection during the preparation process of the test substance, etc.

[0214] The above are only embodiments of the present invention, and thus do not limit the patent scope of the present invention. Any equivalent transformation made using the content of the specification of the present invention, or directly or indirectly applied in related technical fields, shall similarly be included within the patent protection scope of the present invention.

Claims

1. A method for determining a methotrexate liquid preparation, characterized in that: For detecting methotrexate and preservatives, the determination method adopts liquid chromatography, and the conditions of the liquid chromatography include: Chromatographic column: octadecylsilane bonded silica gel column or column with equivalent performance; Mobile phase A: 0.08-0.12% v / v glacial acetic acid solution, mobile phase B: acetonitrile; The elution procedure is: 。 2. The measuring method according to claim 1, characterized in that: The mobile phase A is 0.09-0.11% v / v glacial acetic acid solution.

3. The measuring method according to claim 2, characterized in that The mobile phase A is 0.10% v / v glacial acetic acid solution.

4. The measuring method according to claim 2, characterized in that The elution procedure is: 。 5. The assay method according to any one of claims 1 to 4, characterized in that: The chromatographic conditions of the liquid chromatography also include: a detection wavelength of 249-259 nm.

6. The assay method according to any one of claims 1 to 4, characterized in that: The chromatographic conditions of the liquid chromatography also include: the injection volume is 15-25 μL.

7. The assay method according to any one of claims 1 to 4, characterized in that: The chromatographic conditions of the liquid chromatography also include: a chromatographic column temperature of 25-35° C.; and / or, The flow rate is 0.9-1.1 mL / min; and / or, The chromatographic column is Welch Ultimate XB-C18, 4.6*150mm, 3μm.

8. The assay method according to any one of claims 1 to 4, characterized in that: The temperature of the chromatographic column was 30° C., the flow rate was 1.0 mL / min, the injection volume was 20 μL, and the detection wavelength was 254 nm.

9. The measuring method according to any one of claims 1 to 4, characterized in that The preservative includes at least one of methylparaben, ethylparaben and p-hydroxybenzoic acid.

10. The determination method according to any one of claims 1 to 4, characterized in that: The methotrexate liquid preparation includes methotrexate oral solution.

Images