CN115902043B

CN115902043B - Method for measuring impurity content in 8-bromooctanoic acid sample

Info

Publication number: CN115902043B
Application number: CN202211739005.7A
Authority: CN
Inventors: 陈晓冬; 向小军; 樊天宇; 刘学军
Original assignee: Shanghai Yaotan Pharmaceutical Research And Development Co ltd
Current assignee: Shanghai Yaotan Pharmaceutical Research And Development Co ltd
Priority date: 2022-12-30
Filing date: 2022-12-30
Publication date: 2023-08-18
Anticipated expiration: 2042-12-30
Also published as: CN115902043A

Abstract

The invention discloses a method for measuring impurity content in an 8-bromooctanoic acid sample. Specifically, the method comprises the following steps: (1) Carrying out esterification reaction on the 8-bromooctanoic acid sample and monohydric alcohol to generate ester; (2) Detecting the ester obtained in the step (1) by adopting a gas chromatography method; the impurity contains one or more of 1, 8-suberic acid, sebacic acid and 8-chlorooctanoic acid; the monohydric alcohol is methanol or ethanol. The method has high sensitivity, low detection limit and good separation degree, and meets the requirement of simultaneously measuring a plurality of substances.

Description

Method for measuring impurity content in 8-bromooctanoic acid sample

Technical Field

The invention belongs to the field of analysis and detection, and particularly relates to a method for measuring impurity content in an 8-bromooctanoic acid sample.

Background

Lipid Nanoparticles (LNP) are a lipid vesicle with a homogeneous lipid core, widely used for delivery of small molecule drugs and nucleic acid drugs, and have received great attention due to their remarkable success as a delivery platform for covd-19 mRNA vaccines. Nevertheless, the use of transient protein expression induced by mRNA far exceeds vaccines for preventing infectious diseases, and they are also expected to be gene editing components of cancer vaccines, protein replacement therapies, and rare genetic diseases. However, naked mRNA itself is unstable and is readily degraded and self-hydrolyzed by nucleases. Encapsulation of mRNA within LNP protects mRNA from extracellular ribonucleic acids and aids in the delivery of mRNA within cells. Month 8 of 2021, gaurav Sahay et al published Chemistry of Lipid Nanoparticles for RNADelivery on Accounts of Chemical Research, discussing the central role of LNP in RNA delivery.

Lipid Nanoparticles (LNP) include new lipids as well as other lipids such as phospholipids, structural lipids and PEG lipids. In BRANCHED TAIL LIPID COMPOUNDS AND COMPOSITIONS FOR INTRACELLULAR DELIVERY OF THERAPEUTIC AGENTS (Kerry E.Benenato et al), 8-bromooctanoic acid is used as a key material in the synthesis of new lipids and compositions thereof, and an analysis method with high accuracy, good stability and simple operation is found to have important significance for the synthesis of new lipids.

One synthetic route for 8-bromooctanoic acid is as follows:

1, 8-suberic acid, sebacic acid and 8-chlorooctanoic acid are produced in the reaction of the reaction route. The content of 1, 8-suberic acid, sebacic acid and 8-chlorooctanoic acid in the 8-bromooctanoic acid is required to be not more than 0.05% (in mass percent) and the standard is set according to the identification impurity (0.05% in mass percent) of ICH relative to the content of 8-bromooctanoic acid. There is no report in any literature on the analysis method for measuring the content of 1, 8-suberic acid, sebacic acid and 8-chlorooctanoic acid in an 8-bromooctanoic acid sample.

Disclosure of Invention

The invention aims to solve the technical problems that when the high performance liquid chromatography is adopted to detect the 8-bromooctanoic acid sample, under the limit concentration (0.05 percent, the percent is mass fraction, the content of impurities relative to the 8-bromooctanoic acid), the signal to noise ratio of 1, 8-octanedioic acid, sebacic acid and 8-chlorooctanoic acid is less than 10, the high performance liquid chromatography cannot be optimized by improving the concentration of the 8-bromooctanoic acid sample, and the problems of poor peak shape and low sensitivity exist in the direct sample injection of the gas chromatography are adopted, so that the method for measuring the content of the impurities in the 8-bromooctanoic acid sample is provided. The method has high sensitivity, low detection limit (the minimum detection limit can reach 0.005%), and good separation degree, and meets the requirement of simultaneously measuring various substances.

The invention provides a method for measuring impurity content in an 8-bromooctanoic acid sample, which comprises the following steps:

a. carrying out esterification reaction on the 8-bromooctanoic acid sample and monohydric alcohol to generate ester;

b. detecting the ester obtained in the step (1) by adopting a gas chromatography method;

the impurity contains one or more of 1, 8-suberic acid, sebacic acid and 8-chlorooctanoic acid;

the monohydric alcohol is methanol or ethanol.

Preferably, the chromatographic column used in the gas chromatography is a 100% dimethylpolysiloxane capillary column or a 6% cyanopropylbenzene-94% dimethylsiloxane capillary column.

Preferably, the carrier gas used in the gas chromatography is nitrogen.

