Detection method and kit for determining edetate in liquid preparation

Abstract

The invention relates to the field of medicine analysis and detection, in particular to a detection method and a kit for determining edetate in a liquid preparation. According to the detection method, liquid chromatography is adopted, and a mobile phase contains a derivatization reagent of edetate. The method effectively solves the problem of complex detection operation in the prior art, and has the advantages of strong specificity, wide linear range, good precision and high accuracy, and the stability of the sample to be detected is not influenced by the detection method and is only determined by the stability of the sample, so that the method is suitable for large-scale popularization and application. The content of edetate disodium in liquid preparations such as eye drops and injections can be reliably and effectively measured.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical analysis and detection, and in particular to a detection method and a kit for determining edetate in a liquid preparation. Background Art

[0002] Edetate Disodium (EDTA), also known as ethylenediaminetetraacetic acid disodium salt dihydrate (chemical formula C 10 H 14 Disodium EDTA (N2Na2O8·2H2O), also known as disodium EDTA, is a common organic compound, typically appearing as a white or off-white crystalline powder. As a hexadentate ligand, it can form stable, water-soluble complexes with alkaline earth metals and heavy metal ions, making it widely used as a metal ion complexing agent, acting as a chelating agent in pharmaceutical formulations, cosmetics, and food. Due to its safety, disodium EDTA is listed as a GRAS (Generally Recognized as Safe) substance. As an excipient, it is included in the FDA's Inactive Ingredients Guide (covering dosage forms such as inhalers, injections, ophthalmic preparations, capsules, tablets, solutions, suspensions, syrups, rectal preparations, topical preparations, and vaginal preparations) as well as relevant UK regulations, and can be used in both non-parenteral and parenteral formulations.

[0003] The addition of edetate disodium during the drug production process is intended to regulate the impact of metal ions on drug quality (especially liquid preparations). As an approved pharmaceutical excipient, the presence of edetate disodium in drug prescriptions is compliant. As national drug review standards continue to improve, the control of excipients in drugs has become increasingly stringent. According to the "Technical Guidelines for Pharmaceutical Research of Chemical Generic Drug Solution Eye Drops", the content of functional excipients needs to be quantitatively monitored throughout the shelf life. Therefore, the control of edetate disodium limits has become one of the key means to improve drug quality and a core indicator for quality evaluation.

[0004] Although several methods exist for detecting edetate disodium in pharmaceutical preparations, the mainstream approaches rely on either semi-quantitative titration or the use of copper- or iron-containing salt solutions to derivatize the edetate disodium in the sample before measurement. These methods generally suffer from drawbacks such as poor accuracy and cumbersome operation. Furthermore, liquid chromatography, after derivatization with copper- or iron-containing salt solutions, typically requires the use of tetrabutylammonium hydroxide as the mobile phase. However, tetrabutylammonium hydroxide is highly corrosive and significantly irritating and corrosive to the skin, eyes, and mucous membranes. Inhalation of its solution can also cause laryngeal or bronchial inflammation, spasms, and even lead to occupational health hazards such as chemical pneumonia and pulmonary edema.

[0005] For example, in the "HPLC Determination of Edetate Disodium in Loratadine Syrup," published by Ke Yuehong et al. in 2022, the sample must first be treated with a derivatization reagent before being detected in a mobile phase containing tetrabutylammonium hydroxide, which has the drawbacks mentioned above. Furthermore, this method only exhibits good linearity within the range of 12.56 to 37.68 μg / ml, limiting its practicality. Frequent dilution or concentration is required for high- or low-concentration samples, further increasing operational errors and workload.

[0006] Similarly, in the "HPLC Derivatization Method for the Determination of Edetate Disodium in Clindamycin Phosphate Injection," published by Chen Xi et al. in 2025, the sample was also pre-treated with a derivatization reagent. While this method exhibited good linearity within the 2-100 μg / ml range, it was still limited in its ability to detect higher concentrations of edetate disodium. Summary of the Invention

[0007] The first object of the present invention is to provide a method for determining edetate in a liquid preparation, which adopts liquid chromatography, and the mobile phase contains a derivatization reagent of edetate.

