CN113109497B - Method for determining related substances in 6-bromohexyltrimethylammonium bromide by ion pair chromatography - Google Patents
Method for determining related substances in 6-bromohexyltrimethylammonium bromide by ion pair chromatography Download PDFInfo
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Abstract
The invention discloses a method for determining related substances in 6-bromohexyltrimethylammonium bromide by an ion pair chromatography, which comprises the following steps: (1) preparation of a standard solution: weighing 6-bromohexyltrimethylammonium bromide and hexamethonium bromide standard substances, respectively dissolving with ultrapure water to a constant volume, and shaking up to obtain standard substance solutions of the 6-bromohexyltrimethylammonium bromide and the hexamethonium bromide; (2) preparing a sample solution: weighing a 6-bromohexyltrimethylammonium bromide sample, dissolving the 6-bromohexyltrimethylammonium bromide sample with ultrapure water to a constant volume, and shaking up to obtain a sample solution; (3) determination of standard and sample solutions: according to certain chromatographic conditions, carrying out sample injection analysis on a standard substance and a sample solution by using an ion chromatograph, and simultaneously recording peak areas; (4) calculating the content of the substance to be detected: and calculating the contents of 6-bromohexyltrimethylammonium bromide and hexamethonium bromide according to an external standard method. Compared with mass spectrometry, the method has the advantages of low test cost and no complex pretreatment, and the 6-bromohexyl trimethyl ammonium bromide sample can be directly injected and analyzed after being dissolved by adding water, so that the method is simple and easy to implement.
Description
Technical Field
The invention relates to the field of ion chromatography, relates to a quantitative analysis method for related substances in 6-bromohexyltrimethylammonium bromide, and particularly relates to a method for determining related substances in 6-bromohexyltrimethylammonium bromide by an ion pair chromatography.
Background
6-bromohexyltrimethylammonium bromide, CAS number 191086-27-8, structural formula as shown below, is easily soluble in water and is very deliquescent.
The preparation process of 6-bromohexyltrimethylammonium bromide is usually accompanied by the generation of impurity hexamethonium bromide, the CAS number is 55-97-0, and the structural formula is shown as follows. The two have similar structural formulas and have quaternary ammonium salt groups, and are excellent medical intermediates and medical products. 6-bromohexyltrimethylammonium bromide is an important intermediate in the synthesis process of the medicine colesevelam hydrochloride for treating hyperlipidemia; hexamethonium bromide is a cholinergic receptor antagonist, can be used as a selective blocking medicament for sympathetic ganglia and is clinically used for treating severe hypertension.
At present, the determination methods of 6-bromohexyltrimethylammonium bromide and hexamethonium bromide are rarely reported, and the reports of quaternary ammonium salt detection methods with similar structures mainly include an ultraviolet spectrophotometry method, a high performance liquid chromatography, a liquid chromatography-mass spectrometry method and a gas chromatography-mass spectrometry method. Ultraviolet spectrophotometry and high performance liquid chromatography can detect quaternary ammonium salt with ultraviolet absorption, and cannot detect saturated alkyl quaternary ammonium salts such as 6-bromohexyltrimethylammonium bromide, hexamethonium bromide and the like; the saturated alkyl quaternary ammonium salt can be measured by liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry, but the operation is complex, the analysis time is long, and the cost is expensive.
Therefore, improvements in the art are needed.
Disclosure of Invention
Aiming at the technical problems in the prior art, the invention provides a method for determining related substances in 6-bromohexyltrimethylammonium bromide by an ion pair chromatography. The invention provides a method for determining related substances in 6-bromohexyltrimethylammonium bromide by using an ion-pair chromatography, aiming at the defects of the prior art and achieving the following purpose, the method can be used for carrying out qualitative and quantitative analysis on the 6-bromohexyltrimethylammonium bromide and the impurity hexamethonium bromide by using the ion-pair chromatography, and can provide a quality control method for the synthesis process of the 6-bromohexyltrimethylammonium bromide.
