Method for detecting 5-hydroxymethylfurfural in calcium gluconate injection by liquid chromatography

Abstract

The present invention relates to the field of pharmaceutical analysis technology, and discloses a method for detecting 5-hydroxymethylfurfural in calcium gluconate injection by liquid chromatography, comprising the following steps: S110: preparation of a test solution: taking an appropriate amount of a sample, adding a first solvent to dissolve and dilute, filtering through a 0.22 μm filter membrane, and taking a subsequent filtrate as a test solution; S120: preparation of a reference solution: taking an appropriate amount of a 5-hydroxymethylfurfural reference substance, accurately weighing, adding water to dissolve and quantitatively diluting to prepare a solution containing about 20 μg per 1 ml; S130: injecting the test solution and the reference solution into a liquid chromatograph respectively, recording a chromatogram, and calculating the content of 5-hydroxymethylfurfural by peak area according to an external standard method. The determination method of the present invention has strong specificity, good method precision, high sensitivity, and the solution is stable within 48 hours. Conclusion: The established method is sensitive and rapid, and the results are accurate and reliable, and can be used as a method for checking 5-hydroxymethylfurfural.

Description

Technical Field

[0001] The invention relates to the technical field of medical analysis, in particular to a method for detecting 5-hydroxymethylfurfural in calcium gluconate injection by liquid chromatography. Background Art

[0002] Calcium gluconate injection is used for acute hypocalcemia, magnesium poisoning, and fluoride poisoning. The main ingredient of this product is calcium gluconate, and its chemical name is D-gluconate calcium salt monohydrate. The auxiliary materials are calcium gluconate, hydrochloric acid or sodium hydroxide, and water for injection. The specification is 10ml: 1g (calculated as C12H22CaO14). Among them, glucose as the starting material may remain in the calcium gluconate. Glucose can be isomerized to produce fructose, and fructose is dehydrated under acidic conditions to produce 5-hydroxymethylfurfural. 5-Hydroxymethylfurfural (5-Hydroxymethylfurfural), chemical name is 5-hydroxymethylfurfural, and the chemical structure is:

[0003]

[0004] Fructose is dehydrated under acidic conditions to produce 5-hydroxymethylfurfural. The reaction equation is:

[0005]

[0006] Studies have shown that a certain dose of 5-hydroxymethylfurfural, after being absorbed by the body, will have adverse effects on organs such as the liver, kidneys, and heart, and will also irritate the eye mucosa, upper respiratory tract mucosa, etc.

[0007] At present, there is no national standard for the method of detecting 5-hydroxymethylfurfural in calcium gluconate injection by liquid chromatography. Therefore, it is very important to propose a method for detecting 5-hydroxymethylfurfural with strong specificity and high sensitivity in the present invention. Summary of the invention

[0008] The invention provides a method for detecting 5-hydroxymethylfurfural in calcium gluconate injection, thereby achieving quality control of the preparation.

[0009] The present application embodiment provides a method for detecting 5-hydroxymethylfurfural in calcium gluconate injection by liquid chromatography, comprising the following steps:

[0010] S110: Preparation of test solution: Take an appropriate amount of sample, add the first solvent to dissolve and dilute, filter through a 0.22 μm filter membrane, and take the filtrate as the test solution;

[0011] S120: Preparation of reference solution: Take an appropriate amount of 5-hydroxymethylfurfural reference, weigh accurately, dissolve in water and quantitatively dilute to make a solution containing about 20 μg per 1 ml;

[0012] S130: Inject the test solution and the reference solution into a liquid chromatograph respectively, record the chromatogram, and calculate the content of 5-hydroxymethylfurfural by peak area according to the external standard method.

[0013] Preferably, in S1, the first solvent is a mixture of 0.1% formic acid solution and 0.1% formic acid acetonitrile solution.

[0014] Preferably, in S1, the volume ratio of the formic acid solution to the formic acid-acetonitrile solution is 90:10.

[0015] Preferably, in S130, octadecylsilane bonded silica gel is used as a filler, and the detection wavelength is 284 nm.

[0016] Preferably, in S130, the flow rate is 1.0 ml / min, and the retention time of 5-hydroxymethylfurfural is 5-7 min.

[0017] Preferably, in S130, the column temperature is 30°C, and the injection volume is 20 μl.

[0018] Preferably, in S130, the mobile phase is 0.05 mol / L potassium dihydrogen phosphate solution (pH adjusted to 3.0 with phosphoric acid)-methanol (90:10).

[0019] Preferably, in S130, if there is a chromatographic peak in the test solution with the same retention time as 5-hydroxymethylfurfural, the 5-hydroxymethylfurfural content shall not exceed 0.02% of the labeled amount of calcium gluconate by calculating the peak area according to the external standard method;

[0020] The calculation formula is as follows:

[0021]

[0022] Where:

[0023] As, Ai: the peak area of ​​5-hydroxymethylfurfural in the reference solution and the peak area of ​​5-hydroxymethylfurfural in the test solution, respectively;

[0024] Ws: weight of reference substance, mg;

[0025] Vs: dilution volume of the reference substance, ml;

[0026] Ps: reference substance content, %;

[0027] M: Specification of the preparation (calculated as calcium gluconate), mg / ml.