[0008] The present invention also provides a kit for determining edetate in a liquid preparation, which provides a mobile phase through a combination of one or more reagents, and the mobile phase is as described above.

[0009] The method provided by the present invention effectively solves the problem of complex detection operation in the prior art. The method has the advantages of strong specificity, wide linear range, good precision and high accuracy. In addition, the stability of the sample to be tested is not affected by the detection method, but is only determined by the stability of the sample itself. Therefore, the method can reliably realize the effective determination of the content of disodium edetate in liquid preparations such as eye drops and injections. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0011] Figure 1 This is the linear range test result in Example 1 of the present invention.

[0012] Figure 2 It is the blank solution chromatogram in Example 2 of the present invention.

[0013] Figure 3 The chromatogram of the reference substance solution in Example 2 of the present invention is shown in FIG.

[0014] Figure 4 This is a chromatogram of the test solution in Example 2 of the present invention.

[0015] Figure 5 3 is the blank solution chromatogram in Example 3 of the present invention.

[0016] Figure 6 The chromatogram of the reference substance solution in Example 3 of the present invention is shown in FIG.

[0017] Figure 7 This is a chromatogram of the test solution in Example 3 of the present invention. DETAILED DESCRIPTION

[0018] The following describes specific embodiments of the present invention in detail. It should be understood that the specific embodiments described herein are intended only to illustrate and explain the present invention and are not intended to limit the present invention. Those skilled in the art may make various modifications and variations to the present invention without departing from the scope or spirit of the present invention. For example, features illustrated or described as part of one embodiment may be used in another embodiment to produce further embodiments.

[0019] Unless otherwise indicated, all terms (including technical and scientific terms) used to disclose the present invention have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. By way of further guidance, the following definitions are provided to better understand the teachings of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0020] The terms "and / or", "or / and", and "and / or" used herein include any one of two or more related listed items, and also include any and all combinations of the related listed items, wherein the arbitrary and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical and" and also undoubtedly includes technical solutions connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B.

[0021] As used herein, the terms "comprising," "including," and "comprising" are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps.

[0022] The recitation of numerical ranges herein by endpoints includes all numbers and fractions subsumed within the range, as well as the recited endpoints.

[0023] Numerical values ​​used in this invention include fluctuations within a certain range. For example, fluctuations within a certain precision range are permitted. For example, a value of 1% may be allowed to fluctuate within ±0.05%. For larger values ​​or values ​​that do not require overly precise control, greater fluctuations are also permitted. For example, a value of 75% may be allowed to fluctuate within ±1%, ±2%, ±5%, and so on.

[0024] In the present invention, descriptions such as "plurality" and "multiple" refer to quantities greater than or equal to 2 unless otherwise specified.

[0025] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0026] In the present invention, “preferred”, “better”, “more preferred” and “suitable” are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of the present invention.

[0027] In the present invention, the terms "optionally," "optional," "optionally," "optionally," "optional," and "optional" mean optional or dispensable, i.e., they refer to either option being selected from two parallel options: "with" or "without." If multiple "optional" or "optional" terms appear in a technical solution, unless otherwise specified and there are no contradictions or constraints, each "optional" or "optional" term is independent of the others.

[0028] In the present invention, the term "edetate" refers to salt derivatives of ethylenediaminetetraacetic acid (EDTA).

[0029] In the present invention, the term "liquid preparation" refers to a type of preparation formed by dispersing or dissolving a drug in a liquid state in a suitable solvent (water, organic solvent, etc.).

[0030] In the present invention, the term "derivative reagent of edetate" refers to a reagent that can change the structure of edetate through a chemical reaction to generate a derivative with a specific analytical signal for easy detection.

[0031] The present invention first provides a detection method for determining edetate in a liquid preparation, which adopts liquid chromatography, and the mobile phase contains a derivatization reagent of edetate.

[0032] The present invention finds that by adding a derivatization reagent for edetate to the mobile phase, the detection of edetate in a liquid preparation can be achieved more simply and efficiently, avoiding the shortcomings of the prior art of cumbersome operation (which also requires an additional derivatization pretreatment step), and has the advantages of simple operation and time saving.