Preferably, the detector in the gas chromatography is a flame ion detector (FID detector).

In some embodiments, the monohydric alcohol is methanol.

In some embodiments, the esterification reaction is performed in the presence of a strong acid. The strong acid is preferably an inorganic proton strong acid conventional in the art. Preferably, the strong acid is concentrated sulfuric acid (e.g., 98% strength by mass aqueous solution) or concentrated hydrochloric acid (e.g., 36% -38% strength by mass aqueous solution). The volume fraction of the strong acid relative to the total volume of the esterification reaction system is preferably 1% to 3.5%, more preferably 1%, 3% or 3.5%. When the strong acid is concentrated sulfuric acid, the volume fraction of the concentrated sulfuric acid relative to the total volume of the esterification reaction system is preferably 1% to 3%. When the strong acid is concentrated hydrochloric acid, the volume fraction of the concentrated hydrochloric acid relative to the total volume of the esterification reaction system is preferably 3.5%.

In some embodiments, the strong acid is preferably diluted prior to the esterification reaction. The strong acid is preferably diluted with the monohydric alcohol.

In some embodiments, the concentration of the 8-bromooctanoic acid sample in the esterification reaction system is from 5mg/ml to 10mg/ml, preferably 10mg/ml.

In the present invention, the monohydric alcohol is present in excess in the esterification reaction relative to the 8-bromooctanoic acid sample to ensure that the relevant test material is fully esterified to the desired product.

In some embodiments, the esterification reaction is performed under heating. The reaction temperature of the esterification reaction is preferably 45℃to 55 ℃ (e.g., 50℃or 55 ℃). The reaction time of the esterification reaction is generally selected according to the amount of the reactants fed, preferably at a point where the reaction starting material is completely converted into the desired product, preferably 1 to 3 hours (e.g., 1 hour). The esterification reaction is preferably carried out at 50℃in a water bath for 1 hour.

In some embodiments, the esterification reaction further comprises the following post-treatment steps: extraction (e.g., extraction with aqueous phase/organic phase; the organic phase is n-hexane, dichloromethane or n-heptane; the aqueous phase is preferably ultrapure water; the volume ratio of the aqueous phase to the organic phase is preferably from 2:1 to 1:3, more preferably 1:1). The extraction preferably comprises the steps of: adding ultrapure water with the same volume as the reaction solution, adding n-hexane with the same volume as the ultrapure water, swirling, standing and layering. The swirling time can be appropriately selected by those skilled in the art according to the amount of the reaction liquid, and generally is sufficient to allow the desired substance to be substantially transferred into the corresponding extract liquid phase layer, preferably 20 seconds to 30 seconds (e.g., 30 seconds).

In some embodiments, the 100% dimethylpolysiloxane capillary column model is Agilent VF-1ms 15m 0.25 μm,0.25 μm.

In some embodiments, the 6% cyanopropylbenzene-94% dimethylsiloxane capillary column model is Agilent DB-624.

In some embodiments, the column flow of carrier gas is from 0.8mL/min to 1.2mL/min, for example 1mL/min.

In some embodiments, the split ratio of the carrier gas is 20:1 to 100:1, e.g., 20:1.

In some embodiments, the sample inlet temperature in the gas chromatography is 300 ℃ to 325 ℃, for example 325 ℃.

In some embodiments, the gas chromatography is performed at a loading of 0.5 μl to 1.5 μl, for example 1 μl.

In some embodiments, the column temperature in the gas chromatography is from 80 ℃ to 325 ℃. Preferably, the column temperature in the gas chromatography is 80 ℃, and after the column temperature is kept for 2min, the column temperature is raised to 325 ℃ at 20 ℃/min, and the column temperature is kept for 10min.

In some embodiments, the temperature of the flame ion detector is 330 ℃ to 350 ℃, such as 350 ℃.

In some embodiments, the method for determining the content of impurities in the 8-bromooctanoic acid sample comprises calculating the content of 1, 8-suberic acid, sebacic acid and 8-chlorooctanoic acid in peak area by adopting an external standard method.

In some embodiments, the assay method preferably comprises the steps of:

b. extracting the reaction liquid in the step a;

c. the organic layer of step b was checked by gas chromatography.

In some embodiments, the gas chromatography detection conditions are as follows: chromatographic column: 100% dimethylpolysiloxane capillary column; carrier gas: nitrogen, column flow 1mL/min, split ratio: 20:1; sample inlet temperature: 325 ℃, the sample injection amount is 1 mu L, the column temperature is 80 ℃, and the temperature is kept for 2min; raising the temperature to 325 ℃ at 20 ℃/min, and keeping for 10min; FID detector, detector temperature: 350 ℃.

In some embodiments, the 8-bromooctanoic acid sample is a conventional sample commercially available in the art, and is not limited to a specific manufacturer, preferably a sample of 8-bromooctanoic acid manufactured by synfeitic pharmaceutical chemicals, inc.

On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.

The reagents and materials used in the present invention are commercially available.

