Method for detecting content of 5-HMF (5-hydroxymethylfurfural) in injection by liquid chromatography and application of method

Abstract

The invention relates to the technical field of analytical chemistry, and particularly discloses a method for detecting the content of 5-HMF in an injection through liquid chromatography and application of the method. The method for detecting the content of 5-HMF in the injection through liquid chromatography comprises the following steps: preparing a reference solution, preparing a test solution, and carrying out liquid chromatography detection, liquid chromatography detection: the reference substance solution and the test solution are injected into a liquid chromatograph, a mobile phase gradient elution method is adopted for separation and detection, and the flow velocity of a mobile phase is 1.4-1.6 mL / min; the used chromatographic column is a chromatographic column taking phenyl bonded silica gel as a filler. According to the method for detecting the content of 5-HMF in the injection through liquid chromatography, the content of 5-HMF in a sample can be rapidly and accurately detected, the detection accuracy is good, the sensitivity is high, and the reliability is good.

Description

Technical Field

[0001] The present application relates to the technical field of analytical chemistry, and in particular to a method for detecting the content of 5-HMF in an injection by liquid chromatography and an application thereof. Background Art

[0002] Injections, especially those containing sugar, often contain the impurity 5-hydroxymethylfurfural (5-HMF), which has toxic side effects on human skeletal muscles and internal organs. Therefore, the pharmacopoeia has made clear provisions on the content of 5-HMF in sugar-containing injections. Therefore, in the production process of sugar-containing injections, accurate determination of the content of 5-HMF is of great significance for controlling the content of 5-hydroxymethylfurfural in the injection.

[0003] Liquid chromatography, as an efficient separation and analysis technology, has been widely used in the field of drug analysis. In order to accurately determine the 5-HMF content in the injection, the commonly used detection methods in the existing technology include but are not limited to using a chromatographic column with octadecyl bonded silica gel filler for liquid chromatography separation and detection, and adjusting the type of mobile phase, flow rate, column temperature and other parameters to improve the separation efficiency of the components to be tested. However, when the 5-HMF content in the sample to be tested is extremely low, the deviation of the test result is large, the repeatability of the test result is poor, and the accuracy is poor.

[0004] Therefore, how to accurately design the mobile phase type and the mobile phase ratio change curve to obtain a 5-HMF content detection method with good detection accuracy and high sensitivity is of great significance for the quality control of sugar-containing injections. Summary of the invention

[0005] In order to improve the accuracy and sensitivity of 5-HMF content detection, the present application provides a method for detecting 5-HMF content in an injection by liquid chromatography and its application.

[0006] In the first aspect, the present application provides a method for detecting the content of 5-HMF in an injection by liquid chromatography, using the following technical solution: A method for detecting the content of 5-HMF in an injection by liquid chromatography, comprising the following steps: preparing a reference solution, preparing a test solution, and detecting by liquid chromatography; Liquid chromatography detection: The reference solution and the test solution are injected into the liquid chromatograph, and the mobile phase gradient elution method is used for separation and detection. The flow rate of the mobile phase is 1.4-1.6mL / min; the chromatographic column used is a chromatographic column filled with phenyl bonded silica gel; The mobile phase gradient elution process is as follows: at 0 min, mobile phase A is 100% and mobile phase B is 0%; from 0 to 1.0 min, mobile phase A is linearly reduced to 75% and mobile phase B is linearly increased to 25%; from 1.0 to 2.5 min, mobile phase A is 75% and mobile phase B is 25%; from 2.5 to 12.5 min, mobile phase A is linearly reduced to 65% and mobile phase B is linearly increased to 35%; from 12.5 to 25 min, mobile phase A is 65% and mobile phase B is 35%; from 25 to 30.5 min, mobile phase A is linearly increased to 100% and mobile phase B is linearly reduced to 0%; at 30.5 min, mobile phase A is 100% and mobile phase B is 0%; mobile phase A and mobile phase B are both measured in volume percentages; The mobile phase A is a tetrabutylammonium hydrogen sulfate buffer with a concentration of 1.2-1.6 g / L, and the mobile phase B is methanol.

