Method for detecting 5-hydroxymethylfurfural in compatibility test of tirofiban hydrochloride injection

Abstract

The invention belongs to the field of pharmaceutical analysis, and relates to a method for detecting 5-hydroxymethylfurfural in a compatibility test of tirofiban hydrochloride injection, which is characterized in that a high performance liquid chromatography method is adopted, octadecylsilane chemically bonded silica is used as a chromatographic column of a filler, water is used as a solvent, a 0.005-0.015 mol / L phosphate buffer solution with the pH value of 2.0-2.5 is used as a mobile phase A, and the concentration of 5-hydroxymethylfurfural in the compatibility test of tirofiban hydrochloride injection is detected through a chromatographic column. An organic solvent is used as a mobile phase B, isocratic elution is performed, a detector is an ultraviolet detector, the detection wavelength is 272-290 nm, and the column temperature is 25-35 DEG C. The detection method provided by the invention can effectively detect the content of 5-hydroxymethylfurfural in the compatibility test of the tirofiban hydrochloride injection, has the advantages of strong specificity, high sensitivity and accuracy, good repeatability and durability, simple operation and reliable result, and is suitable for the detection of 5-hydroxymethylfurfural in the compatibility test of the tirofiban hydrochloride injection.

Description

Technical Field

[0001] The invention belongs to the field of drug analysis, and particularly relates to an accurate quantitative detection method for 5-HMF in a compatibility test of tirofiban hydrochloride injection. Background Art

[0002] Tirofiban hydrochloride Chemical name: N-(butylsulfonyl)-O-[4-(4-piperidinyl)butyl]-L-tyrosine hydrochloride monohydrate, molecular formula: C 22 H 36 N 2 O 5 S·HCl·H 2 O, molecular weight: 495.08, structural formula:

[0003]

[0004] Tirofiban hydrochloride was developed by Merck in the United States and was first launched in the United States in May 1998. It has now been launched in many countries. Tirofiban hydrochloride is a highly effective and selective GPⅡb / Ⅲa receptor antagonist. Tirofiban hydrochloride injection is used to treat patients with unstable angina or non-Q wave myocardial infarction to prevent cardiac ischemic events. It is also suitable for patients with coronary ischemic syndrome undergoing coronary angioplasty or intracoronary plaque resection to prevent cardiac ischemic complications related to sudden occlusion of the treated coronary artery.

[0005] In recent years, due to the emergence of new drugs and the need for clinical treatment, tirofiban hydrochloride injection often needs to be used in combination with other injectable drugs, such as dopamine hydrochloride injection, epinephrine hydrochloride injection, lidocaine hydrochloride injection, midazolam injection, nitroglycerin injection, and potassium chloride injection. Tirofiban hydrochloride injection needs to be diluted with glucose or saline before being injected into the human body. 5-HMF is an aldehyde compound produced by the dehydration of monosaccharide compounds such as glucose under high temperature or weak acid conditions, which is harmful to human skeletal muscles and internal organs. Therefore, it is very necessary to control and detect 5-HMF in the compatibility experiment after tirofiban hydrochloride injection is diluted with glucose.

[0006] At present, the 2020 edition of the Chinese Pharmacopoeia includes the inspection method for 5-HMF "Chinese Pharmacopoeia 2020 Edition <Part II [Glucose Injection]>". The specific method is: Use UV-visible spectrophotometry (Chinese Pharmacopoeia 2020 Edition Part IV General Rules 0401), accurately measure approximately 1.0g of sample equivalent to glucose, place it in a 100ml volumetric flask, dilute to the scale with water, shake well, and measure at a wavelength of 284nm. The absorbance shall not be greater than 0.32.

[0007] In actual operation, it was found that the above-mentioned pharmacopoeia method had the following deficiencies: this detection method is a rough detection of 5-HMF in glucose, which is not suitable for quantitative and qualitative detection of 5-HMF in the compatibility test of tirofiban hydrochloride injection with glucose and other small injections, and it is difficult to accurately control the content and limit of 5-HMF in quality control.