The technical scheme provided by the invention is as follows:
a method for measuring related substances in 6-bromohexyltrimethylammonium bromide by using an ion-pair chromatography method is used for detecting main components of the 6-bromohexyltrimethylammonium bromide and impurities of hexamethonium bromide, and comprises the following steps:
(1) preparation of Standard solutions
Weighing 6-bromohexyltrimethylammonium bromide and hexamethonium bromide standard substances, respectively dissolving with ultrapure water to a constant volume, and shaking up to obtain standard substance solutions of the 6-bromohexyltrimethylammonium bromide and the hexamethonium bromide;
(2) preparation of sample solution
Weighing a 6-bromohexyltrimethylammonium bromide sample, dissolving the 6-bromohexyltrimethylammonium bromide sample with ultrapure water to a constant volume, and shaking up to obtain a sample solution;
(3) Determination of Standard and sample solutions
According to certain chromatographic conditions, carrying out sample injection analysis on the standard substance and the sample solution by using an ion chromatograph, and simultaneously recording peak areas;
the chromatographic conditions are as follows: the chromatographic column is a reversed phase C18 column; the eluent of the chromatographic column is a mixed solution consisting of perchloric acid solution, acetonitrile and water; the temperature of the chromatographic column is 25-45 ℃; the detector is a bipolar pulse conductance detector, and the temperature of the detector is 30-45 ℃; the suppressor is a cation suppressor, the current of the suppressor is 15-60 mA, the regeneration liquid of the suppressor is 30mmol/L sodium hydroxide, and the flow rate is 1.0-2.0 mL/min;
(4) calculating the content of the substance to be detected
And calculating the contents of 6-bromohexyltrimethylammonium bromide and hexamethonium bromide according to an external standard method.
Under the conditions of this technical scheme, the quaternary ammonium salt groups (R) of 6-bromohexyltrimethylammonium bromide and hexamethonium bromide4N+) Will react with ion pairing reagent (ClO)4 -) Forming neutral "ion pairs" distributed between the stationary phase (hydrophobic environment) and the mobile phase (hydrophilic environment). The hexamethonium bromide has two quaternary ammonium salt groups, so that the hydrophilicity is stronger, the elution is easier, while the retention of the 6-bromohexyltrimethylammonium bromide is stronger, the content of an organic reagent in a mobile phase needs to be increased, the adsorption of ions on the reagent on a stationary phase is reduced, and the elution is accelerated. The two are separated by chromatography, and converted into quaternary ammonium base (R) by cation inhibitor 4N-OH) as measured by a conductivity detector.
The method for determining related substances in 6-bromohexyltrimethylammonium bromide by using the ion pair chromatography is characterized in that in the leacheate, a 50mmol/L perchloric acid solution, acetonitrile and water are added according to a volume ratio of 10: 5: 85-5: 20: 75, and mixing the components in a ratio of 75.
The method for determining related substances in 6-bromohexyltrimethylammonium bromide by using the ion pair chromatography is characterized in that the elution mode of the eluent is gradient elution, the elution time is 0-4.1min, and the volume ratio of 50mmol/L perchloric acid solution to acetonitrile to water is 10: 5: 85; 4.1-14min, wherein the volume ratio of 50mmol/L perchloric acid solution to acetonitrile to water is 5: 20: 75; 14-20min, wherein the volume ratio of 50mmol/L perchloric acid solution to acetonitrile to water is 5: 20: and 75, adjusting to 10: 5: 85.
a method for determining related substances in 6-bromohexyltrimethylammonium bromide by using ion pair chromatography as described above, wherein the flow rate of the leacheate is 1.0 mL/L.
Under the selection of the types, gradient elution conditions and flow rates of the leacheate, the weak retention component of the hexamethonium bromide can obtain good separation effect with other weak retention components, and the strong retention component of the 6-bromohexyltrimethylammonium bromide can be kept for 12.629min under the action of acetonitrile, so that the test time is saved.