[0007] The present application screens the types of chromatographic columns and mobile phases, and designs the mobile phase gradient elution conditions, thereby developing a method for detecting the content of 5-HMF in an injection by liquid chromatography, which can quickly and accurately detect the content of 5-HMF in a sample, and has good detection accuracy, high sensitivity, and good reliability. The above detection method is used in the production of sugar-containing injections, which can effectively control the quality of sugar-containing injections and improve pharmaceutical safety. The detection limit of the detection method provided in the present application for detecting 5-HMF is 0.39 ng / mL, and the quantitative limit is 1.2 ng / mL.

[0008] In some embodiments, the flow rate of the mobile phase A can be 1.4-1.5 mL / min or 1.5-1.6 mL / min.

[0009] In a specific embodiment, the flow rate of the mobile phase A can also be 1.4 mL / min, 1.5 mL / min or 1.6 mL / min.

[0010] Optionally, the concentration of tetrabutylammonium bisulfate in the tetrabutylammonium bisulfate buffer is 1.3-1.5 g / L.

[0011] In some embodiments, the concentration of tetrabutylammonium bisulfate in the tetrabutylammonium bisulfate buffer may be 1.2-1.3 g / L, 1.2-1.4 g / L, 1.2-1.5 g / L, 1.2-1.6 g / L, 1.3-1.4 g / L, 1.3-1.5 g / L, 1.3-1.6 g / L, 1.4-1.5 g / L, 1.4-1.6 g / L, or 1.5-1.6 g / L.

[0012] In a specific embodiment, the concentration of tetrabutylammonium hydrogen sulfate in the tetrabutylammonium hydrogen sulfate buffer may be 1.2 g / L, 1.3 g / L, 1.4 g / L, 1.5 g / L or 1.6 g / L.

[0013] Optionally, the tetrabutylammonium hydrogen sulfate buffer solution is prepared by adding 1.2-1.6 g of tetrabutylammonium hydrogen sulfate, 0.8-1.4 g of potassium dihydrogen phosphate and 1.5-3 mL of phosphoric acid into water for dissolution, and then adding water to make the volume to 1000 mL to prepare the tetrabutylammonium hydrogen sulfate buffer solution.

[0014] Optionally, the specific steps of preparing the reference solution are: taking a 5-HMF reference substance, adding a diluent to dilute it, and obtaining a reference solution; the preparation method of the diluent is: dissolving anhydrous sodium sulfite in a tetrabutylammonium hydrogen sulfate buffer to obtain a diluent with an anhydrous sodium sulfite concentration of 18-22 mg / L.

[0015] Optionally, the test solution is a solution obtained by mixing moxifloxacin hydrochloride and sodium chloride injection and 5% glucose injection.

[0016] Optionally, the volume ratio of the moxifloxacin hydrochloride and sodium chloride injection to the 5% glucose injection is 10:1.

[0017] Optionally, in the liquid chromatography detection, the column temperature is 40-50°C.

[0018] Optionally, in the liquid chromatography detection, the detection wavelength is 284 nm.

[0019] Optionally, in the liquid chromatography detection, the chromatographic column is Kromasil 100-5-phenyl, 250mm×4.6mm, 5μm.

[0020] In a second aspect, the method for detecting the content of 5-HMF in an injection by liquid chromatography provided in the present application is used for detecting the content of 5-HMF in a solution obtained by combining glucose injection, moxifloxacin hydrochloride and sodium chloride injection with glucose injection.

[0021] In summary, this application has the following beneficial effects: 1. The present application provides a method for detecting the content of 5-HMF in an injection by liquid chromatography. The method uses a chromatographic column filled with phenyl bonded silica gel, and adopts a specific mobile phase and gradient elution procedure, so that the 5-HMF content in the sample can be detected quickly and accurately, and the detection has good accuracy, high sensitivity and good reliability.

[0022] 2. 5-HMF was detected using the method provided in the present application for detecting the content of 5-HMF in the injection by liquid chromatography. The detection limit of 5-HMF was 0.39 ng / mL and the quantification limit was 1.2 ng / mL.