[0008] Therefore, in order to accurately monitor the content of 5-HMF in tirofiban hydrochloride injection, a simple, specific and accurate HPLC analysis method is urgently needed to achieve qualitative and quantitative analysis of 5-HMF in injection compatibility tests. Summary of the invention

[0009] The purpose of the present invention is to establish a high performance liquid phase analysis method for determining 5-HMF in the compatibility of tirofiban hydrochloride injection, which can achieve qualitative and accurate quantitative analysis of 5-HMF in the compatibility of tirofiban hydrochloride injection. The method has strong specificity, high sensitivity and accuracy, good repeatability and durability, and stable method.

[0010] To achieve the purpose of the present invention, the inventors conducted a large number of experiments and finally obtained the following solution:

[0011] A method for detecting 5-HMF in a compatibility test of tirofiban hydrochloride injection using high performance liquid chromatography, wherein the chromatographic conditions of the high performance liquid chromatography are:

[0012] Chromatographic column: chromatographic column with octadecylsilane bonded silica gel as filler;

[0013] Mobile phase: composed of mobile phase A and mobile phase B, mobile phase A is phosphate buffer, mobile phase B is acetonitrile, methanol or one or more; wherein the concentration of the buffer substance in the buffer is 0.005 mol / L to 0.015 mol / L, and the pH is adjusted to 2.0 to 2.5;

[0014] Linear isocratic elution, the volume ratio of the mobile phase A to the mobile phase B is 70:30 to 73:27;

[0015] Column temperature: 30℃~40℃;

[0016] Flow rate: 0.8ml / min~1.2ml / min;

[0017] Detection wavelength: 272nm~290nm;

[0018] Injection volume: 30~70μl.

[0019] Preferably, in the above chromatographic conditions, the octadecylsilane bonded silica gel chromatographic column is a YMC-Triart C18, 4.6×250 mm, 5 μm or an Agela Venusil MP, 4.6×250 mm, 5 μm chromatographic column.

[0020] Preferably, in the above chromatographic conditions, mobile phase A buffer is potassium dihydrogen phosphate buffer.

[0021] Preferably, in the above chromatographic conditions, the concentration of potassium dihydrogen phosphate buffer is 0.01 mol / L, and phosphoric acid is used to adjust the pH of the solution to 2.3.

[0022] Preferably, in the above chromatographic conditions, mobile phase B is acetonitrile.

[0023] Preferably, the volume ratio of mobile phase A to mobile phase B in the above chromatographic conditions is 71:29.

[0024] Preferably, the column temperature in the above chromatographic conditions is 35°C.

[0025] Preferably, the flow rate of the mobile phase in the above chromatographic conditions is 1.0 ml / min.

[0026] Preferably, the detection wavelength in the above chromatographic conditions is 284 nm.

[0027] Preferably, the injection volume in the above chromatographic conditions is 50 μl.

[0028] Specifically, the liquid chromatography method of the present invention is:

[0029] Chromatographic column: chromatographic column with octadecylsilane bonded silica as filler (YMC-Triart C18, 4.6×250mm, 5μm);

[0030] Mobile phase: mobile phase A (0.01 mol / L potassium dihydrogen phosphate buffer solution, pH adjusted to 2.3 with phosphoric acid) - mobile phase B (acetonitrile) = 71:29, linear isocratic elution.