The invention has the positive progress effects that: the method has the advantages of low equipment requirement, simple and convenient operation, high sensitivity, low detection limit and good separation degree, and can meet the purity requirement of detecting the 8-bromooctanoic acid, and the impurity in the method can meet the detection requirement.

Drawings

FIG. 1 is a GC spectrum of a sensitivity solution in the detection method of example 1.

FIG. 2 is a GC spectrum of the labeled test sample solution of the test method of example 1.

FIG. 3 is a GC spectrum of the control solution in the test method of example 1.

FIG. 4 is a GC spectrum of a sample solution in the test method of example 1.

FIG. 5 is a GC spectrum of the labeled test sample solution of the test method of example 2.

FIG. 6 is a GC spectrum of the control solution in the test method of example 2.

FIG. 7 is a GC spectrum of a test solution in the test method of example 2.

FIG. 8 is a GC spectrum of the labeled test sample solution of the test method of example 3.

FIG. 9 is a GC spectrum of the control solution in the test method of example 3.

FIG. 10 is a GC spectrum of a sample solution in the test method of example 3.

FIG. 11 is a GC spectrum of the labeled test sample solution of the test method of example 4.

FIG. 12 is a GC spectrum of the control solution in the test method of example 4.

FIG. 13 is a GC spectrum of a test solution in the test method of example 4.

FIG. 14 is a GC spectrum of the labeled test solution of example 5.

FIG. 15 is a GC spectrum of a control solution at a sulfuric acid concentration of 1% in example 6.

FIG. 16 is a GC spectrum of a test solution in example 6 when the sulfuric acid concentration is 1%.

FIG. 17 is a GC spectrum of the control solution in example 6 at a sulfuric acid concentration of 3%.

FIG. 18 is a GC spectrum of a test solution in example 6 when the sulfuric acid concentration is 3%.

FIG. 19 is a GC spectrum of a control solution at a hydrochloric acid concentration of 1% in example 7.

FIG. 20 is a GC spectrum of a test solution in example 7 when the hydrochloric acid concentration is 1%.

FIG. 21 is a GC spectrum of the control solution in example 7 at a hydrochloric acid concentration of 3.5%.

FIG. 22 is a GC spectrum of a test solution in example 7 at a hydrochloric acid concentration of 3.5%.

FIG. 23 is a GC spectrum of the labeled test sample solution of the test method of example 8.

FIG. 24 is a GC spectrum of the labeled test sample solution of the test method of example 9.

FIG. 25 is a GC spectrum of a blank solution in the test method of comparative example 1.

FIG. 26 is a GC spectrum of the control solution in the test method of comparative example 1.

FIG. 27 is a GC spectrum of a test solution in the test method of comparative example 1.

FIG. 28 is a GC spectrum of a blank solution in the test method of comparative example 2.

FIG. 29 is a GC spectrum of the control solution in the test method of comparative example 2.

FIG. 30 is an HPLC chart of the test solution and the control solution in the test method of comparative example 3.

FIG. 31 is a GC spectrum of the labeled test solution of comparative example 4.

FIG. 32 is a GC spectrum of the labeled test sample solution of the test method of comparative example 5.

FIG. 33 is a GC spectrum of the control solution in the test method of comparative example 6.

FIG. 34 is a GC spectrum of the control solution in the test method of comparative example 7.

Detailed Description

The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

In the following examples, the recovery rate calculation formula is: recovery = (measured concentration in labeled sample solution-measured concentration in sample)/concentration of impurities in impurity stock solution added in labeled sample solution

Measured concentration in the labeled sample solution = area of impurity peak in the labeled sample solution x concentration of impurity in the reference solution ≡area of impurity in the reference solution

Measured concentration in sample solution = area of impurity peak in sample solution x concentration of impurity in reference solution ≡area of impurity peak in reference solution

When the recovery rate is more than 100%, errors may be caused by various factors such as errors in the sample configuration process or instrument systems, the recovery rate of 80% -120% belongs to a reasonable normal range in the analysis method verification, and the Chinese pharmacopoeia prescribes that if the recovery rate of the method is between 80% -120%, the accuracy of the method is good. The same samples were used and the same method was used, and recovery was different due to batch-to-batch.

In the following examples, the degree of separation is the ratio of the difference between the retention times of adjacent chromatographic peaks to the peak-to-peak average value of the two chromatographic peaks, and represents the degree of separation of the adjacent two peaks.

In the present invention, the minimum detection limit is calculated by the concentration of the impurity in the sensitivity solution of example 1/the concentration of the 8-bromooctanoic acid sample in the test sample solution, i.e., 0.0005/10=0.005%.

Example 1

Gas chromatography conditions:

chromatographic column: 100% Dimethicone capillary column (Agilent VF-1ms 15 mX0.25 μm,0.25 μm).

Carrier gas: nitrogen, column flow 1mL/min, split ratio: 20:1.

Sample inlet temperature: 325 ℃, sample injection amount: 1 mul.

The column temperature is 80 ℃ and kept for 2min; raising the temperature to 325 ℃ at 20 ℃/min and keeping the temperature for 10min.