[0023] 3. The present application further controls the concentration of tetrabutylammonium hydrogen sulfate in mobile phase A within the range of 1.3-1.5 g / L, and the obtained detection method has better repeatability, which can reduce the RSD value of the detection result to below 0.35%.

[0024] 4. The method for detecting the content of 5-HMF in an injection by liquid chromatography provided in the present application can be used in the production of sugar-containing injections, and can effectively control the quality of sugar-containing injections, thereby improving the safety of pharmaceutical use. DETAILED DESCRIPTION

[0025] The present application provides a method for detecting the content of 5-HMF in an injection by liquid chromatography, comprising the following steps: (1) preparing a reference solution: accurately weighing 10 mg of a 5-HMF reference substance, placing it in a 20 ml volumetric flask, adding methanol to dissolve and dilute to the mark, shaking well, and obtaining a 5-hydroxymethylfurfural stock solution; accurately measuring 1 mL of the 5-hydroxymethylfurfural stock solution, placing it in a 10 ml volumetric flask, adding a diluent to dilute to the mark, shaking well, and obtaining a reference solution; The preparation method of the diluent is as follows: dissolving anhydrous sodium sulfite in tetrabutylammonium hydrogen sulfate buffer to obtain a diluent with an anhydrous sodium sulfite concentration of 18-22 mg / L; The preparation method of the tetrabutylammonium hydrogen sulfate buffer is as follows: 1.2-1.6g of tetrabutylammonium hydrogen sulfate, 0.8-1.4g of potassium dihydrogen phosphate and 1.5-3mL of phosphoric acid are added into water to dissolve, and water is added to make the volume to 1000mL to prepare the tetrabutylammonium hydrogen sulfate buffer (2) Preparation of the test solution: Moxifloxacin hydrochloride sodium chloride injection and 5% glucose injection are mixed in a volume ratio of 10:1, and shaken to obtain the test solution.

[0026] (3) Liquid chromatography detection: 30 μL of the reference solution and the test solution were respectively taken and injected into the liquid chromatograph. The content of 5-HMF was calculated based on the peak areas of the standard solution and the test solution. The liquid chromatography detection conditions were as follows: Kromasil 100-5-phenyl column, specification 250 mm × 4.6 mm, 5 μm, flow rate 1.4-1.6 mL / min, injection volume 25 μL, column temperature 40-50 °C, detection wavelength 284 nm.

[0027] The mobile phase gradient elution program is as follows: at 0 min, mobile phase A is 100%, and mobile phase B is 0%; from 0 to 1.0 min, mobile phase A is linearly reduced to 75%, and mobile phase B is linearly increased to 25%; from 1.0 to 2.5 min, mobile phase A is 75%, and mobile phase B is 25%; from 2.5 to 12.5 min, mobile phase A is linearly reduced to 65%, and mobile phase B is linearly increased to 35%; from 12.5 to 25 min, mobile phase A is 65%, and mobile phase B is 35%; from 25 to 30.5 min, mobile phase A is linearly increased to 100%, and mobile phase B is linearly reduced to 0%; at 30.5 min, mobile phase A is 100%, and mobile phase B is 0%; mobile phase A and mobile phase B are both measured in volume percentage; The mobile phase A is a tetrabutylammonium hydrogen sulfate buffer with a concentration of 1.2-1.6 g / L, and the mobile phase B is methanol; further, the concentration of tetrabutylammonium hydrogen sulfate in the tetrabutylammonium hydrogen sulfate buffer is 1.3-1.5 g / L.

[0028] In the present application, the batch number of moxifloxacin hydrochloride sodium chloride injection is 2001131, the specification is 250mL, containing 0.4g moxifloxacin and 2.0g sodium chloride; the batch number of 5% glucose injection is 20051921, the specification is 250mL, containing 12.5g glucose; 5-HMF reference substance is purchased from China Food and Drug Inspection Institute; the CAS number of tetrabutylammonium hydrogen sulfate is 250-27-8; the CAS number of impurity A is 151213-15-9; the CAS number of impurity F is 1179992-99-4; the CAS number of impurity I is 172426-88-9; the CAS number of impurity J is 1322062-57-6; The structural formula of impurity B is: The structural formula of impurity C is: The structural formula of impurity D is: The structural formula of impurity E is: The raw materials, reagents, solvents, etc. used in this application can all be obtained commercially.

