CN110887913B

CN110887913B - HPLC detection method of biotin intermediate diamino substance

Info

Publication number: CN110887913B
Application number: CN201911373716.5A
Authority: CN
Inventors: 吕国锋; 王昌泽; 单国红; 杭姣; 王振振; 李小军; 段文杰; 韦念想
Original assignee: Zhejiang Nhu Pharmaceutical Co ltd; Shangyu Nhu Biochemical Industry Co ltd
Current assignee: Zhejiang Nhu Pharmaceutical Co ltd; Shangyu Nhu Biochemical Industry Co ltd
Priority date: 2019-12-27
Filing date: 2019-12-27
Publication date: 2020-10-20
Anticipated expiration: 2039-12-27
Also published as: WO2021128665A1; CN110887913A

Abstract

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.

Description

HPLC detection method of biotin intermediate diamino substance

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.

In the methods in the prior art, biotin is used as a target product for detection, so that double amino substances capable of being converted into biotin are ignored. Since the diamino species appear near the dead time, its residue is not readily determined, thereby affecting the overall yield of biotin. At present, the biotin detection methods mainly include a microbiological method, a liquid chromatography method, an enzyme-linked immunoassay method, a spectroscopic method, an electrochemical method and the like. The operation of the microbiological method is time-consuming and labor-consuming, and the cost is high; although the enzyme-linked immunosorbent assay and the spectroscopic method have simple operation, high analysis speed and high accuracy, the method has high market cost and is difficult to popularize and apply in production. The methods are all based on the detection of biotin, but have poor accuracy and sensitivity in the detection of impurities.

Through retrieval, the Chinese patent document CN 200810162138-D-biotin and the HPLC determination method of the impurity content thereof disclose a method for quantitatively detecting D-biotin and the impurity content thereof. The chromatographic conditions when the high performance liquid chromatography is adopted for detection are as follows: adopting a reversed phase C18 column, a trifluoroacetic acid aqueous solution-acetonitrile mobile phase with the mass concentration of 0.05 percent, the flow rate of 0.5-1.5ml/min, the detection wavelength of 210-220nm, and the volume ratio of the trifluoroacetic acid aqueous solution to the acetonitrile in the mobile phase is 60-80: 40-20; dissolving d-biotin containing impurities with a mobile phase, diluting, introducing a sample for determination, and detecting the content of the d-biotin and each impurity by a high performance liquid chromatograph. The structures of these impurities A-H are shown below:

the method meets the requirement of biotin quantitative detection, has higher sensitivity (the detection limit is as low as 0.5ng), and is beneficial to improving the quality control of biotin.

However, in the embodiment provided by the invention, the sample is prepared by dissolving biotin and various impurities, and a mobile phase is directly used as a solvent. As can be seen from the peak appearance, the impurity diamino compound (structure C) appears early (about 1.5-2 min). The detection method is suitable for detecting the quality of a final product, and in the actual production process, the reaction solvent for synthesizing the biotin is usually methanol, toluene, hydrobromic acid and the like. When the method is adopted to directly detect the reaction solution, the problem that the content of the diamino compound C cannot be accurately detected due to the coincidence of the double amino compound C and a solvent peak can occur. Therefore, the above method is not directly applicable to the direct detection of the reaction solution in the biotin preparation process.

In the process of biotin preparation, the content of intermediates and products needs to be monitored at any time to improve the product yield. Therefore, a method for quantitative detection of diamino compounds with specificity is needed.

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, peak area corresponding to the diamino 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.

Example 1

1. Instruments and reagents

The instrument model is as follows: dean liquid chromatograph, U3000

Reagent: acetonitrile is used as chromatographic pure reagent, and other reagents are used as analytical pure reagents

2. Preparation of standard solution

Preparing a diamino substance standard solution: dissolving appropriate amount of amino substance standard substance in mobile phase, and optionally ultrasonic treating to dissolve completely.

Preparing a buffer solution: 0.015moL of dipotassium hydrogen phosphate is weighed and dissolved in 1L of deionized water, and the mixture is filtered by a 0.22um microporous filter membrane for standby and is ready for use.

3. Preparation of sample to be tested

0.05g of reaction solution after biotin synthesis is taken by a capillary tube into a 15mL sampling bottle, 10mL of mobile phase is added for dissolution, and the solution is reserved after ultrasonic treatment.