[0031] Column temperature: 35°C;

[0032] Flow rate: 1.0ml / min;

[0033] Detection wavelength: 284nm;

[0034] Injection volume: 50 μl

[0035] The method of the present invention can effectively separate and quantitatively determine 5-HMF in the compatibility test of tirofiban hydrochloride injection, and the specific operation steps are as follows:

[0036] a. Take an appropriate amount of 5-hydroxymethylfurfural reference substance, weigh it accurately, dissolve it in water and quantitatively dilute it to make a solution with a concentration of about 15μg / ml as the reference substance solution;

[0037] b. Dilute the tirofiban hydrochloride injection with glucose injection, and shake well with the corresponding injection to be used as the test solution;

[0038] c. Take 50 μl of the reference solution and the test solution and inject them into the high performance liquid chromatograph respectively, and complete the analysis and detection of 5-HMF in the compatibility experiment of tirofiban hydrochloride injection according to the following conditions;

[0039] The chromatographic conditions are:

[0040] Chromatographic column: chromatographic column with octadecylsilane bonded silica as filler (YMC-Triart C18, 4.6×250mm, 5μm);

[0041] Mobile phase: mobile phase A (0.01 mol / L potassium dihydrogen phosphate buffer solution, pH adjusted to 2.3 with phosphoric acid) - mobile phase B (acetonitrile) = 71:29, linear gradient elution.

[0042] Column temperature: 35°C;

[0043] Flow rate: 1.0ml / min;

[0044] Detection wavelength: 284nm;

[0045] Injection volume: 50 μl

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] The analytical detection method disclosed by the present invention can effectively detect the content of 5-HMF in the compatibility test of tirofiban hydrochloride injection, and the method has good repeatability and durability, low detection limit, simple operation, and stable and reliable results, so that the method can be used for quantitative control of 5-HMF in the compatibility test of tirofiban hydrochloride injection, and effectively guarantee the quality of the final product. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 HPLC chromatogram of blank solvent

[0049] Figure 2 HPLC chromatogram of mixed solution of tirofiban hydrochloride injection, glucose injection and 5-HMF

[0050] Figure 3 HPLC chromatogram of 5-hydroxymethylfurfural peak positioning solution

[0051] Figure 4 HPLC chromatogram of Tirofiban hydrochloride peak localization solution

[0052] Figure 5 HPLC chromatogram of glucose peak positioning solution

[0053] Figure 6 HPLC chromatogram of dopamine hydrochloride injection localization solution

[0054] Figure 7 HPLC chromatogram of adrenaline hydrochloride injection localization solution

[0055] Figure 8 HPLC chromatogram of lidocaine hydrochloride injection localization solution

[0056] Fig. 9 HPLC chromatogram of the localized solution of midazolam injection

[0057] Fig.10 HPLC chromatogram of nitroglycerin injection localization solution

[0058] Fig.11 HPLC chromatogram of potassium chloride injection localized solution

[0059] Fig.12 Quantitation limit solution HPLC spectrum

[0060] Fig.13 Detection limit solution HPLC spectrum

[0061] Fig.14 Example 2 HPLC chromatogram of 5-HMF solution

[0062] Fig.15 Example 2 HPLC chromatogram of mixed solution of tirofiban hydrochloride injection, glucose injection and 5-HMF

[0063] Fig.16 Comparative Example 1 Tirofiban Hydrochloride Injection HPLC Spectrum DETAILED DESCRIPTION

[0064] The following are specific embodiments of the present invention, which further describe the technical solutions of the present invention, but the protection scope of the present invention is not limited to these embodiments. Without departing from the above technical premise of the present invention, the corresponding replacement or modification made according to the common technical knowledge and conventional means in the field are all included in the present invention.

[0065] Example 1

[0066] 1. Instruments and conditions: Waters liquid chromatography system, 2998 detector, chromatographic column: YMC-Triart C18 (4.6×250 mm, 5 μm); 0.01 mol / L potassium dihydrogen phosphate buffer (adjusted to pH 2.3 with phosphoric acid)-acetonitrile (71:29) as the mobile phase, linear isocratic elution, column temperature 35°C, flow rate 1.0 ml / min, detection wavelength 284 nm, injection volume 50 μl.

[0067] 2. Test steps:

[0068] 2.1 Exclusivity

[0069] 1) Blank solvent interference test

[0070] Take an appropriate amount of water, filter, take the filtrate as the blank solvent, measure according to the above chromatographic conditions, record the chromatogram, and the HPLC spectrum is shown in Figure 1 .