[0033] In some embodiments, the detection method does not include a step of performing pre-derivative treatment on the sample.

[0034] In some embodiments, the detection method does not include any pre-treatment steps for the sample.

[0035] The detection method of the present invention is based on the common characteristics of edetate in liquid phase systems. Through systematic optimization of the derivatization method of edetate (especially edetate disodium) (and optionally the mobile phase system), the applicability of the method is ensured to be independent of the physical form or formulation differences of specific dosage forms. In some embodiments, the detection method of the present invention is applicable to aqueous solutions (such as eye drops or injections), suspensions containing suspended solids, emulsions, and other liquid preparations containing edetate in flowable forms.

[0036] In some embodiments, the liquid preparation is an eye drop or an injection. The present invention does not specifically limit other components in the eye drop or injection formulation, and is applicable to the detection of edetate, especially edetate disodium, in eye drops or injections of different formulations.

[0037] In some embodiments, the edetate salt is selected from one or more of edetate disodium, edetate calcium sodium, edetate sodium, and edetate tetrasodium.

[0038] In some specific embodiments, the edetate is edetate disodium. The method of the present invention has a better detection effect when applied to the detection of edetate disodium.

[0039] In some embodiments, the derivatization reagent contains Cu 2+ and / or Fe 3+ .

[0040] In some embodiments, the derivatization reagent contains Cu 2+ .

[0041] In some embodiments, the derivatization reagent contains Fe 3+ .

[0042] In some embodiments, the derivatization reagent contains Cu 2+ and Fe 3+ .

[0043] In some embodiments, the derivatization reagent contains one or more selected from copper sulfate, copper acetate, copper chloride, copper nitrate, ferric chloride, ferric sulfate, ferric nitrate, and ferric acetate.

[0044] In some embodiments, the derivatization reagent contains copper nitrate and / or ferric chloride.

[0045] In some embodiments, the mobile phase contains 5 to 30 mmol / L of Cu 2+ and / or Fe 3+ When the derivatization reagent is used in the mobile phase at this dosage, the detection effect of edetate in liquid preparations (especially eye drops) is better.

[0046] As an example, in some embodiments, the mobile phase contains the following concentrations of Cu 2+ and / or Fe 3+ : 5mmol / L, 8mmol / L, 10mmol / L, 11mmol / L, 12mmol / L, 13mmol / L, 14mmol / L, 15mmol / L, 16mmol / L, 17mmol / L, 18mmol / L, 19mmol / L, 20mmol / L, 22mmol / L, 25mmol / L, 27mmol / L, 30mmol / L, or any value within the range of 5-30mmol / L.

[0047] In some embodiments, the mobile phase contains 70-80 vol% (e.g., 70 vol%, 71 vol%, 72 vol%, 73 vol%, 74 vol%, 75 vol%, 76 vol%, 77 vol%, 78 vol%, 79 vol%, 80 vol%, or any value within the range of 70-80 vol%) of nitrate buffer, and the nitrate buffer contains 20±5 mmol / L (e.g., 15 mmol / L, 16 mmol / L, 17 mmol / L, 18 mmol / L, 19 mmol / L, 20 mmol / L, 21 mmol / L, 22 mmol / L, 23 mmol / L, 24 mmol / L, 25 mmol / L, or any value within the range of 20±5 mmol / L) of Cu. 2+ and / or Fe 3+ , 51±10mmol / L (such as 41mmol / L, 43mmol / L, 45mmol / L, 47mmol / L, 49mmol / L, 50mmol / L, 51mmol / L, 52mmol / L, 53mmol / L, 55mmol / L, 57mmol / L, 59mmol / L, 61mmol / L, or any value within the range of 51±10mmol / L)- and H of 11±2mmol / L (e.g., 9mmol / L, 9.5mmol / L, 10mmol / L, 10.5mmol / L, 11mmol / L, 11.5mmol / L, 12mmol / L, 12.5mmol / L, 13mmol / L, or any value within the range of 11±2mmol / L) + By adding the above-mentioned nitrate buffer to the mobile phase, the detection effect of edetate in liquid preparations is better.