FID detector, detector temperature: 350 ℃.

The operation steps are as follows:

dilution: and measuring about 10mL of concentrated sulfuric acid (98%) and adding methanol to dilute to 1000mL, and shaking uniformly to obtain the diluent.

Blank solution: adding 4mL of diluent into a 20mL headspace bottle, capping, heating in a water bath at 50 ℃ for 1 hour, taking out, cooling, adding 4mL of ultrapure water, 4mL of n-hexane, swirling for 30 seconds, standing for layering, and taking the upper layer solution as a blank solution.

Control solution: precisely weighing 1, 8-suberic acid, sebacic acid and 8-chlorooctanoic acid into 50 mg-100 mL measuring bottles respectively, adding a diluent to dissolve and dilute to scale, and taking the mixture as an impurity stock solution. Precisely measuring 1mL to 100mL of impurity stock solution in a measuring flask, diluting to a scale with a diluent, shaking uniformly, taking 4mL of the solution, adding the solution into a 20mL headspace bottle, capping, heating in a water bath at 50 ℃ for 1 hour, taking out and cooling, adding 4mL of ultrapure water, 4mL of n-hexane, swirling for 30 seconds, standing for layering, and taking the upper layer solution as a reference substance solution.

Sensitivity solution: precisely measuring the impurity stock solution in a measuring flask with 0.1mL to 100mL, diluting to a scale with a diluent, shaking uniformly, taking 4mL of the solution, adding the solution into a 20mL headspace bottle, capping, heating in a water bath at 50 ℃ for 1 hour, taking out and cooling, adding 4mL of ultrapure water, 4mL of n-hexane, swirling for 30 seconds, standing for layering, and taking the upper layer solution as a sensitivity solution.

Test solution: 40mg of the sample (i.e., 8-bromooctanoic acid sample) is weighed and placed in a 20mL headspace bottle, 4mL of diluent is added, the cap is added, the mixture is heated in a water bath at 50 ℃ for 1 hour, the mixture is taken out and cooled, 4mL of ultrapure water, 4mL of n-hexane is added, vortex is carried out for 30 seconds, the mixture is static and layered, and the upper layer solution is taken as the sample solution.

Adding a labeled test sample solution: precisely measuring 1mL to 100mL of impurity stock solution in a measuring flask, diluting to a scale with a diluent, shaking uniformly, weighing 40mg of a test sample, placing the test sample in a 20mL headspace bottle, adding 4mL of the solution, capping, heating in a water bath at 50 ℃ for 1 hour, taking out and cooling, adding 4mL of ultrapure water, 4mL of n-hexane, swirling for 30 seconds, standing and layering, and taking the upper layer solution as the solution for adding the standard test sample.

Testing the solution by adopting the gas chromatography conditions, wherein a gas chromatogram measured for the sensitivity solution is shown in figure 1; the gas chromatogram measured for the labeled test solution is shown in FIG. 2; the gas chromatogram measured for the control solution is shown in FIG. 3; the gas chromatogram measured for the test solution is shown in FIG. 4.

The signal to noise ratios of 8-chlorooctanoic acid, 1, 8-suberic acid and sebacic acid in the sensitive solution shown in the figure 1 are respectively 20, 12 and 20, and are all more than 10 (see table 1 in particular), so that the quantitative requirements are met.

The peaks in the labeled test solution in FIG. 2 are 8-chlorooctanoic acid, 1, 8-suberic acid, 8-bromooctanoic acid and sebacic acid in sequence from left to right, and the separation degree between the peaks is more than 1.5 (see in particular Table 2).

The prepared blank solution is injected, and no peak exists at the positions of an impurity peak and a main peak, which indicates that the blank solution has no interference at the positions of 8-chlorooctanoic acid, 1, 8-suberic acid, 8-bromooctanoic acid and sebacic acid at the peak positions.

Meanwhile, the recovery rate of the method is examined, the recovery rates of the 8-chlorooctanoic acid, the 1, 8-suberic acid and the sebacic acid are respectively 90%, 101% and 97% (the data required by the recovery rate calculation are shown in the table 3), the detection requirement of the method is met, and the method can sensitively and accurately detect the 1, 8-suberic acid, the 8-bromooctanoic acid and the sebacic acid in the 8-bromooctanoic acid.

Table 1 gas chromatography data for example 1 sensitivity solution measurements

Table 2 gas chromatography data for example 1 labeled test solutions

Peak number	Name of the name	Retention time	Degree of separation	Area percent
					1	8-Chlorooctanoic acid	6.461min	N/A	0.11
2	1, 8-suberic acid	6.774min	9.2	0.04
					3	8-Bromooctanoic acid	7.073min	5.8	99.78
4	Sebacic acid	7.970min	19	0.07

TABLE 3 recovery data Table for example 1

Example 2

In the diluent preparation step, about 35mL of concentrated hydrochloric acid (36% -38%) is measured, methanol is added for dilution to 1000mL, and the diluent is obtained after shaking. The other conditions were the same as in example 1.