[0071] Depend on Figure 1 It can be seen that there is no impurity peak in the blank solvent that interferes with the detection of 5-HMF.

[0072] 2) Interference test between tirofiban hydrochloride injection and glucose injection

[0073] After diluting the tirofiban hydrochloride injection with glucose injection, add the 5-HMF reference solution and mix well to obtain a mixed solution of tirofiban hydrochloride injection, glucose injection and 5-HMF. Measure under the above chromatographic conditions and record the chromatogram. The HPLC spectrum is shown in Figure 2 .

[0074] Depend on Figure 2 It can be seen that after the tirofiban hydrochloride injection was diluted with glucose and added to the mixed solution of the 5-HMF reference substance, there was no impurity peak that interfered with the detection of 5-HMF.

[0075] 2.2 Peak positioning test

[0076] The 5-HMF reference solution, tirofiban hydrochloride injection and the injection solution used in combination with tirofiban hydrochloride injection were used for positioning test.

[0077] Injection positioning solution for compatibility: Take appropriate amount of glucose injection, dopamine hydrochloride injection, epinephrine hydrochloride injection, lidocaine hydrochloride injection, midazolam injection, nitroglycerin injection, and potassium chloride injection, place them in 10ml volumetric flasks, dilute with water to the scale, and use them as the injection positioning solution. The results are shown in Table 1. HPLC spectrum is shown in Figure 3-11 .

[0078] Table 1 Localization results of 5-HMF, tirofiban hydrochloride injection and combined solutions

[0079] name Retention time (min) 5-HMF 3.532 Tirofiban Hydrochloride 11.402 glucose 5.928 Dopamine Hydrochloride 2.211 Epinephrine Hydrochloride 2.140 Lidocaine Hydrochloride 3.992 Midazolam 11.655 Nitroglycerin No UV absorption, no retention time Potassium chloride No UV absorption, no retention time

[0080] Depend on Figure 3-11 As shown in Table 1, tirofiban hydrochloride injection diluted with glucose and its combination with dopamine hydrochloride injection, epinephrine hydrochloride injection, lidocaine hydrochloride injection, midazolam injection, nitroglycerin injection and potassium chloride injection did not interfere with the detection of 5-HMF.

[0081] 2.3 Limit of quantification and limit of detection

[0082] 1) Quantitative limit solution: Take an appropriate amount of 5-HMF reference substance, accurately weigh it, dissolve it in water and quantitatively dilute it to make a solution with a concentration of about 0.015 μg / ml as the quantitative limit solution. Determine it according to the chromatographic conditions of Example 1, record the chromatogram, and the HPLC spectrum is shown in Fig.12 .

[0083] Depend on Fig.12 It can be seen that the retention time of 5-HMF is 3.547 min and the S / N is 12.97.

[0084] 2) Detection limit solution: Take an appropriate amount of 5-HMF reference substance, weigh it accurately, dissolve it in water and quantitatively dilute it to make a solution with a concentration of about 0.0045 μg / ml as the detection limit solution. Determine according to the chromatographic conditions of Example 1, record the chromatogram, and the HPLC spectrum is shown in Fig.13 .

[0085] Depend on Fig.13 It can be seen that the retention time of 5-HMF is 3.549 min and the S / N is 5.01.

[0086] 2.4 Linearity and range tests

[0087] Take an appropriate amount of 5-HMF reference substance, quantitatively dilute with water to prepare solutions of 0.015 μg / ml, 3.06 μg / ml, 7.65 μg / ml, 12.24 μg / ml, 15.30 μg / ml, 18.36 μg / ml, 22.95 μg / ml concentration series, inject respectively, measure according to the chromatographic conditions of implementation case 1, record the chromatogram, and obtain the linear equation. The results are shown in Table 2.