[0029] The present application is further described in detail below in conjunction with embodiments and performance testing experiments.

[0030] Example 1 Example 1 provides a method for detecting the content of 5-HMF in an injection by liquid chromatography, comprising the following steps: (1) preparing a reference solution: accurately weighing 10 mg of a 5-HMF reference substance, placing it in a 20 ml volumetric flask, adding methanol to dissolve and dilute to the mark, shaking well, and obtaining a 5-hydroxymethylfurfural stock solution; accurately measuring 1 mL of the 5-hydroxymethylfurfural stock solution, placing it in a 10 ml volumetric flask, adding a diluent to dilute to the mark, shaking well, and obtaining a reference solution; The preparation method of the diluent is as follows: dissolving anhydrous sodium sulfite in 1.4 g / L tetrabutylammonium hydrogen sulfate buffer to obtain a diluent with an anhydrous sodium sulfite concentration of 20 mg / L; The preparation method of the tetrabutylammonium hydrogen sulfate buffer is as follows: 1.4 g of tetrabutylammonium hydrogen sulfate, 1 g of potassium dihydrogen phosphate and 2 mL of phosphoric acid are added into water to dissolve, and water is added to make the volume to 1000 mL to prepare a 1.4 g / L tetrabutylammonium hydrogen sulfate buffer.

[0031] (2) Preparation of test solution: Mix moxifloxacin hydrochloride sodium chloride injection and 5% glucose injection in a volume ratio of 10:1 and shake well to obtain a test solution.

[0032] (3) Liquid chromatography detection: 30 μL of the reference solution and the test solution were respectively taken and injected into the liquid chromatograph. The content of 5-HMF was calculated based on the peak areas of the standard solution and the test solution. The liquid chromatography detection conditions were as follows: Kromasil 100-5-phenyl column, 250 mm × 4.6 mm, 5 μm, flow rate 1.5 mL / min, injection volume 25 μL, column temperature 45 °C, detection wavelength 284 nm.

[0033] The mobile phase gradient elution program is as follows: at 0 min, mobile phase A is 100%, and mobile phase B is 0%; from 0 to 1.0 min, mobile phase A is linearly reduced to 75%, and mobile phase B is linearly increased to 25%; from 1.0 to 2.5 min, mobile phase A is 75%, and mobile phase B is 25%; from 2.5 to 12.5 min, mobile phase A is linearly reduced to 65%, and mobile phase B is linearly increased to 35%; from 12.5 to 25 min, mobile phase A is 65%, and mobile phase B is 35%; from 25 to 30.5 min, mobile phase A is linearly increased to 100%, and mobile phase B is linearly reduced to 0%; at 30.5 min, mobile phase A is 100%, and mobile phase B is 0%; mobile phase A and mobile phase B are both measured in volume percentage; The mobile phase A is a tetrabutylammonium hydrogen sulfate buffer with a concentration of 1.4 g / L, and the mobile phase B is methanol.

[0034] 1. Repeatability Six test sample solutions (1-6) were prepared in parallel, and the reference sample solution and the test sample solution were injected into a liquid chromatograph. The test was performed according to the method provided in Example 1, and the chromatogram was recorded. The results are shown in Table 1 below.

[0035] Table 1 Repeatability test results According to the test results in Table 1, 6 groups of test solutions were tested in parallel using the test method provided in Example 1 of the present application, and the average value of the test results was 0.004535%, and the RSD value of the test results was 0.21%. Therefore, it is shown that the method for detecting the content of 5-HMF in injection by liquid chromatography provided in the present application can be used to determine the content of 5-HMF in sugar-containing injections, and the detection repeatability is good.

[0036] (II) Instrument precision The reference solution was accurately measured and injected into a liquid chromatograph, and the sample was injected six times continuously according to the chromatographic conditions of step (3) in Example 1, and the chromatogram was recorded. The test results are shown in Table 2 below.