The preparation process of biotin in the patent is as follows: bromine on uric acid produces monobenzyl and is rapidly converted to biotin, but at the same time, the ring is opened by an excess reaction to produce a diamino, which regenerates biotin by photochemical cyclization. The reaction solution after biotin synthesis is a reaction solution (photochemical reaction solution) at the end of photochemical cyclization reaction. Specific preparation examples of biotin are given below:

in a 500ml four-neck flask with a rectifying tower, dibenzyl biotin (30g, 70mmol) and 300ml of hydrobromic acid with a certain concentration are added, stirring reaction is carried out at 130 ℃, and benzyl bromide is separated from the top of the rectifying tower in time during the reaction. After the reaction was completed, the reaction mixture was cooled to 80 ℃ by HPLC, 100ml of toluene was added to extract and layer the resulting aqueous layer containing biotin and the diamino compound formed by the excess reaction, and hydrobromic acid was recovered under reduced pressure. After the recovery, adding a sodium hydroxide solution to adjust the pH to 9-10, dripping a solid phosgene toluene solution (50ml, 16mmol) at the temperature of-5 ℃ in an ice bath, and continuously adding the sodium hydroxide solution to maintain the pH of the reaction system to 9-10 in the process of dripping the solid phosgene. After the dropwise addition, stirring is continued for 30min under heat preservation, and a reaction result is monitored by sampling. The sample is photochemical reaction liquid, needs to stand for 5-10min, and is detected by a water layer.

4. Selection of detection wavelength

Weighing the diamino standard substance, preparing a solution with a proper concentration by using a mobile phase, and scanning in the wavelength range of ultraviolet 190-400nm, wherein the result shows that the diamino standard substance has the highest response value at 200nm, so the detection wavelength is preferably 200 nm. Chromatograms of high performance liquid chromatography detection results of the diamino compounds at different wavelengths are shown in fig. 1-4.

5. Chromatographic column conditions

A chromatographic column: acclaim TM Mixed-Mode WCX-1;

flow rate: 1.0 mL/min;

column temperature: 30 ℃;

and (3) testing time: 20 min;

sample introduction volume: 20 uL;

mobile phase: acetonitrile-potassium dihydrogen phosphate buffer (70%: 30%).

6. And (3) an elution mode: and (4) eluting at equal concentration.

7. And (3) detection results: chromatograms of the high performance liquid chromatography results of the reaction solution at different wavelengths are shown in fig. 5-8.

As is evident from comparison of FIGS. 5-8, the peak-off time of the diamino compound is stabilized at 10-12min under the conditions of wavelength 195-220nm, and the diamino compound is well separated from other impurities. And all impurities in the reaction solution completely peak at about 16 min. The detection time is short.

The results of measuring the contents of the respective impurities in the reaction solution as described above are shown in table 1 below.

195nm responded better, but the baseline began to deteriorate; at 220nm, the various peak responses begin to change, and overall the 200nm test results are better.

Methodology investigation

1. Precision survey

The diamino standard substance diluted by 100 times is taken and subjected to 7 times of repeatability tests under the detection conditions, and the relative standard deviation ranges of the chromatographic peak area and the retention time of the obtained diamino are 0.31-1.20% and 0.14-1.94% respectively, which shows that the method has good precision.

2. Detection limit

Under the chromatographic condition, the diamino standard solution is gradually dissolved by a mobile phase at room temperature, sample injection detection and analysis are carried out, and the calculated minimum detection limit of the diamino substance is 1.2 ng.

Finally, it should be noted that the above-mentioned contents are only used for illustrating the technical solutions of the present invention, and not for limiting the protection scope of the present invention, and that the simple modifications or equivalent substitutions of the technical solutions of the present invention by those of ordinary skill in the art can be made without departing from the spirit and scope of the technical solutions of the present invention.

Claims

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; the structural formula of the biotin is shown as the following (a), and the structural formula of the diamino compound is shown as the following (b):

the volume ratio of acetonitrile to phosphate buffer in the mobile phase acetonitrile-phosphate is as follows: 60% -80%: 40% -20%; the phosphate buffer solution is 0.01-0.03 mol/L; isoconcentration elution is adopted in the HPLC detection process;

the phosphate buffer is potassium dihydrogen phosphate buffer.

2. An HPLC detection method according to claim 1, wherein the mobile phase acetonitrile-phosphate has a volume ratio of acetonitrile to phosphate buffer of 70%: 30 percent.

3. The HPLC detection method according to claim 1, wherein the ratio of the cyclization reaction solution to the mobile phase in the biotin synthesis process is as follows: 0.05 g: 10 mL.

4. 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.

5. 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_iChromatogram of sample solutionThe sum of the peak areas corresponding to the components in (1).