[0088] Table 2 5-HMF linearity and range test results

[0089]

[0090] It can be seen from Table 2 that the linear relationship between the concentration and the peak area of ​​5-HMF is good within the range of 0.015 to 22.95 μg / ml.

[0091] 2.5 Repeatability test

[0092] Take an appropriate amount of 5-HMF reference substance, quantitatively dilute with water to prepare solutions with concentrations of 7.65 μg / ml, 15.30 μg / ml, and 22.95 μg / ml, prepare 3 portions for each concentration, measure according to the chromatographic conditions of Example 1, record the chromatogram, measure the peak area, and calculate the relative standard deviation. The results are shown in Table 3.

[0093] Table 3 5-HMF precision test results

[0094]

[0095]

[0096] It can be seen from Table 3 that at low, medium and high concentrations, the RSD of the 5-HMF peak area was less than 2.0%, indicating that the method had good precision.

[0097] 2.6 Accuracy test

[0098] 4 ml of glucose injection was accurately measured and placed in a 10 ml volumetric flask, 5-HMF reference substance was accurately added, and the solution containing 7.65 μg / ml, 15.30 μg / ml and 22.95 μg / ml of 5-HMF reference substance was quantitatively diluted with water, respectively, as low, medium and high concentrations of test samples, and three copies of each concentration were prepared in parallel, and the chromatographic conditions of implementation case 1 were measured, the chromatogram was recorded, the peak area was measured, and the recovery rate was calculated by the external standard method. The results are shown in Table 4.

[0099] Table 4 5-HMF inspection accuracy test results

[0100]

[0101] It can be seen from Table 4 that this method has high accuracy.

[0102] 2.7 Intermediate precision test

[0103] Accurately measure 4 ml of glucose injection, place in a 10 ml volumetric flask, accurately add 5-HMF reference substance, quantitatively dilute with water to make a solution containing about 15.30 μg / ml of 5-HMF reference substance, and prepare 6 copies in parallel.

[0104] Two experimenters prepared each test solution and used different instruments to measure the samples. The measured amount of 5-HMF in the test solution was calculated by the external standard method. Each instrument measured 6 copies and calculated the standard deviation of the 12 measurement results. The results are shown in Table 5.

[0105] Table 5 5-HMF inspection intermediate precision test results

[0106]

[0107]

[0108] It can be seen from Table 5 that this method has good intermediate precision.

[0109] 2.8 Durability test

[0110] Durability test solution:

[0111] Durability test reference solution: Take an appropriate amount of 5-HMF reference substance and dilute it quantitatively with water to prepare a solution containing about 15.30 μg / ml as the reference solution.

[0112] Durability test test solution: Accurately measure 4 ml of tirofiban hydrochloride injection, place it in a 10 ml volumetric flask, add glucose injection to dilute to the scale, shake well, and use it as the test solution.

[0113] 1) Durability flow rate change

[0114] ①Instruments and conditions: Waters liquid chromatography system, 2998 detector, chromatographic column: YMC-Triart C18 (4.6×250mm, 5μm); 0.01mol / L potassium dihydrogen phosphate buffer (adjusted to pH 2.3 with phosphoric acid)-acetonitrile (71:29) as the mobile phase, linear isocratic elution, column temperature 35℃, flow rate 0.9ml / min, detection wavelength 284nm, injection volume 50μl.

[0115] Determination: Accurately measure 50 μl of each sample and inject it into the liquid chromatograph, record the chromatogram, and calculate the 5-HMF content detected in the test sample by the external standard method.

[0116] Under this condition, there was no impurity peak interfering with the detection of 5-HMF in the chromatogram of the test solution, and the 5-HMF content in the test sample was 0.0060%.

[0117] ②Instruments and conditions: Waters liquid chromatography system, 2998 detector, chromatographic column: YMC-Triart C18 (4.6×250mm, 5μm); 0.01mol / L potassium dihydrogen phosphate buffer (adjusted to pH 2.3 with phosphoric acid)-acetonitrile (71:29) as the mobile phase, linear isocratic elution, column temperature 35℃, flow rate 1.1ml / min, detection wavelength 284nm, injection volume 50μl.