[0037] Table 2 Precision test results Injection times Retention time (min) Peak area 1 3.304 126065 2 3.308 124407 3 3.305 122018 4 3.303 120874 5 3.298 119947 6 3.297 119751 RSD(%) 0.13 2.09 From the test results in Table 2, it can be seen that the reference solution was injected 6 times continuously, the RSD value of the retention time was 0.13% (≤1.0%), and the RSD value of the peak area was 2.09% (≤5.0%). Therefore, it is shown that the instrument precision of the 5-HMF content detection method provided in Example 1 is good.

[0038] (III) Limit of quantitation, detection limit and repeatability of limit of quantitation (1) Limit of quantification and limit of detection: A reference solution with a concentration of 0.05 mg / mL was prepared according to the method in Example 1, and then the reference solution was diluted step by step according to the peak height signal-to-noise ratio of the reference solution. When the peak height signal-to-noise ratio S / N ≥ 10:1, this concentration was used as the limit of quantification; when the peak height signal-to-noise ratio S / N ≥ 3:1, this concentration was used as the limit of detection. The results are shown in Table 3 below. (2) Repeatability of limit of quantitation: Six solutions of limit of quantitation (1-6) were prepared in parallel and tested under the liquid chromatography conditions of Example 1, and the RSD values ​​of the peak areas were calculated. The results are shown in Table 4 below.

[0039] Table 3 Test results of quantitation limit and detection limit Table 4 Test results of repeatability of quantitation limit name Peak area Quantitative limit solution-1 1540 Quantitative limit solution-2 1609 Quantitative limit solution-3 1563 Quantitative limit solution-4 1592 Quantitative limit solution-5 1576 Quantitative limit solution-6 1569 RSD(%) 1.52 From the test results in Table 3 and Table 4, it can be seen that the detection method provided in the present application has high sensitivity and good repeatability, and the detection limit of 5-HMF can be as low as 0.39 ng / mL, and the quantitative limit can be as low as 1.2 ng / mL.

[0040] (IV) Exclusivity (1) Solution preparation: Preparation of 5-hydroxymethylfurfural stock solution: accurately weigh 10 mg of 5-HMF reference substance, place in a 20 ml volumetric flask, dissolve in methanol and dilute to scale, shake well to obtain 5-hydroxymethylfurfural stock solution Preparation of moxifloxacin hydrochloride and impurity stock solutions: take about 2 mg of moxifloxacin hydrochloride reference substance, accurately weigh, place in a 25 ml volumetric flask, dissolve in diluent and dilute to scale, shake well to obtain moxifloxacin hydrochloride stock solution. Prepare impurity A, B, C, D, E, F, I, J stock solutions in the same way; Prepare each impurity localization solution: accurately measure 1 ml of impurity A stock solution, place it in a 10 ml volumetric flask, dilute to the mark with diluent, shake well, and obtain impurity A localization solution; prepare impurity B, C, D, E, F, I, and J localization solutions in the same way; Prepare mixed reference solution: accurately measure 1 ml each of impurity A, B, C, D, E, F, I, J localization solution, moxifloxacin hydrochloride stock solution and 5-hydroxymethylfurfural stock solution, place in a 10 ml volumetric flask, shake well to obtain a mixed reference solution.

[0041] (2) The mixed reference solution and the test solution were injected into a liquid chromatograph, and the detection was performed according to the liquid chromatography detection method provided in Example 1. The chromatogram was recorded. The results are shown in Table 5 below.

[0042] Table 5 Test results of specificity verification From the detection results in Table 5, it can be seen that in the chromatogram, the separation between the 5-HMF peak and the adjacent peak (impurity J peak) is 12.88 (>1.5). Therefore, it is shown that the method for detecting the content of 5-HMF in the injection by liquid chromatography provided in the present application is used to detect the solution obtained by combining moxifloxacin hydrochloride sodium chloride injection and glucose injection. Neither moxifloxacin nor other impurity peaks interfere with the detection of 5-HMF, indicating that the specificity of the detection method is good.

[0043] Embodiment 2-5 Examples 2-5 respectively provide a method for detecting the content of 5-HMF in an injection solution by liquid chromatography.