Testing the labeled sample solution, and obtaining a gas chromatogram shown in figure 5; testing the reference substance solution, and obtaining a gas chromatogram shown in figure 6; the test sample solution was tested and the resulting gas chromatogram is shown in FIG. 7.

FIG. 3 shows that the separation degree between peaks in the labeled test sample solution is greater than 1.5 (see Table 4).

The recovery rates of the 8-chlorooctanoic acid, the 1, 8-suberic acid and the sebacic acid are 103%, 100% and 100% respectively (the data required by the recovery rate calculation are shown in Table 5), and the detection requirements are met.

Table 4 gas chromatography data for example 2 labeled test solutions

Peak number	Name of the name	Retention time	Degree of separation	Area percent
					1	8-Chlorooctanoic acid	6.453min	N/A	0.81
2	1, 8-suberic acid	6.766min	9.2	0.04
					3	8-Bromooctanoic acid	7.066min	6.1	99.08
4	Sebacic acid	7.963min	19.9	0.07

TABLE 5 recovery data Table for example 2

Example 3

The water bath heating temperature in the operation step was 55℃and the other conditions were the same as in example 1.

Testing the labeled sample solution, wherein the obtained gas chromatogram is shown in figure 8, and the reference sample solution is tested, and the obtained gas chromatogram is shown in figure 9; the test sample solution was tested and the resulting gas chromatogram is shown in FIG. 10.

FIG. 8 shows that the separation degree between peaks in the labeled test sample solution is greater than 1.5 (see Table 6).

The recovery rates of the 8-chlorooctanoic acid, the 1, 8-suberic acid and the sebacic acid are respectively 94%, 105% and 101% (the data required by the recovery rate calculation are shown in Table 7), and the detection requirements are met.

TABLE 6 determination of gas chromatography data for example 3 labeled test sample solutions

Peak number	Name of the name	Retention time	Degree of separation	Peak area	Area percent
						1	8-Chlorooctanoic acid	6.457min	N/A	5.67	0.12
2	1, 8-suberic acid	6.769min	9.4	2.07	0.04
						3	8-Bromooctanoic acid	7.069min	5.9	4658.04	99.77
4	Sebacic acid	7.966min	19.0	3.31	0.07

TABLE 7 recovery data sheet

Example 4

The water bath heating time in the operation step was 3 hours, and the other conditions were the same as in example 1.

Testing the labeled sample solution, the obtained gas chromatogram is shown in figure 11, and the reference sample solution is tested, and the obtained gas chromatogram is shown in figure 12; the test sample solution was tested and the resulting gas chromatogram is shown in FIG. 13.

FIG. 11 shows that the separation degree between peaks in the labeled test sample solution is greater than 1.5 (see Table 8).

The recovery rates of the 8-chlorooctanoic acid, the 1, 8-suberic acid and the sebacic acid are respectively 101%, 107% and 100% (the data required by the recovery rate calculation are shown in Table 9), and the detection requirements are met.

Table 8 gas chromatography data for example 4 labeled test solutions

TABLE 9 recovery data sheet

Example 5

The rest of the conditions in the diluent preparation step were the same as in example 1 except that methanol was replaced with ethanol. The labeled sample solution was tested, the obtained gas chromatogram is shown in fig. 14, and specific data are shown in table 10.

Table 10 gas chromatography data for example 5 labeled test solutions

Peak number	Name of the name	Retention time	Degree of separation	Peak area	Area percent
						1	8-Chlorooctanoic acid	6.924min	N/A	5.26	0.10
2	8-Bromooctanoic acid	7.511min	12.6	5288.13	99.77
						3	1, 8-suberic acid	7.616min	2.3	3.16	0.06
4	Sebacic acid	8.706min	36.7	3.93	0.07

Example 6

And (3) examining gas chromatography data when the concentration of the sulfuric acid (98%) is 1% and the concentration of the sulfuric acid (3%) are respectively in the diluent preparation step, namely respectively measuring about 10mL or 30mL of concentrated sulfuric acid (98%) and adding methanol to dilute to 1000mL and shaking uniformly in the diluent preparation step to obtain the diluent. The other conditions were the same as in example 1.

When the concentration of sulfuric acid is 1%, the reference substance solution is tested, and the obtained gas chromatogram is shown in FIG. 15; when the concentration of sulfuric acid is 1%, testing the sample solution, and obtaining a gas chromatogram shown in FIG. 16; when the concentration of sulfuric acid is 3%, the reference substance solution is tested, and the obtained gas chromatogram is shown in FIG. 17; when the sulfuric acid concentration was 3%, the test sample solution was tested, and the obtained gas chromatogram was shown in fig. 18.

It was found that when the sulfuric acid concentration was 1% and 3% respectively, the separation degree between the peaks was 1.5 or more, and the recovery rate was all able to meet the detection requirements, as shown in Table 11 below.