[0118] Determination: Accurately measure 50 μl of each sample and inject it into the liquid chromatograph, record the chromatogram, and calculate the 5-HMF content detected in the test sample by the external standard method.

[0119] Under this condition, there was no impurity peak interfering with the detection of 5-HMF in the chromatogram of the test solution, and the 5-HMF content in the test sample was 0.0060%.

[0120] 2) Durability column temperature change

[0121] ①Instruments and conditions: Waters liquid chromatography system, 2998 detector, chromatographic column: YMC-Triart C18 (4.6×250mm, 5μm); 0.01mol / L potassium dihydrogen phosphate buffer (adjusted to pH 2.3 with phosphoric acid)-acetonitrile (71:29) as the mobile phase, linear isocratic elution, column temperature 30℃, flow rate 1.0ml / min, detection wavelength 284nm, injection volume 50μl.

[0122] Determination: Accurately measure 50 μl of each sample and inject it into the liquid chromatograph, record the chromatogram, and calculate the 5-HMF content detected in the test sample by the external standard method.

[0123] Under this condition, there was no impurity peak interfering with the detection of 5-HMF in the chromatogram of the test solution, and the 5-HMF content in the test sample was 0.0060%.

[0124] ②Instruments and conditions: Waters liquid chromatography system, 2998 detector, chromatographic column: YMC-Triart C18 (4.6×250mm, 5μm); 0.01mol / L potassium dihydrogen phosphate buffer (adjusted to pH 2.3 with phosphoric acid)-acetonitrile (71:29) as the mobile phase, linear isocratic elution, column temperature 40℃, flow rate 1.0ml / min, detection wavelength 284nm, injection volume 50μl.

[0125] Determination: Accurately measure 50 μl of each sample and inject it into the liquid chromatograph, record the chromatogram, and calculate the 5-HMF content detected in the test sample by the external standard method.

[0126] Under this condition, there was no impurity peak interfering with the detection of 5-HMF in the chromatogram of the test solution, and the 5-HMF content in the test sample was 0.0060%.

[0127] 2) Durability Changes in Mobile Phase Ratio

[0128] ①Instruments and conditions: Waters liquid chromatography system, 2998 detector, chromatographic column: YMC-Triart C18 (4.6×250mm, 5μm); 0.01mol / L potassium dihydrogen phosphate buffer (adjusted to pH 2.3 with phosphoric acid)-acetonitrile (70:30) as the mobile phase, linear isocratic elution, column temperature 35℃, flow rate 1.0ml / min, detection wavelength 284nm, injection volume 50μl.

[0129] Determination: Accurately measure 50 μl of each sample and inject it into the liquid chromatograph, record the chromatogram, and calculate the 5-HMF content detected in the test sample by the external standard method.

[0130] Under this condition, there was no impurity peak interfering with the detection of 5-HMF in the chromatogram of the test solution, and the 5-HMF content in the test sample was 0.0060%.

[0131] ②Instruments and conditions: Instruments and conditions: Waters liquid chromatography system, 2998 detector, chromatographic column: YMC-Triart C18 (4.6×250mm, 5μm); 0.01mol / L potassium dihydrogen phosphate buffer (adjusted to pH 2.3 with phosphoric acid)-acetonitrile (72:28) as the mobile phase, linear isocratic elution, column temperature 35℃, flow rate 1.0ml / min, detection wavelength 284nm, injection volume 50μl.

[0132] Determination: Accurately measure 50 μl of each sample and inject it into the liquid chromatograph, record the chromatogram, and calculate the 5-HMF content detected in the test sample by the external standard method.

[0133] Under this condition, there was no impurity peak interfering with the detection of 5-HMF in the chromatogram of the test solution, and the 5-HMF content in the test sample was 0.0060%.