[0048] In order to further optimize the detection effect of edetate in liquid preparations, the present invention further optimizes other components in the mobile phase and obtains the following implementation scheme:

[0049] In some embodiments, the mobile phase further contains 20-30 vol% acetonitrile.

[0050] In some embodiments, the mobile phase further contains acetonitrile in an amount of 20 vol%, 21 vol%, 22 vol%, 23 vol%, 24 vol%, 25 vol%, 26 vol%, 27 vol%, 28 vol%, 29 vol%, 30 vol%, or any value within the range of 20 to 30 vol%.

[0051] In some embodiments, the mobile phase further contains 0.05-0.15 vol% trifluoroacetic acid.

[0052] In some embodiments, the mobile phase further contains trifluoroacetic acid in the following amounts: 0.05 vol%, 0.06 vol%, 0.07 vol%, 0.08 vol%, 0.09 vol%, 0.10 vol%, 0.11 vol%, 0.12 vol%, 0.13 vol%, 0.14 vol%, 0.15 vol%, or any value within the range of 0.05 to 0.15 vol%.

[0053] In some embodiments, the mobile phase further contains 20-30 vol% acetonitrile and 0.05-0.15 vol% trifluoroacetic acid.

[0054] In some embodiments, the mobile phase contains nitrate buffer, acetonitrile and trifluoroacetic acid in a volume ratio of (70-80):(20-30):0.1.

[0055] As an example, in some embodiments, the mobile phase comprises nitrate buffer, acetonitrile, and trifluoroacetic acid in a volume ratio of 70:30:0.1. In some embodiments, the mobile phase comprises nitrate buffer, acetonitrile, and trifluoroacetic acid in a volume ratio of 80:20:0.1. In some embodiments, the mobile phase comprises nitrate buffer, acetonitrile, and trifluoroacetic acid in a volume ratio of 77:23:0.1. In some embodiments, the mobile phase comprises nitrate buffer, acetonitrile, and trifluoroacetic acid in a volume ratio of 73:27:0.1.

[0056] In some preferred embodiments, the mobile phase contains nitrate buffer, acetonitrile and trifluoroacetic acid in a volume ratio of 75:25:0.1.

[0057] In some embodiments, the mobile phase does not contain tetrabutylammonium hydroxide.

[0058] In some embodiments, an anion exchange chromatography column is used as a filler.

[0059] The present invention does not particularly limit the specific column type of the anion exchange chromatography column. Anion exchange chromatography columns obtained from commercial sources by those skilled in the art in combination with common sense can achieve the detection effect of the present invention. As an example, in some specific embodiments, the anion exchange column can be selected from the following column types: strong anion exchange columns (quaternary ammonium type) such as AS11-HC, AS19, and AS23 of the Thermo Fisher Scientific IonPac series, A Supp 4-250 and A Supp 7-250 of the Mettler-Toledo Metrosep series, and Agilent ZORBAX SAX, etc. Such columns use quaternary ammonium stationary phases to achieve efficient separation of EDTA polyvalent anions; weak anion exchange columns (tertiary amine type) such as Waters IC Pak AX, Shimadzu Shim-pack SAX, etc.; polymer matrix columns such as Hamilton PRP-X100 and Thermo Fisher Scientific IonPac NG1, etc. In some embodiments, the anion exchange column can be a Waters Spherisorb SAX 4.6 mm×250 mm, 5 μm or a chromatography column of equivalent performance.

[0060] In some embodiments, the flow rate of the mobile phase is controlled to be 1.0±0.2 mL / min.

[0061] As an example, in some embodiments, the flow rate of the mobile phase is controlled to be 0.8 mL / min, 0.9 mL / min, 1.0 mL / min, 1.1 mL / min, 1.2 mL / min, or 1.0±0.2 mL / min.

[0062] In some embodiments, the column temperature is controlled at 35±5°C, preferably 35±3°C.

[0063] As an example, in some embodiments, the column temperature is controlled to be 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, or any value within the range of 35±5°C.

[0064] In some embodiments, the injection volume is 10 to 50 μL.

[0065] As an example, in some embodiments, the injection volume is 10 μL, 15 μL, 20 μL, 25 μL, 30 μL, 35 μL, 40 μL, 45 μL, 50 μL, or 10 to 50 μL.