TABLE 11 recovery of sulfuric acid (98%) at 1% and 3% concentration, respectively, in the diluent preparation step

Example 7

And (3) examining gas chromatography data when the concentration of hydrochloric acid (36% -38%) is 1% and the concentration of hydrochloric acid is 3.5% in the diluent preparation step, namely respectively measuring about 10mL or 35mL of hydrochloric acid (36% -38%) in the diluent preparation step, adding methanol to dilute to 1000mL, and shaking uniformly to obtain the diluent. The other conditions were the same as in example 1.

When the concentration of hydrochloric acid is 1%, testing the reference substance solution, and obtaining a gas chromatogram shown in FIG. 19; when the concentration of the hydrochloric acid is 1%, testing the sample solution, and obtaining a gas chromatogram shown in FIG. 20; when the concentration of hydrochloric acid is 3.5%, the control solution is tested, and the obtained gas chromatogram is shown in FIG. 21; when the concentration of hydrochloric acid was 3.5%, the test sample solution was tested, and the obtained gas chromatogram was shown in fig. 22.

It was found that the degree of separation between peaks was 1.5 or more at 1% and 3.5% hydrochloric acid concentration, respectively, and that the recovery rate of hydrochloric acid at 3.5% was satisfactory (80% -120%), but that the recovery rate of 8-chlorooctanoic acid was-206% at 1% hydrochloric acid concentration (see Table 12, in particular).

TABLE 12 recovery of hydrochloric acid (36% -38%) concentrations at 1% and 3.5% respectively in the diluent preparation step

Example 8

The chromatographic column was replaced with a 6% cyanopropylbenzene-94% dimethylsiloxane capillary column (Agilent DB-624), the remaining conditions were the same as in example 1, and the addition of the standard sample solution was tested, and the gas chromatogram obtained was shown in FIG. 23 (see Table 13 for specific data), and found to be similar to the separation effect using a 100% dimethylpolysiloxane capillary column.

Table 13 gas chromatography data for example 8 labeled test solutions

Peak number	Name of the name	Retention time	Degree of separation	Peak area	Area percent
						1	8-Chlorooctanoic acid	11.713min	N/A	5.67	0.09
2	1, 8-suberic acid	12.031min	6.2	2.54	0.04
						3	8-Bromooctanoic acid	12.500min	8.6	6133.22	99.80
4	Sebacic acid	13.844min	20.5	3.81	0.06

Example 9

In the steps of preparing the blank solution, the reference solution, the sensitivity solution, the test sample solution and the labeled test sample solution, the steps of adding 4mL of ultrapure water, 4mL of normal hexane, swirling for 30 seconds, standing for layering and taking the upper layer solution are reduced, the other conditions are the same as in example 1, the labeled test sample solution is tested, and the measured gas chromatogram is shown in FIG. 24.

Comparative example 1

Chromatographic conditions:

Carrier gas: nitrogen, column flow 1mL/min, split ratio: 20:1.

Sample inlet temperature: 325 ℃, sample injection amount: 1 mul.

FID detector, detector temperature: 350 ℃.

Operating procedure

Diluent (which is also blank solution): methanol.

Control solution: precisely weighing 1, 8-suberic acid, sebacic acid and 8-chlorooctanoic acid into 50 mg-100 mL measuring bottles respectively, adding a diluent to dissolve and dilute to scale, and taking the mixture as an impurity stock solution. Accurately measuring the impurity stock solution in a 1 mL-100 mL measuring flask, diluting to a scale with a diluent, and shaking uniformly to obtain a reference substance solution.

Test solution: taking a proper amount of 8-bromooctanoic acid sample and preparing into a solution of 10mg/ml by using methanol.

The method adopts a conventional gas phase direct sample injection mode, chromatographic conditions are the same as those of the embodiment 1, a gas phase chromatogram of a blank solution is shown in fig. 25 (wherein 1-2 min is a methanol peak, and the rest peaks are impurity peaks in methanol), a gas phase chromatogram of a reference solution is shown in fig. 26, and the gas phase chromatograms of the reference solution show that no peak exists in 1, 8-suberic acid, sebacic acid and 8-chlorooctanoic acid under the limit concentration (0.05%), the gas phase chromatograms are basically consistent with the gas phase chromatogram of the blank solution, so that the sensitivity requirement of quantitative detection cannot be met, and the retention time of the main peak 8-bromooctanoic acid is 7.412min, the peak is asymmetric, so that the direct sample injection mode cannot reach the sensitivity detection requirement under the direct sample injection mode, and the symmetrical peak type cannot be obtained at the same time.

In this example, the limit concentration was calculated by the concentration of impurities in the control solution/the concentration of 8-bromooctanoic acid sample in the test solution, i.e., 0.005/10=0.05%.

Comparative example 2

Operating procedure

A diluent: methanol.

Control solution: precisely weighing 1, 8-suberic acid, sebacic acid and 8-chlorooctanoic acid into 50 mg-100 mL measuring bottles respectively, adding a diluent to dissolve and dilute to scale, and taking the mixture as an impurity stock solution. Accurately measuring the impurity stock solution in a 1 mL-10 mL measuring flask, diluting to a scale with a diluent, and shaking uniformly to obtain a reference substance solution.