A method for the determination of related substances in 6-bromohexyltrimethylammonium bromide by ion-pair chromatography as described above, the temperature of the column being 30 ℃. Under the condition of the technical scheme, the separation degree and the peak area response of the component to be detected are good. Experiments prove that the separation degree of the hexamethonium bromide is gradually reduced along with the temperature of the chromatographic column rising from 25 ℃ to 45 ℃, a low-temperature region is preferably 25-30 ℃, and the peak area response of the 6-bromohexyltrimethylammonium bromide at 25 ℃ is obviously lower than 30 ℃, so that the temperature of the chromatographic column is selected to be 30 ℃ as the most preferable temperature.
A method for the determination of related substances in 6-bromohexyltrimethylammonium bromide by ion-pair chromatography as described above, the temperature of the detector being 32 ℃. Because the suppressor constantly heats in the continuous work of impressed current, under the condition of this technical scheme, detector temperature colorimetric column temperature is slightly higher, can reduce the influence of suppressor thermal effect to the detector.
As described aboveThe method for measuring related substances in 6-bromohexyltrimethylammonium bromide by using the ion pair chromatography is characterized in that the suppressor current is 30 mA. Under the conditions of the technical scheme, H in the mobile phase+The type cation pair reagent can be completely inhibited, and the background conductance is reduced to 3.99 mu S/cm.
A method for measuring related substances in 6-bromohexyltrimethylammonium bromide by ion pair chromatography as described above, wherein the flow rate of the suppressor regenerant was 1.7 mL/min. Under the condition of the technical scheme, NaOH solution is used as cation suppressor regeneration solution, OH-Can pass through the anion exchange membrane and H in the mobile phase+Combined with generated water, reduces background conductivity and simultaneously reacts with ion-pairing reagent ClO4 -Exchange is carried out, and the detection sensitivity is improved. But ClO4 -Has strong affinity to anion exchange membrane for increasing OH-And ClO4 -The flow rate of the regeneration liquid is adjusted to be 1.7mL/min by the driving force of the anion exchange membrane, so that OH can be ensured-Has a regeneration capacity of about 10 times that of ClO4 -. In the present invention, OH is-The regeneration capacity of (1.7 mL/min) is the product of the concentration of the regeneration liquid and the flow rate, ClO4 -Has a capacity of ClO4 -Concentration times flow rate (5 mmol/L1.0 mL/min).
In the method for determining related substances in 6-bromohexyltrimethylammonium bromide by using the ion-pair chromatography method, the sample amount of the standard solution and the sample solution is 5 mu L. Under the condition of the technical scheme, the chromatographic peak symmetry of the 6-bromohexyltrimethylammonium bromide serving as the main component is good, the baseline is stable, and the noise is small.
The method for measuring related substances in 6-bromohexyltrimethylammonium bromide by using the ion pair chromatography is characterized in that the filler of the chromatographic column is octadecylsilane chemically bonded silica with the particle size of 5 mu m; the chromatographic column has an inner diameter of 4mm and a length of 250 mm. Under the condition of the technical scheme, the 6-bromohexyltrimethylammonium bromide and the hexamethonium bromide have good separation performance on a reversed phase C18 chromatographic column. If a polymer chromatographic column with polystyrene-divinylbenzene matrix as a filler is used as an analytical column, the strongly-retained component 6-bromohexyltrimethylammonium bromide needs to be eluted by selecting higher-concentration acetonitrile (50%), and considering the influence of an organic solvent on the service life of a cation suppressor, a reversed-phase C18 chromatographic column is selected.
The technical scheme provided by the invention has the beneficial effects that:
1. the method for determining related substances in 6-bromohexyltrimethylammonium bromide by using the ion pair chromatography provided by the invention successfully establishes the ion chromatography method for determining the contents of the main component 6-bromohexyltrimethylammonium bromide and the impurity hexamethonium bromide, is simple, rapid, accurate and reliable, lays a foundation for development and utilization of the quality control level of the synthetic process of 6-bromohexyltrimethylammonium bromide, and provides an effective reference for application of 6-bromohexyltrimethylammonium bromide.