[0044] The difference between the above embodiment and embodiment 1 is that the concentration of mobile phase A is specifically shown in Table 6 below.

[0045] Table 6 Concentration of mobile phase A in the detection method provided in Examples 2-5 Example Tetrabutylammonium hydrogen sulfate concentration in mobile phase A (g / L) 2 1.2 3 1.3 4 1.5 5 1.6 Example 6 Example 6 provides a method for detecting the content of 5-HMF in an injection solution by liquid chromatography.

[0046] The difference between the above embodiment and embodiment 1 is that the diluent used to prepare the reference solution is 1.4 g / L tetrabutylammonium hydrogen sulfate buffer.

[0047] Example 7 Example 7 provides a method for detecting the content of 5-HMF in an injection solution by liquid chromatography.

[0048] The difference between the above embodiment and embodiment 1 is that in the liquid chromatography detection, the flow rate of the mobile phase is 1.4 mL / min.

[0049] Example 8 Example 8 provides a method for detecting the content of 5-HMF in an injection solution by liquid chromatography.

[0050] The difference between the above embodiment and embodiment 1 is that in the liquid chromatography detection, the flow rate of the mobile phase is 1.6 mL / min.

[0051] Comparative Example 1 Comparative Example 1 provides a method for detecting the content of 5-HMF in an injection solution by liquid chromatography.

[0052] The difference between the comparative example and Example 1 is that the concentration of tetrabutylammonium hydrogen sulfate in the mobile phase A is 1.0 g / L.

[0053] Comparative Example 2 Comparative Example 2 provides a method for detecting the content of 5-HMF in an injection solution by liquid chromatography.

[0054] The difference between the comparative example and Example 1 is that the concentration of tetrabutylammonium hydrogen sulfate in the mobile phase A is 1.8 g / L.

[0055] Comparative Example 3 Comparative Example 3 provides a method for detecting the content of 5-HMF in an injection solution by liquid chromatography.

[0056] The difference between the comparative example and Example 1 is that in the liquid chromatography detection, the flow rate of the mobile phase is 1.2 mL / min.

[0057] Comparative Example 4 Comparative Example 4 provides a method for detecting the content of 5-HMF in an injection solution by liquid chromatography.

[0058] The difference between the comparative example and Example 1 is that: during the liquid chromatography detection process, isocratic elution of the mobile phase is adopted; the ratio of the mobile phase is: 0.5 g / L tetrabutylammonium hydrogen sulfate buffer: methanol = 70:30.

[0059] Comparative Example 5 Comparative Example 5 provides a method for detecting the content of 5-HMF in an injection solution by liquid chromatography.

[0060] The difference between the comparative example and Example 1 is: the mobile phase gradient elution process.

[0061] The mobile phase gradient elution process of Comparative Example 2 is as follows: at 0-40min, mobile phase A is 70% and mobile phase B is 25%; at 40-60min, mobile phase A is linearly reduced to 40% and mobile phase B is linearly increased to 60%; at 60-70min, mobile phase A is 40% and mobile phase B is 60%; at 70-75min, mobile phase A is linearly increased to 75% and mobile phase B is linearly reduced to 25%; at 75-95min, mobile phase A is 75% and mobile phase B is 25%; mobile phase A and mobile phase B are both expressed in volume percentage; mobile phase A is a tetrabutylammonium hydrogen sulfate buffer with a concentration of 1.4 g / L, and mobile phase B is methanol.

[0062] Test results Prepare a mixed reference solution and a test solution (6 portions in parallel) respectively, and then inject the mixed reference solution and the test solution into a liquid chromatograph, and detect according to the liquid chromatography detection method provided in Examples 1-8 and Comparative Examples 1-5, respectively. The 5-HMF content in the test solution is obtained according to the chromatogram, and the average value and RSD value of the test results obtained by each method, as well as the separation between the 5-HMF peak and the adjacent peak (impurity J) are taken. The results are shown in Table 7 below.