[0028] Compared with the prior art, the technical solution of this application has the following beneficial effects:

[0029] The determination method of the present invention has strong specificity, good method precision, high sensitivity, and the solution is stable within 48 hours. Conclusion: The established method is sensitive and rapid, and the results are accurate and reliable, and can be used as a method for detecting 5-hydroxymethylfurfural. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0031] Figure 1 It is a linear relationship graph. DETAILED DESCRIPTION

[0032] The present invention is further described below in conjunction with the accompanying drawings and embodiments, but they are not intended to limit the present invention.

[0033] The method described in this patent for determining 5-hydroxymethylfurfural in calcium gluconate injection by high performance liquid chromatography uses octadecylsilane bonded silica gel as a filler; potassium dihydrogen phosphate solution (pH adjusted with phosphoric acid)-methanol as a mobile phase; the pH value of the mobile phase aqueous phase is 2.8-3.2; the ratio of the mobile phase components is 87:13-93:7; the flow rate is 0.8-1.2 ml per minute; the column temperature is 25-35°C; the detection wavelength is 284 nm; and the injection volume is 20 μl.

[0034] The flow rate was 1.0 ml per minute; the column temperature was 30°C; the detection wavelength was 284 nm; the injection volume was 20 μl, and the mobile phase was 0.05 mol / L potassium dihydrogen phosphate solution (pH adjusted to 3.0 with phosphoric acid)-methanol (90:10).

[0035] Analytical method confirmation test:

[0036] 1. Exclusivity

[0037] (1) Test solution: this product;

[0038] (2) Reference solution: Take an appropriate amount of 5-hydroxymethylfurfural reference substance, weigh it accurately, dissolve it in water and quantitatively dilute it to make a solution containing about 20 μg per 1 ml.

[0039] (3) Blank excipient solution: blank excipient (without calcium gluconate).

[0040] Accurately measure 20 μl of blank solvent, blank excipient solution, test solution and reference solution, inject them into liquid chromatography, and record the chromatogram. From the chromatogram, it can be seen that the blank solvent and blank excipient do not interfere with the detection of 5-hydroxymethylfurfural, indicating that this method has good specificity.

[0041] 2. System precision

[0042] Take the reference solution under the specificity item and measure it 6 times continuously, and record the chromatogram. The results are shown in Table 1.

[0043] Table 1 shows the system precision test results:

[0044]

[0045] The test results show that the RSD of the peak area of ​​5-hydroxymethylfurfural is less than 5.0%, and the RSD of the retention time is less than 2.0%, indicating that the system has good precision and can meet the test requirements.

[0046] 3. Limit of Quantitation

[0047] Take the reference solution under the specificity item and dilute it step by step until the signal-to-noise ratio (S / N) is about 10, which corresponds to the quantitative limit concentration. Accurately measure 20 μl of the above solution, inject it 6 times in parallel, inject it into the liquid chromatograph, and record the chromatogram. The results are shown in Table 2.

[0048] Table 2 shows the results of the quantitation limit test:

[0049] 4. Detection Limit

[0050] Take the quantitative limit solution and dilute it step by step until the signal-to-noise ratio (S / N) is about 3, which corresponds to the detection limit concentration. Accurately measure 20 μl, inject it into the liquid chromatograph, and record the chromatogram. The results are shown in Table 3.

[0051] Table 3 shows the detection limit test results:

[0052]

[0053] 5. Linearity and range

[0054] Take 5-hydroxymethylfurfural reference substance, dilute to limit concentration LOQ~200% solution, accurately measure 20μl of each solution, inject into liquid chromatograph, record chromatogram. In the concentration range of LOQ~200%, linear regression analysis is performed with concentration as horizontal axis (X) and peak area as vertical axis (Y). The results are shown in Table 4.

[0055] Table 4 shows the linear results:

[0056] In the range of 0.004 μg / ml to 43.133 μg / ml, the linear equation was: y=162.7196x-2.8098, r was 1.0000, the residual sum of squares (RSS) was 893.0989, and the percentage of the residual standard deviation relative to the 100% response value was 0.46%.

[0057] 6. Repeatability

[0058] Take an appropriate amount of 5-hydroxymethylfurfural reference substance, accurately weigh it, dissolve it in this product and quantitatively dilute it to make a solution containing about 20μg per 1ml, as the spiked test solution, and prepare 6 copies in parallel. Take an appropriate amount of 5-hydroxymethylfurfural reference substance, accurately weigh it, dissolve it in water and quantitatively dilute it to make a solution containing about 20μg per 1ml, as the reference solution. Accurately measure 20μl of each of the spiked test solution and the reference solution, inject them into the liquid chromatograph, and record the chromatogram. The results are shown in Table 5.

[0059] Table 5 shows the results of the repeatability test:

[0060]

[0061] The test results show that when 6 samples were measured in parallel, the RSD of 5-hydroxymethylfurfural content was less than 10.0%, indicating that the method has good repeatability.