2. According to the method for determining related substances in 6-bromohexyltrimethylammonium bromide by using the ion pair chromatography, on one hand, because the saturated alkyl quaternary ammonium salt has no chromophoric group and has good conductance response, the conductivity detector is used for detecting the saturated alkyl quaternary ammonium salt, so that the limitation of detection of a mass spectrum detector is broken; on the other hand, compared with the mass spectrometry, the method has the advantages of low test cost and no complex pretreatment, the 6-bromohexyl trimethyl ammonium bromide sample can be directly injected and analyzed after being dissolved by adding water, and the method is simple and easy to implement.
3. The method has high precision, and by adopting the detection method, the RSD of the 6-bromohexyltrimethylammonium bromide is less than 0.80 percent and the RSD of the hexamethylenamine bromide is less than 1.10 percent by comparing the peak areas after 6 times of accurate sample injection analysis and simultaneously recording the peak areas, thereby showing that the method has good reproducibility.
4. The method performs a standard addition recovery experiment, the standard addition recovery rate of the 6-bromohexyltrimethylammonium bromide is between 98.6 and 103.3 percent, and the standard addition recovery rate of the hexamethonium bromide is between 95.8 and 99.1 percent, so that the method has high accuracy of the test result.
Drawings
FIG. 1 is a chromatogram for detecting 6-bromohexyltrimethylammonium bromide standard in example 3 of the present invention;
FIG. 2 is a chromatogram for detecting hexamethonium bromide as an impurity in a sample of 6-bromohexyltrimethylammonium bromide # 1 in accordance with example 4 of the present invention;
FIG. 3 is a chromatogram obtained when hexamethonium bromide as an impurity in a 6-bromohexyltrimethylammonium bromide 2# sample is detected in example 4 of the present invention;
FIG. 4 is a chromatogram of comparative example 1 of the present invention, which is a chromatogram obtained by detecting a mixed standard of 6-bromohexyltrimethylammonium bromide and hexamethonium bromide using a polymer chromatographic column filled with polystyrene-divinylbenzene matrix;
FIG. 5 is a chromatogram of a mixed standard of 6-bromohexyltrimethylammonium bromide and hexamethonium bromide after adjusting the acetonitrile content by using a polymer chromatographic column filled with a polystyrene-divinylbenzene matrix in comparative example 1 of the present invention;
in fig. 1, 2, 3, 4 and 5, the abscissa represents time and the ordinate represents conductance values.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the contents in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items. It is to be understood that the various starting materials of the present invention may be commercially available, unless otherwise specified.
The ion chromatograph adopted by the invention is an ion chromatograph of which the model is CIC-D180 and is produced by Qingdaoshan chromatography technology Limited company and is an existing product.
EXAMPLE 16 Leaching solution conditions for Bromohexyltrimethylammonium Bromide and Hexamethylene Bromide
The method adopts an ion chromatograph to carry out detection, and comprises the following specific steps:
(1) preparing 6-bromohexyltrimethylammonium bromide and hexamethonium bromide standard stock solutions
102.04mg of 6-bromohexyltrimethylammonium bromide standard substance (with the purity of 98%) is precisely weighed into a 100mL volumetric flask, dissolved by ultrapure water, and is subjected to constant volume to reach a scale, and shaken up to prepare 1000 mg/L6-bromohexyltrimethylammonium bromide standard stock solution.
Precisely weighing 101.01mg of a hexamethonium bromide standard substance (with the purity of 99%) in a 100mL volumetric flask, dissolving with ultrapure water, fixing the volume to a scale, shaking up, and preparing into 1000mg/L hexamethonium bromide standard stock solution.
(2) Preparing a mixed standard solution of 6-bromohexyltrimethylammonium bromide and hexamethonium bromide
Accurately transferring 1.0mL, 0.5mL 6-bromohexyltrimethylammonium bromide and hexamethonium bromide standard stock solutions into a 10mL volumetric flask, dissolving with ultrapure water, fixing the volume to a scale, and shaking up to obtain a mixed standard solution of 50 mg/L6-bromohexyltrimethylammonium bromide and 100mg/L hexamethonium bromide.