Test solution: taking a proper amount of 8-bromooctanoic acid sample and preparing the sample into a solution with the concentration of 100mg/ml by using methanol.

The other conditions were the same as in example 1.

Since the sensitivity detection requirement cannot be met in comparative example 1, on the basis of the detection requirement, we consider that the sample concentration is increased to meet the detection requirement, and the sample concentration is increased from 10mg/ml to 100mg/ml in comparative example 1 by adopting the same sample injection mode and detection condition as in comparative example 1, the gas phase chromatogram of the blank solution is shown in fig. 28 (wherein 1-2 min is a methanol peak, and the rest peaks are impurity peaks in methanol), the gas phase chromatogram of the reference solution is shown in fig. 29, and the gas phase chromatogram of the reference solution shows that the 1, 8-suberic acid, sebacic acid and 8-chlorooctanoic acid still do not show peaks under the condition, which are basically consistent with the gas phase chromatogram of the blank solution, so that the requirement of detecting sensitivity cannot be met in increasing the sample concentration.

Comparative example 3

Chromatographic conditions:

instrument: a high performance liquid chromatograph,

Chromatographic column: SHIMDZU Shim-pack GIST C18, 4.6X105 mm,5 μm

Mobile phase a:0.05% aqueous trifluoroacetic acid solution

Mobile phase B:0.05% acetonitrile solution of trifluoroacetic acid

Detector and wavelength: UV/200nm

Mobile phase flow rate: 1.0ml/min

Column temperature: 35 DEG C

Sample injection volume: 20 μl of

Gradient elution was used, see table below:

operating procedure

A diluent: methanol.

Precisely weighing 1, 8-suberic acid, sebacic acid and 8-chlorooctanoic acid into 50 mg-100 mL measuring bottles respectively, adding a diluent to dissolve and dilute to scale, and taking the mixture as an impurity stock solution. Accurately measuring the impurity stock solution in a measuring flask with the volume of 10mL to 100mL, diluting to a scale with a diluent, and shaking uniformly to obtain a reference substance solution.

As shown in the graph of FIG. 30, the comparative example adopts an HPLC-UV method, the ultraviolet absorption of 1, 8-suberic acid, sebacic acid and 8-chlorooctanoic acid is weak, no peak is shown at a low wavelength of 200nm, and the main peak 8-bromooctanoic acid peak is flat-headed, which shows that under the condition that the sample amount is overloaded, the 1, 8-suberic acid, sebacic acid and 8-chlorooctanoic acid with the limit concentration (0.05%) still cannot be detected at the low wavelength of 200nm, and the HPLC-UV method cannot meet the detection sensitivity requirement.

Comparative example 4

The diluent preparation step was replaced with isopropanol, and the rest conditions were the same as in example 1, and the labeled sample solution was tested, and the obtained gas chromatogram was shown in fig. 31, and found to have underderived 8-bromooctanoic acid, wherein the 7.4min chromatographic peak was underderived 8-bromooctanoic acid.

Comparative example 5

The methanol in the diluent preparation step was replaced with isopropanol, the heating time in the water bath was changed to 6 hours, and the other conditions were the same as in example 1, and the labeled sample solution was tested, and the obtained gas chromatogram was shown in fig. 32, and it was found that there was still underderived 8-bromooctanoic acid, in which the 7.4min chromatographic peak was underderived 8-bromooctanoic acid.

Comparative example 6

The control solution was tested under the same conditions as in example 1 except that the diluent was replaced with methanol (without concentrated sulfuric acid), and the gas chromatogram obtained was shown in fig. 33.

Comparative example 7

The chromatographic column is replaced by a polyethylene glycol capillary column modified by nitroterephthalic acid, the other conditions are the same as those of the example 1, the control solution is tested, the detected gas chromatogram is shown in fig. 34, and a derivative product peak cannot be found in the chromatogram.

Claims

1. The method for measuring the impurity content in the 8-bromooctanoic acid sample is characterized by comprising the following steps of:

(1) Carrying out esterification reaction on the 8-bromooctanoic acid sample and monohydric alcohol to generate ester;

(2) Detecting the ester obtained in the step (1) by adopting a gas chromatography method;

the monohydric alcohol is methanol or ethanol;

the chromatographic column adopted in the gas chromatography is a 100% dimethyl polysiloxane capillary column or a 6% cyanopropylbenzene-94% dimethyl siloxane capillary column;

the detector in the gas chromatography is a flame ion detector;

the esterification reaction also comprises the following post-treatment steps: extracting with aqueous and organic phases; the organic phase is n-hexane or n-heptane; the water phase is ultrapure water;

the column temperature in the gas chromatography is 80 ℃, and after the column temperature is kept for 2min, the temperature is raised to 325 ℃ at 20 ℃/min, and the column temperature is kept for 10min.