[0134] 4) Durability of mobile phase buffer pH changes

[0135] ①Instruments and conditions: Waters liquid chromatography system, 2998 detector, chromatographic column: YMC-Triart C18 (4.6×250mm, 5μm); 0.01mol / L potassium dihydrogen phosphate buffer (adjusted to pH 2.5 with phosphoric acid)-acetonitrile (71:29) as the mobile phase, linear isocratic elution, column temperature 35℃, flow rate 1.0ml / min, detection wavelength 284nm, injection volume 50μl.

[0136] Determination: Accurately measure 50 μl of each sample and inject it into the liquid chromatograph, record the chromatogram, and calculate the 5-HMF content detected in the test sample by the external standard method.

[0137] Under this condition, there was no impurity peak interfering with the detection of 5-HMF in the chromatogram of the test solution, and the 5-HMF content in the test sample was 0.0055%.

[0138] ②Instruments and conditions: Waters liquid chromatography system, 2998 detector, chromatographic column: YMC-Triart C18 (4.6×250mm, 5μm); 0.01mol / L potassium dihydrogen phosphate buffer (adjusted to pH 2.1 with phosphoric acid)-acetonitrile (71:29) as the mobile phase, linear isocratic elution, column temperature 35℃, flow rate 1.0ml / min, detection wavelength 284nm, injection volume 50μl.

[0139] Determination: Accurately measure 50 μl of each sample and inject it into the liquid chromatograph, record the chromatogram, and calculate the 5-HMF content detected in the test sample by the external standard method.

[0140] Under this condition, there was no impurity peak interfering with the detection of 5-HMF in the chromatogram of the test solution, and the 5-HMF content in the test sample was 0.0054%.

[0141] The durability results are summarized in Table 6.

[0142] Table 6 5-HMF durability test results

[0143]

[0144]

[0145] It can be seen from Table 6 that this method has good durability.

[0146] Example 2

[0147] 1. Instruments and conditions: Waters liquid chromatography system, 2998 detector, chromatographic column: Agela Venusil MPC18 (4.6×250 mm, 5 μm); 0.01 mol / L potassium dihydrogen phosphate buffer (adjusted to pH 2.3 with phosphoric acid)-acetonitrile (71:29) as the mobile phase, linear isocratic elution, column temperature 35°C, flow rate 1.0 ml / min, detection wavelength 284 nm, injection volume 50 μl.

[0148] 2. Experimental steps:

[0149] Experiment: 1) Take an appropriate amount of 5-HMF reference substance, weigh it accurately, dissolve it in water and quantitatively dilute it to make a solution with a concentration of about 15μg / ml. Accurately measure 50μl and inject it into the liquid chromatograph, record the chromatogram, and the HPLC spectrum is shown in Fig.14 .

[0150] Depend on Fig.14 It can be seen that the retention time of 5-HMF is 3.555 min, and there is no impurity peak that interferes with the detection of 5-HMF.

[0151] 2) After diluting the tirofiban hydrochloride injection with glucose injection, add the 5-HMF reference solution and mix well to obtain a mixed solution of tirofiban hydrochloride injection, glucose injection and 5-HMF. Measure according to the above chromatographic conditions and record the chromatogram. The HPLC spectrum is shown in Fig.15 .

[0152] Depend on Fig.15 It can be seen that after the tirofiban hydrochloride injection was diluted with glucose and added to the mixed solution of the 5-HMF reference substance, there was no impurity peak that interfered with the detection of 5-HMF.

[0153] It can be seen from the above test results that the detection method of the present invention can effectively detect the content of 5-HMF in the compatibility experiment of tirofiban hydrochloride injection, and the blank solvent and the compatible injection have no interference in its detection. Therefore, this method can be used for the quantitative analysis of 5-HMF in tirofiban hydrochloride injection.

