阅读说明：本技术 一种生物素中间体双氨基物的hplc检测方法 (HPLC detection method of biotin intermediate diamino substance ) 是由 吕国锋 王昌泽 单国红 杭姣 王振振 李小军 段文杰 韦念想 于 2019-12-27 设计创作，主要内容包括：本发明属于生物素中间体含量检测技术领域,具体涉及一种生物素合成中的过程中间体双氨基物的HPLC检测方法。该方法包括如下步骤：用流动相溶解生物素合成过程中的环合反应液后在以下色谱条件下进行检测：色谱柱C18-WCX混合柱；检测波长为195-220nm,测试时间为16-20min,流动相为乙腈-磷酸盐缓冲液。本发明提供的检测方法可以在使待测物质双氨基物的出峰时间稳定在10-12min,解决了双氨基物与溶剂峰出峰重合的问题,使得监测更加准确、可靠。(The invention belongs to the technical field of biotin intermediate content detection, and particularly relates to an HPLC (high performance liquid chromatography) detection method of a diamino compound as an intermediate in a biotin synthesis process. The method comprises the following steps: after the cyclization reaction solution in the biotin synthesis process is dissolved by using a mobile phase, detection is carried out under the following chromatographic conditions: chromatographic column C18-WCX mixed column; the detection wavelength is 195-220nm, the detection time is 16-20min, and the mobile phase is acetonitrile-phosphate buffer solution. The detection method provided by the invention can stabilize the peak emergence time of the diamino substance to be detected within 10-12min, solve the problem of peak emergence coincidence of the diamino substance and the solvent, and ensure that the monitoring is more accurate and reliable.)

1. An HPLC detection method of biotin intermediate diamino substances comprises the following steps:

after the cyclization reaction solution in the biotin synthesis process is dissolved by using a mobile phase, detection is carried out under the following chromatographic conditions: chromatographic column C18-WCX mixed column;

the detection wavelength is 195-220nm, the detection time is 16-20min, and the mobile phase is acetonitrile-phosphate buffer solution.

2. The HPLC detection method according to claim 1, wherein the phosphate buffer is a potassium dihydrogen phosphate buffer.

3. The HPLC detection method according to claim 1, wherein the phosphate buffer is 0.01-0.03 mol/L.

4. An HPLC detection method according to claim 1, wherein the volume ratio of acetonitrile to phosphate buffer in mobile phase acetonitrile-phosphate is: 60% -80%: 40% -20%, and preferably the volume ratio of acetonitrile-phosphate is 70%: 30 percent.

5. An HPLC detection method according to claim 1, wherein isoconcentration elution is adopted during HPLC detection.

6. The HPLC detecting method according to claim 1, wherein the structural formula of biotin is (a) and the structural formula of bisamino is (b):

7. the HPLC detection method of claim 1, wherein the ratio of the sample containing biotin and diamino compounds to the mobile phase is: 0.05 g: 10 mL.

8. An HPLC detection method according to claim 1, wherein the sample volume: 20uL and the column temperature is 30 ℃; flow rate: 1.0-1.5 mL/min.

9. The HPLC detecting method according to claim 1, wherein the content of the diamino compound is calculated by the formula:

in the formula: a. the_{Diamino radical}Peak areas corresponding to the diamino compounds in the chromatogram of the sample solution; sigma A_iAnd the sum of the peak areas corresponding to all the components in the chromatogram of the sample solution.

Technical Field

The invention belongs to the technical field of biotin intermediate content detection, and particularly relates to an HPLC (high performance liquid chromatography) detection method of a diamino compound as an intermediate in a biotin synthesis process.

Background

A diamino substance (shown as the following formula b) is generated in the synthesis process of biotin (shown as the following formula a), and the substance is an intermediate product for generating biotin from uric acid, and d-biotin can be obtained through carbonylation and post-treatment.

Disclosure of Invention

In order to solve the above problems in the prior art, the present invention aims to provide a method for detecting biotin intermediate bis-amino compound by HPLC. The method has accurate and reliable detection result and simple and convenient operation. Moreover, the content of the diamino compound can be better mastered by using the detection method, so that the conversion rate can be effectively controlled, and the overall yield of the biotin can be improved.

In order to achieve the above purpose, the invention provides the following technical scheme:

an HPLC detection method of biotin intermediate diamino substances comprises the following steps: after dissolving the sample containing biotin and diamino substances by using a mobile phase, detecting under the following chromatographic conditions: chromatographic column C18-WCX mixed column;

the detection wavelength is 195-220nm, the detection time is 16-20min, and the mobile phase is acetonitrile-phosphate buffer solution.

The phosphate buffer is potassium dihydrogen phosphate buffer.

Preferably, the phosphate buffer is 0.01-0.03 mol/L. Further preferably 0.015 mol/L.

Preferably, the ratio of acetonitrile to phosphate buffer in the mobile phase acetonitrile-phosphate is as follows: 60% -80%: 40% -20%, and preferably the proportion of acetonitrile-phosphate is 70%: 30 percent.

Preferably, isoconcentrate elution is used during the HPLC assay.

Preferably, the detection wavelength is 200 nm.

Preferably, the structural formula of biotin is as follows (a), and the structural formula of the diamino is as follows (b):

preferably, the ratio of the sample containing biotin and diamino compounds to the mobile phase is: 0.05 g: 10 mL.

Preferably, the injection volume is: 10uL and the column temperature is 30 ℃; flow rate: 1.0-1.5 mL/min.

Preferably, the content of the diamino compound is calculated by the following formula:

in the formula: a. the_{Diamino radical}Peak areas corresponding to the diamino compounds in the chromatogram of the sample solution; sigma Ai is the sum of the peak areas corresponding to each component in the chromatogram of the sample solution.

Compared with the prior art, the invention has the following beneficial effects:

(1) the detection method provided by the invention can stabilize the peak emergence time of the diamino substance to be detected within 10-12min, solve the problem of peak emergence coincidence of the diamino substance and the solvent, and ensure that the monitoring is more accurate and reliable.

(2) The detection method provided by the invention can more accurately master the content of the impurity diamino substance in the biotin, can better control the reaction end point, effectively converts the diamino substance into the biotin and improves the yield of the biotin by about 10 percent on average.

(3) The detection method provided by the invention has high precision, and the relative standard deviation ranges of chromatographic peak area and retention time of the diamino compound are respectively 0.31-1.20% and 0.14-1.94% as verified by a repeatability test.

(4) The method has low detection limit, and the minimum detection limit of the diamino compound is 1.2 ng.

Drawings

FIG. 1 is a chromatogram of HPLC detection result of the diamino compound standard substance at 195 nm;

FIG. 2 is a chromatogram of HPLC detection result of the diamino compound standard at 200 nm;

FIG. 3 is a chromatogram of HPLC detection at 210nm of the diamino standard of the present invention;

FIG. 4 is a chromatogram of HPLC detection results at 220nm for the diamino standard of the present invention;

FIG. 5 is a chromatogram of HPLC detection result of the diamino compound at 195nm in the reaction solution;

FIG. 6 is a chromatogram of HPLC detection result of the diamino at 200nm in the reaction solution;

FIG. 7 is a chromatogram of HPLC detection result of the diamino compound at 210nm in the reaction solution;

FIG. 8 is a chromatogram of HPLC detection results of the bisamide at 220nm in the reaction solution.

Detailed Description

The method of the present invention is described below with reference to specific examples to make it easier to understand and understand the technical solution of the present invention, but the present invention is not limited thereto. The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.