[0062] 7. Intermediate precision

[0063] Another tester takes an appropriate amount of 5-hydroxymethylfurfural reference substance, weighs it accurately, dissolves it in water and dilutes it quantitatively to make a solution containing about 20μg per 1ml as the spiked test solution, and prepares 6 portions in parallel. Take an appropriate amount of 5-hydroxymethylfurfural reference substance, weigh it accurately, dissolve it in water and dilute it quantitatively to make a solution containing about 20μg per 1ml as the reference solution. Accurately measure 20μl of each spiked test solution and reference solution, inject them into the liquid chromatograph, and record the chromatogram. The results are shown in Table 6.

[0064] Table 6 shows the results of the intermediate precision experiment:

[0065]

[0066] The test results show that: this method was used to measure 12 samples in parallel, and the RSD of 5-hydroxymethylfurfural content was less than 10.0%, indicating that this method has good intermediate precision.

[0067] 8. Solution stability

[0068] Take an appropriate amount of 5-hydroxymethylfurfural reference substance, accurately weigh it, dissolve it in this product and quantitatively dilute it to make a solution containing about 20μg per 1ml as the spiked test solution. Take an appropriate amount of 5-hydroxymethylfurfural reference substance, accurately weigh it, dissolve it in water and quantitatively dilute it to make a solution containing about 20μg per 1ml as the reference solution. Place it at room temperature, and accurately measure 20μl of the reference solution and the spiked test solution at 0h, 4h, 8h, 12h, 24h, 36h, and 48h, respectively, inject it into the liquid chromatograph, and record the chromatogram. The results are shown in Tables 7 and 8.

[0069] Table 7 shows the stability results of the reference solution:

[0070] Table 8 shows the stability results of the spiked test solution.

[0071]

[0072] Conclusion: Under room temperature conditions, the RSD of the peak areas of the reference solution and the spiked test solution was less than 5.0%, and they were stable after being placed for 48 hours.

[0073] The specific embodiments described above provide a series of detailed descriptions of the technical problems, technical solutions and beneficial effects solved by the present invention. However, the description is only for the specific embodiments of the present invention, and any equivalent embodiments or changes that do not deviate from the technical basis, principle and spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting 5-hydroxymethylfurfural in calcium gluconate injection by liquid chromatography, characterized in that: The following steps are involved: S110: Preparation of test solution: Take an appropriate amount of sample, add the first solvent to dissolve and dilute, filter through a 0.22 μm filter membrane, and take the filtrate as the test solution; S120: Preparation of reference solution: Take an appropriate amount of 5-hydroxymethylfurfural reference, weigh accurately, dissolve in water and quantitatively dilute to make a solution containing about 20 μg per 1 ml; S130: Inject the test solution and the reference solution into a liquid chromatograph respectively, record the chromatogram, and calculate the content of 5-hydroxymethylfurfural by peak area according to the external standard method.

2. The method for detecting 5-hydroxymethylfurfural in calcium gluconate injection by liquid chromatography according to claim 1, characterized in that: In S1, the first solvent is a mixture of 0.1% formic acid solution and 0.1% formic acid acetonitrile solution.

3. The method for detecting 5-hydroxymethylfurfural of calcium gluconate injection by liquid chromatography according to claim 2, characterized in that: In S1, the volume ratio of the formic acid solution to the formic acid acetonitrile solution is 90:

10.

4. The method for detecting 5-hydroxymethylfurfural in calcium gluconate injection by liquid chromatography according to claim 1, characterized in that: In the S130, octadecylsilane bonded silica gel is used as a filler, and the detection wavelength is 284 nm.

5. The method for detecting 5-hydroxymethylfurfural in calcium gluconate injection by liquid chromatography according to claim 1, characterized in that: In the S130, the flow rate is 1.0 ml / min, and the retention time of 5-hydroxymethylfurfural is 5-7 min.

6. The method for detecting 5-hydroxymethylfurfural in calcium gluconate injection by liquid chromatography according to claim 1, characterized in that: In the S130, the column temperature is 30°C, and the injection volume is 20 μl.

7. The method for detecting 5-hydroxymethylfurfural in calcium gluconate injection by liquid chromatography according to claim 1, characterized in that: In the S130, the mobile phase is 0.05 mol / L potassium dihydrogen phosphate solution (the pH value is adjusted to 3.0 with phosphoric acid)-methanol (90:10).

8. The method for detecting 5-hydroxymethylfurfural in calcium gluconate injection by liquid chromatography according to claim 1, characterized in that: In S130, if there is a chromatographic peak in the test solution with the same retention time as 5-hydroxymethylfurfural, the 5-hydroxymethylfurfural content shall not exceed 0.02% of the labeled amount of calcium gluconate by calculating the peak area according to the external standard method; The calculation formula is as follows: Where: As, Ai: the peak area of ​​5-hydroxymethylfurfural in the reference solution and the peak area of ​​5-hydroxymethylfurfural in the test solution, respectively; Ws: weight of reference substance, mg; Vs: dilution volume of the reference substance, ml; Ps: reference substance content, %; M: Specification of the preparation (calculated as calcium gluconate), mg / ml.

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