2. The method according to claim 1, wherein the carrier gas used in the gas chromatography is nitrogen.

3. The assay of claim 1 wherein the monohydric alcohol is methanol.

4. The assay of claim 1 wherein,

the esterification reaction is carried out under strong acid; the strong acid is an inorganic proton strong acid.

5. The assay of claim 1 wherein,

the concentration of the 8-bromooctanoic acid sample in the esterification reaction system is 5mg/ml-10mg/ml.

6. The assay of claim 1 wherein,

the reaction temperature of the esterification reaction is 45 ℃ to 55 ℃.

7. The assay of claim 1 wherein,

the reaction time of the esterification reaction is 1 to 3 hours.

8. The method according to claim 4, wherein the strong acid is concentrated sulfuric acid or concentrated hydrochloric acid.

9. The method according to claim 8, wherein the concentrated sulfuric acid is a concentrated sulfuric acid aqueous solution having a mass concentration of 98%; the concentrated hydrochloric acid is a concentrated hydrochloric acid aqueous solution with the mass concentration of 36% -38%.

10. The assay of claim 4 wherein,

when the esterification reaction is carried out under a strong acid, the volume fraction of the strong acid relative to the total volume of the esterification reaction system is 1% to 3.5%.

11. The assay of claim 4 wherein,

when the esterification reaction is carried out under a strong acid, the strong acid is diluted and then participates in the esterification reaction.

12. The assay of claim 6 wherein,

the reaction temperature of the esterification reaction is 50 ℃ or 55 ℃.

13. The assay of claim 7 wherein,

the reaction time of the esterification reaction was 1 hour.

14. The assay of claim 10 wherein when the esterification reaction is carried out in the presence of a strong acid, the volume fraction of the strong acid relative to the total volume of the esterification reaction system is 1%, 3%, or 3.5%.

15. The assay of claim 10 wherein,

when the esterification reaction is performed under a strong acid, and the strong acid is concentrated sulfuric acid, the volume fraction of the concentrated sulfuric acid relative to the total volume of the esterification reaction system is 1% -3%.

16. The assay of claim 10 wherein,

when the esterification reaction is carried out under a strong acid, and the strong acid is concentrated hydrochloric acid, the volume fraction of the concentrated hydrochloric acid relative to the total volume of the esterification reaction system is 3.5%.

17. The assay of claim 11 wherein said strong acid is diluted with said monohydric alcohol when said esterification reaction is carried out in the presence of a strong acid and said strong acid is diluted prior to being involved in the esterification reaction.

18. The assay of claim 10 wherein,

the esterification reaction was carried out for 1 hour in a water bath at 50 ℃.

19. The assay of claim 1 wherein the volume ratio of the aqueous phase to the organic phase is from 2:1 to 1:3.

20. The assay of claim 19 wherein the volume ratio of the aqueous phase to the organic phase is 1:1.

21. The assay of claim 1, wherein the extraction comprises the steps of: adding ultrapure water with the same volume as the reaction solution, adding n-hexane with the same volume as the ultrapure water, swirling, standing and layering.

22. The assay of claim 21 wherein the vortexing time is from 20 seconds to 30 seconds.

23. The assay of claim 2 wherein,

when the chromatographic column is a 100% dimethyl polysiloxane capillary column, the 100% dimethyl polysiloxane capillary column is of the model Agilent VF-1ms 15m x 0.25 μm,0.25 μm.

24. The assay of claim 2 wherein,

when the chromatographic column is a 6% cyanopropylbenzene-94% dimethylsiloxane capillary column, the 6% cyanopropylbenzene-94% dimethylsiloxane capillary column is Agilent DB-624.

25. The assay of claim 2 wherein,

the column flow rate of the carrier gas is 0.8mL/min-1.2mL/min.

26. The assay of claim 2 wherein,

the split ratio of the carrier gas is 20:1-100:1.

27. The assay of claim 2 wherein the sample inlet temperature in the gas chromatography is 300 ℃ to 325 ℃.

28. The assay of claim 2 wherein,

the sample injection amount in the gas chromatography is 0.5-1.5 mu L.

29. The assay of claim 2 wherein,

the temperature of the flame ion detector is 330-350 ℃.

30. The assay of claim 25 wherein,

the column flow of the carrier gas was 1mL/min.

31. The assay of claim 26 wherein,

the split ratio of the carrier gas was 20:1.

32. The assay of claim 27 wherein,

the temperature of the sample inlet in the gas chromatography is 325 ℃.

33. The assay of claim 28 wherein,

the sample injection amount in the gas chromatography is 1 mu L.

34. The assay of claim 29 wherein,

the temperature of the flame ion detector was 350 ℃.

35. The assay of claim 1, wherein the gas chromatography detection conditions are as follows: chromatographic column: 100% dimethylpolysiloxane capillary column; carrier gas: nitrogen, column flow 1mL/min, split ratio: 20:1; sample inlet temperature: 325 ℃, the sample injection amount is 1 mu L, the column temperature is 80 ℃, and the temperature is kept for 2min; raising the temperature to 325 ℃ at 20 ℃/min, and keeping for 10min; FID detector, detector temperature: 350 ℃.