[0066] In some embodiments, the detection wavelength is 250 ± 3 nm.

[0067] As an example, in some embodiments, the detection wavelength is 247 nm, 248 nm, 249 nm, 250 nm, 251 nm, 252 nm, 253 nm, or any value within the range of 250 ± 3 nm.

[0068] By setting the parameters according to the above scheme during the detection, the detection effect of the chelate with ultraviolet signal derived in real time in the present invention can be further improved.

[0069] In some embodiments, the detection method of the present invention is used to determine the presence and / or content of edetate in a liquid preparation.

[0070] In some specific embodiments, the detection method further comprises: using a reference solution of known concentration, drawing a standard curve with concentration as the horizontal axis and peak area as the vertical axis; and then calculating the concentration of edetate in the sample to be tested using the peak area of ​​edetate detected and the standard curve.

[0071] During specific implementation, those skilled in the art may combine the above-mentioned embodiments with common sense to obtain more embodiments of the detection method of the present invention.

[0072] The present invention also provides a kit for determining edetate in a liquid preparation, which provides a mobile phase through a combination of one or more reagents, and the mobile phase is as described in any of the above embodiments.

[0073] In some embodiments, the kit further comprises an anion exchange chromatography column.

[0074] In some embodiments, the kit further comprises a reference substance (standard substance) and / or a quality control substance (QC sample).

[0075] In some embodiments, the kit further comprises a column flushing reagent.

[0076] Example

[0077] The embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. For experimental methods in the following examples where specific conditions are not specified, reference is made to the guidance provided in the present invention, and may also be made to experimental manuals or conventional conditions in the art, other experimental methods known in the art, or conditions recommended by the manufacturer.

[0078] In the following examples, the anion exchange column is a Waters Spherisorb SAX 4.6 mm×250 mm, 5 μm. In specific implementations, those skilled in the art may also use other chromatographic columns with equivalent performance.

[0079] In order to facilitate comparison, copper nitrate is used as the derivatization reagent in the following examples. 2+ and / or Fe 3+ All derivatization reagents can achieve similar or equivalent detection effects.

[0080] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.

[0081] Example 1 Detection method and methodological investigation

[0082] This embodiment provides a method for determining edetate in a liquid preparation, wherein:

[0083] The chromatographic conditions were as follows: an anion exchange column was used as the filler; the mobile phase was nitrate buffer (3.75 g of copper nitrate was dissolved in 11 mmol / L nitric acid and diluted to 1000 mL)-acetonitrile-trifluoroacetic acid in a volume ratio of 75:25:0.1; the flow rate was 1.0 mL / min; the detection wavelength was 250 nm; the column temperature was 35°C; and the injection volume was 10 μL.

[0084] The following is a methodological review of the detection method:

[0085] 1.1 Linear range:

[0086] Accurately measure the edetate disodium reference substance, dissolve it in water and dilute it to the concentration shown in Table 1, and prepare standard solutions of different concentrations. Measure according to the above-mentioned "chromatographic conditions" and record the peak area of ​​each standard solution. The results are shown in Table 1 below. Linear regression is performed with the edetate disodium concentration as the horizontal axis (x) and the peak area as the vertical axis (y). The results are as follows Figure 1 As shown in the results, the method of the present invention has a good linear relationship in the range of 0.05006~0.5006mg / mL, R 2 is 0.9991.

[0087] Table 1

[0088] Concentration (mg / mL) Peak area 0.05006 345.486 0.1251 684.890 0.2503 1356.930 0.3004 1637.314 0.3755 2074.548 0.5006 2696.873

[0089] 1.2 Accuracy:

[0090] Accuracy was assessed using recovery results. Three concentration levels, 50%, 100%, and 150% of the limit concentration (i.e., the limit concentration of edetate disodium in eye drops), were used as accuracy solutions. Recovery was calculated using the ratio of the measured value to the theoretical value, expressed as a percentage (%): recovery (%) = (measured value - background value) / theoretical value × 100%. A blank excipient solution (i.e., eye drops without edetate disodium) and a limit-level edetate disodium reference substance were accurately measured and dissolved to prepare a 100% limit concentration accuracy solution. The 100% limit concentration accuracy solution was diluted with water to a 50% limit concentration to obtain a 50% limit concentration accuracy solution. The 100% limit concentration accuracy solution was concentrated to a 150% limit concentration to obtain a 150% limit concentration accuracy solution. Accuracy solutions with limited concentrations of 50%, 100%, and 150% were measured according to the above-mentioned "chromatographic conditions", the peak area of ​​each solution was recorded, and the recovery rate of each solution was calculated based on the peak area. The results are shown in Table 2.