[0063] Table 7 Test results obtained by the methods provided in Examples 1-8 and Comparative Examples 1-5 According to the test results in Table 7, the test solution was detected by the method provided in Examples 1-8, and the 5-HMF content in the obtained test solution was 0.0045117-0.004535%, the RSD value was 0.27-0.50%, and the separation between the 5-HMF peak and the adjacent peak in the detection spectrum was 11.58-12.04 (>1.5); therefore, it is shown that the method for detecting the 5-HMF content in the injection by liquid chromatography provided in the present application has high detection accuracy, good repeatability and good separation, and can be used to detect the 5-HMF content in the sugar-containing injection to achieve effective quality control.

[0064] The test results of Examples 1-5 and Comparative Examples 1-2 show that in the method of Examples 1-5, a tetrabutylammonium hydrogen sulfate buffer solution with a concentration of 1.2-1.6 g / L of tetrabutylammonium hydrogen sulfate is used as mobile phase A, and the 5-HMF content in the test solution obtained by the test is 0.0045267-0.004535%, the RSD value is 0.27-0.50% (≤0.5%), and the separation between the 5-HMF peak and the adjacent peak in the test spectrum is The results are shown in Table 1. The results show that the content of 5-HMF in the sample solution is 0.0044817-0.0045%, the RSD value is 0.74-0.93%, and the separation degree between the 5-HMF peak and the adjacent peak in the detection spectrum is only 1.35-1.44. It is explained that the present application uses a tetrabutylammonium hydrogen sulfate buffer solution with a concentration of 1.2-1.6 g / L as mobile phase A and methanol as mobile phase B, and the obtained detection method has good repeatability and good separation; further comparison shows that the RSD value of the 5-HMF content in the test solution obtained by detection in Example 1 and Example 3-4 is 0.27-0.32% (≤0.35%), indicating that the present application further controls the concentration of tetrabutylammonium hydrogen sulfate in mobile phase A within the range of 1.3-1.5 g / L, and the obtained detection method has better repeatability.

[0065] It can be seen from the test results of Example 1 and Example 6 that Example 1 uses anhydrous sodium sulfite and 1.4g / L tetrabutylammonium hydrogen sulfate buffer as a diluent to prepare the reference solution, and the RSD value of the test result obtained is 0.27%; while Example 6 uses 1.4g / L tetrabutylammonium hydrogen sulfate buffer as a diluent to prepare the reference solution, and the RSD value of the test result obtained is 0.43%. Therefore, it is explained that the present application further uses a solution prepared by anhydrous sodium sulfite and tetrabutylammonium hydrogen sulfate buffer as a diluent to prepare the reference solution, which can make the test repeatability better.

[0066] From the test results of Example 1, Examples 7-8, and Comparative Example 3, it can be seen that the methods of Example 1 and Examples 7-8 control the flow rate of the mobile phase at 1.4-1.6 mL / min, and the 5-HMF content in the test solution obtained by detection is 0.0045183-0.004535%, the RSD value is 0.27-0.43%, and the separation degree between the 5-HMF peak and the adjacent peaks in the detection spectrum is 11.72-11.97; while the method of Comparative Example 3 controls the flow rate of the mobile phase at 1.2 mL / min, the 5-HMF content in the test solution obtained by detection is 0.004425%, the RSD value is 4.33%, and the separation degree between the 5-HMF peak and the adjacent peaks in the detection spectrum is only 1.16. Therefore, it is shown that the present application controls the flow rate of the mobile phase in the liquid chromatography detection method within the range of 1.4-1.6 mL / min, which can effectively separate 5-HMF in the test solution from other substances, thereby accurately detecting the 5-HMF content in the test solution, and the detection repeatability is good.

[0067] The method provided in Comparative Example 4 was used to detect the test solution. The symmetry of the 5-HMF peak in the obtained detection spectrum was poor, and the 5-HMF content in the test solution obtained by the detection was quite different. Therefore, it is explained that the isocratic elution method provided in Comparative Example 4 has poor reproducibility for the results of 5-HMF, and it is difficult to achieve accurate detection of the 5-HMF content.

[0068] The test solution was detected by the method provided in Comparative Example 5. The baseline in the obtained detection spectrum was not stable, the 5-HMF peak had obvious tailing, the detection result repeatability was poor, and the separation between the peaks in the detection spectrum was only 1.21, indicating that the mobile phase elution procedure provided in Comparative Example 5 could not achieve stable and accurate detection of the 5-HMF content in the solution obtained by combining moxifloxacin hydrochloride and sodium chloride injection with glucose injection.