[0154] Comparative Example 1

[0155] 1. Instruments and conditions: The 5-HMF detection method and chromatographic column in the "Chinese Pharmacopoeia 2020 Edition <Part II [Metronidazole Glucose Injection]>" were adopted. Instruments and conditions: Waters liquid chromatography system, 2998 detector, chromatographic column: YMC-TriartC18 (4.6×250mm, 5μm); methanol-water (20:80) was used as the mobile phase; the detection wavelength was 284nm, and the injection volume was 20μl.

[0156] 2. Experimental steps:

[0157] test:

[0158] Take tirofiban hydrochloride injection, accurately measure 50 μl and inject it into the liquid chromatograph, record the chromatogram, and the HPLC spectrum is shown in Fig.16 .

[0159] Depend on Fig.16 It can be seen that tirofiban hydrochloride does not produce a peak, and this method cannot detect the content of 5-HMF in the compatibility test of tirofiban hydrochloride injection.

[0160] The experimental results show that the detection method of the present invention can effectively detect the content of 5-hydroxymethylfurfural in the compatibility test of tirofiban hydrochloride injection, and the method has strong specificity, high sensitivity and accuracy, good repeatability and durability, simple operation, and stable and reliable results.

[0161] The preferred embodiments are described, and relevant persons can obviously modify or appropriately change and combine the methods and applications described herein within the content, ideas and scope of the present invention to implement and apply the technology of the present invention. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.

Claims

1. A method for detecting 5-hydroxymethylfurfural (5-HMF) in a compatibility test of tirofiban hydrochloride injection, characterized in that: The detection method is to use high performance liquid chromatography, and the chromatographic conditions of the high performance liquid chromatography are: Chromatographic column: chromatographic column with octadecylsilane bonded silica gel as filler; Mobile phase: composed of mobile phase A and mobile phase B, mobile phase A is phosphate buffer, mobile phase B is acetonitrile, methanol or one or more; wherein the concentration of the buffer substance in the buffer is 0.005 mol / L to 0.015 mol / L, and the pH is adjusted to 2.0 to 2.

5.

2. The method according to claim 1, characterized in that The chromatographic conditions of the described high performance liquid chromatography are: Linear isocratic elution, the volume ratio of the mobile phase A to the mobile phase B is 70:30 to 73:27; Column temperature: 30℃~40℃; Flow rate: 0.8ml / min~1.2ml / min; Detection wavelength: 272nm~290nm; Injection volume: 30~70μl.

3. The detection method according to claim 1, characterized in that: The octadecylsilane bonded silica gel chromatographic column is a YMC-Triart C18 or Agela Venusil MP chromatographic column.

4. The detection method according to claim 2, characterized in that: The mobile phase A buffer is potassium dihydrogen phosphate buffer.

5. The detection method according to claim 4, characterized in that: The concentration of the potassium dihydrogen phosphate buffer solution is 0.01 mol / L, and phosphoric acid is used to adjust the pH of the solution to 2.

3.

6. The detection method according to claim 2, characterized in that: The mobile phase B is acetonitrile.

7. The detection method according to claim 2, characterized in that: The volume ratio of the mobile phase A to the mobile phase B is 71:

29.

8. The detection method according to claim 2, characterized in that: The column temperature is 35°C; the flow rate of the mobile phase is 1.0 ml / min.

9. The detection method according to claim 2, characterized in that: The detection wavelength is 284 nm; the injection volume is 50 μl.

10. The detection method according to any one of claims 1 to 9, characterized in that: The specific operation steps of the high performance liquid phase method are as follows: a. Take an appropriate amount of 5-hydroxymethylfurfural reference substance, weigh it accurately, dissolve it in water and quantitatively dilute it to make a solution with a concentration of about 15μg / ml as the reference substance solution; b. Dilute the tirofiban hydrochloride injection with glucose injection, and shake well with the corresponding injection to be used as the test solution; c. Take 50 μl of the reference solution and the test solution respectively and inject them into the high performance liquid chromatograph to complete the analysis and detection of 5-HMF in the compatibility experiment of tirofiban hydrochloride injection according to the following conditions.

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