[0091] Table 2

[0092]

[0093] 1.3 Repeatability and Reproducibility:

[0094] Repeatability Study: Prepare six replicates of the 100% accuracy solution as described in "1.2 Accuracy." Measure the solution using the aforementioned "Chromatographic Conditions." Record the peak area for each solution and calculate the RSD of the recovery based on the peak area. The calculated RSD is 0.2% (n = 6).

[0095] Reproducibility Study: On different days, different analysts prepared test samples using the 100% accuracy solution defined under "1.2 Accuracy." Repeatability testing was performed on different laboratory instruments using different chromatographic columns. Measurements were made under the aforementioned "Chromatographic Conditions." The peak area of ​​each solution was recorded, and the RSD of the recoveries for the 12 solutions was calculated based on the peak area. The calculated RSD was 0.2% (n=12).

[0096] Example 2 Detection of the content of disodium edetate in eye drops

[0097] Test solution: Take this product and shake well. The pH of the preparation has been confirmed to be 8.4.

[0098] Reference solution: Take an appropriate amount of edetate disodium reference substance, accurately weigh it, dissolve it in water and dilute it to make a solution containing approximately 0.21 mg per 1 mL, namely STD1 and STD2.

[0099] Blank solution: water.

[0100] Chromatographic conditions: an anion exchange column was used as the filler; the mobile phase was nitrate buffer (3.75 g of copper nitrate was dissolved in 11 mmol / L nitric acid and diluted to 1000 mL)-acetonitrile-trifluoroacetic acid in a volume ratio of 75:25:0.1; the flow rate was 1.0 mL / min; the detection wavelength was 250 nm; the column temperature was 35°C; and the injection volume was 10 μL.

[0101] Determination method: Accurately measure the test solution, reference solution and blank solution, inject them into liquid chromatograph respectively, and record the chromatogram.

[0102] Results: As shown in Tables 3 to 4 and Figures 2-4 As shown, Figure 2 is the blank solution chromatogram, Figure 3 is the chromatogram of the reference solution. Figure 4 The chromatogram of the test solution.

[0103] Table 3 Test results of reference solution

[0104]

[0105] Table 4 Test results of test solution

[0106]

[0107] Example 3 Detection of the content of disodium edetate in eye drops

[0108] Test solution: Take this product and shake it well, and use it directly as the test solution. The pH of this preparation has been confirmed to be 5.9.

[0109] Reference solution: Accurately weigh an appropriate amount of edetate disodium reference substance, dissolve it in water and dilute it to make a solution containing approximately 0.25 mg of edetate disodium per 1 mL, namely STD1 and STD2.

[0110] Blank solution: water.

[0111] Chromatographic conditions: an anion exchange column was used as the filler; the mobile phase was nitrate buffer (3.75 g of copper nitrate was dissolved in 11 mmol / L nitric acid and diluted to 1000 mL)-acetonitrile-trifluoroacetic acid in a volume ratio of 75:25:0.1; the flow rate was 1.0 mL / min; the detection wavelength was 250 nm; the column temperature was 35°C; the injection volume was 10 μL, and the run time was 10 minutes.

[0112] Determination method: Accurately measure the test solution, reference solution and blank solution, inject them into liquid chromatograph respectively, and record the chromatogram.

[0113] Results: As shown in Tables 5-6 and Figures 5-7 As shown, Figure 5 is the blank solution chromatogram, Figure 6 is the chromatogram of the reference solution. Figure 7 The chromatogram of the test solution is shown in Table 5. The retest of STD1 shows that the peak area is not significantly different from the first test, indicating that the solution is relatively stable.