[0069] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for detecting the content of 5-HMF in an injection by liquid chromatography, characterized in that: The following steps are involved: Prepare reference solution, prepare test solution, and perform liquid chromatography detection; Liquid chromatography detection: The reference solution and the test solution are injected into the liquid chromatograph, and the mobile phase gradient elution method is used for separation and detection. The flow rate of the mobile phase is 1.4-1.6mL / min; the chromatographic column used is a chromatographic column filled with phenyl bonded silica gel; The mobile phase gradient elution process is as follows: at 0 min, mobile phase A is 100%, and mobile phase B is 0%; from 0 to 1.0 min, mobile phase A is linearly reduced to 75%, and mobile phase B is linearly increased to 25%; from 1.0 to 2.5 min, mobile phase A is 75%, and mobile phase B is 25%; from 2.5 to 12.5 min, mobile phase A is linearly reduced to 65%, and mobile phase B is linearly increased to 35%; from 12.5 to 25 min, mobile phase A is 65%, and mobile phase B is 35%; from 25 to 30.5 min, mobile phase A is linearly increased to 100%, and mobile phase B is linearly reduced to 0%; at 30.5 min, mobile phase A is 100%, and mobile phase B is 0%; mobile phase A and mobile phase B are both measured in volume percentages; The mobile phase A is a tetrabutylammonium hydrogen sulfate buffer with a concentration of 1.2-1.6 g / L, and the mobile phase B is methanol.

2. The method for detecting 5-HMF content in injection by liquid chromatography according to claim 1, characterized in that: The concentration of tetrabutylammonium hydrogen sulfate in the tetrabutylammonium hydrogen sulfate buffer is 1.3-1.5 g / L.

3. The method for detecting 5-HMF content in injection by liquid chromatography according to claim 1, characterized in that: The preparation method of the tetrabutylammonium hydrogen sulfate buffer is as follows: 1.2-1.6 g of tetrabutylammonium hydrogen sulfate, 0.8-1.4 g of potassium dihydrogen phosphate and 1.5-3 mL of phosphoric acid are added into water to dissolve, and water is added to make the volume to 1000 mL to prepare the tetrabutylammonium hydrogen sulfate buffer.

4. The method for detecting 5-HMF content in injection by liquid chromatography according to claim 1, characterized in that: The specific steps of preparing the reference solution are: taking a 5-HMF reference substance, adding a diluent to dilute it, and obtaining a reference solution; The preparation method of the diluent is as follows: dissolving anhydrous sodium sulfite in tetrabutylammonium hydrogen sulfate buffer to obtain a diluent with an anhydrous sodium sulfite concentration of 18-22 mg / L.

5. The method for detecting the content of 5-HMF in injection by liquid chromatography according to claim 1, characterized in that: The test solution is a solution obtained by mixing moxifloxacin hydrochloride and sodium chloride injection and 5% glucose injection.

6. The method for detecting the content of 5-HMF in injection by liquid chromatography according to claim 5, characterized in that: The volume ratio of the moxifloxacin hydrochloride and sodium chloride injection to the 5% glucose injection is 10:

1.

7. The method for detecting the content of 5-HMF in injection by liquid chromatography according to claim 1, characterized in that: In the liquid chromatography detection, the column temperature is 40-50°C.

8. The method for detecting the content of 5-HMF in injection by liquid chromatography according to claim 1, characterized in that: In the liquid chromatography detection, the detection wavelength is 284 nm.

9. The method for detecting the content of 5-HMF in injection by liquid chromatography according to claim 1, characterized in that: In the liquid chromatography detection, the chromatographic column is Kromasil 100-5-phenyl, 250mm×4.6mm, 5μm.

10. Application of the method for detecting the content of 5-HMF in an injection by liquid chromatography according to any one of claims 1 to 9 in detecting the content of 5-HMF in a solution obtained by combining glucose injection, moxifloxacin hydrochloride and sodium chloride injection with glucose injection.

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