[0114] Table 5 Test results of reference solution

[0115]

[0116]

[0117] Table 6 Test results of test solution

[0118]

[0119] Figures 2 to 7 The chromatogram shows that the test solution has a main peak with a retention time consistent with that of the edetate disodium reference substance, and is well separated from adjacent impurity peaks. Meanwhile, the blank solution chromatogram has no interfering peak at this position. This shows that the method of the present invention can effectively detect edetate disodium from eye drops.

[0120] Example 4

[0121] The only difference between this embodiment and embodiment 1 is that the Cu 2+ The concentration was adjusted to 5 mmol / L and detected according to the method of the present invention. The theoretical EDTA content of the test sample was 0.25 mg / mL, and the actual detection result was 0.251 mg / mL.

[0122] Example 5

[0123] The only difference between this embodiment and embodiment 1 is that the Cu 2+ The concentration was adjusted to 30 mmol / L and detected according to the method of the present invention. The theoretical EDTA content of the test sample was 0.25 mg / mL, and the actual detection result was 0.250 mg / mL.

[0124] The above-described embodiments merely illustrate several implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that variations and modifications are possible without departing from the spirit of the present invention, and all such variations and modifications fall within the scope of the present invention.

Claims

1. A method for determining edetate in a liquid preparation, which uses liquid chromatography and the mobile phase contains a derivatization reagent for edetate.

2. The method for detecting edetate in a liquid preparation according to claim 1, wherein The liquid preparation is eye drops or injection.

3. The method for detecting edetate in a liquid preparation according to claim 1 or 2, wherein: The edetate salt is selected from one or more of edetate disodium, edetate calcium sodium, edetate sodium, and edetate tetrasodium.

4. The method for detecting edetate in a liquid preparation according to any one of claims 1 to 3, wherein: The derivatization reagent contains Cu 2+ and / or Fe 3+ .

5. The method for detecting edetate in a liquid preparation according to claim 4, wherein: The derivatization reagent contains one or more selected from copper sulfate, copper acetate, copper chloride, copper nitrate, ferric chloride, ferric sulfate, ferric nitrate, and ferric acetate.

6. The method for detecting edetate in a liquid preparation according to claim 4 or 5, wherein: The mobile phase contains 5 to 30 mmol / L of Cu 2+ and / or Fe 3+ .

7. The method for detecting edetate in a liquid preparation according to claim 4, wherein: The mobile phase contains 70-80 vol% nitrate buffer, and the nitrate buffer contains 20±5 mmol / L Cu 2+ and / or Fe 3+ , 51±10mmol / L NO3 - and 11±2mmol / L H + .

8. The method for detecting edetate in a liquid preparation according to any one of claims 1 to 7, wherein The mobile phase also contains 20-30 vol% of acetonitrile and 0.05-0.15 vol% of trifluoroacetic acid.

9. The method for detecting edetate in a liquid preparation according to claim 7, wherein: The mobile phase contains nitrate buffer, acetonitrile and trifluoroacetic acid in a volume ratio of (70-80):(20-30):0.

1.

10. The method for detecting edetate in a liquid preparation according to any one of claims 1 to 9, wherein Anion exchange chromatography column was used as filler.

11. The method for detecting edetate in a liquid preparation according to any one of claims 1 to 10, wherein: The flow rate of the mobile phase was controlled at 1.0 ± 0.2 mL / min.

12. The method for detecting edetate in a liquid preparation according to any one of claims 1 to 11, wherein The column temperature was controlled at 35±5℃.

13. The method for detecting edetate in a liquid preparation according to any one of claims 1 to 12, wherein: The injection volume is 10-50 μL.

14. The method for detecting edetate in a liquid preparation according to any one of claims 1 to 13, wherein: The detection wavelength is 250±3nm.

15. A kit for determining edetate in a liquid preparation, which provides a mobile phase by a combination of one or more reagents, wherein the mobile phase is as described in any one of claims 1 and 3 to 9. 16 . The kit for determining edetate in a liquid preparation according to claim 15 , further comprising an anion exchange chromatography column.

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