(3) Preparing 6-bromohexyl trimethyl ammonium bromide sample solution
Precisely weighing 19.20mg of 6-bromohexyltrimethylammonium bromide sample No. 1 in a 10mL volumetric flask, dissolving with ultrapure water, and fixing the volume to the scale to prepare a sample solution No. 1.
(3) Setting of ion chromatography conditions
The chromatographic conditions used were as follows:
a chromatographic column: the reverse phase C18 column, wherein the filler of the chromatographic column is octadecylsilane chemically bonded silica with the diameter of 5 mu m, the inner diameter of the chromatographic column is 4mm, and the length of the chromatographic column is 250 mm; temperature of the column: 30 ℃; a detector: a bipolar pulse conductance detector with the model number of SHD-6, which is produced by Qingdao Han chromatography technology Limited company is adopted; detector temperature: at 32 ℃; a suppressor: a 4mm cation suppressor with the model of SHY-C-5, which is produced by Qingdao Han chromatography technology Limited company is adopted; suppressor current: 30 mA; suppressor regeneration liquid: 30mmol/L sodium hydroxide with flow rate of 1.7 mL/min; the sample injection amount is 5 mu L; leacheate: mixing 50mmol/L perchloric acid solution, acetonitrile and water in a certain proportion, and performing isocratic elution or gradient elution respectively at the flow rate of 1.0 mL/min.
(4) Detecting with ion chromatograph
And (3) carrying out sample injection analysis on the mixed standard solution of 6-bromohexyltrimethylammonium bromide and hexamethonium bromide, and taking the retention time of the two solutions as a qualitative basis. And (3) under the same leacheate condition, carrying out sample injection analysis on the 1# sample solution, and inspecting the retention time and the separation degree of the component to be detected in the actual sample. The results are shown in Table 1.
TABLE 1 Effect of leacheate concentration on Retention time and separation of Components to be tested
As can be seen from Table 1, with the increase of the perchloric acid concentration, the retention time of the component to be detected is increased, and the separation degree is increased, because the perchloric acid is used as an ion pair reagent, the higher the concentration is, the neutral ion pair is formed by the action of the perchloric acid and the component to be detected, and the retention is stronger; with the increase of the acetonitrile concentration, the retention time of the component to be detected is shortened, the separation degree is reduced, particularly, the component 6-bromohexyltrimethylammonium bromide with strong retention can not be eluted under the action of an eluent of 5% acetonitrile, because the higher the acetonitrile content is, the higher the acetonitrile content competes with an ion pair reagent for the adsorption position on the surface of a stationary phase, the effective capacity of a column is reduced, and the component to be detected is easier to elute. In order to improve the separation degree of the weakly-retained component, namely the hexamethylamine bromide and accelerate the elution of the 6-bromohexyltrimethylammonium bromide, the gradient elution condition is finally determined to be 0-4.1min, and the volume ratio of 50mmol/L perchloric acid solution to acetonitrile to water is 10: 5: 85 parts by weight; 4.1-14min, wherein the volume ratio of 50mmol/L perchloric acid solution to acetonitrile to water is 5: 20: 75; 14-20min, wherein the volume ratio of 50mmol/L perchloric acid solution to acetonitrile to water is 5: 20: and 75, regulating to 10: 5: 85.
Example 26 Linear parameters and detection limits for Hexyltrimethylammonium Bromide and Hexamethylamine Bromide
The method adopts an ion chromatograph to carry out detection, and comprises the following specific steps:
(1) preparing 6-bromohexyltrimethylammonium bromide and hexamethonium bromide standard stock solutions
Standard stock solutions of 6-bromohexyltrimethylammonium bromide and hexamethonium bromide were prepared as in example 1.
(2) Standard solution series of 6-bromohexyltrimethylammonium bromide and hexamethonium bromide are prepared
Accurately transferring 0.1mL, 0.5mL, 1.0mL, 2.5mL and 5.0mL of 6-bromohexyltrimethylammonium bromide standard solution into a 10mL volumetric flask, dissolving with ultrapure water, fixing the volume to a scale, and shaking up to obtain 6-bromohexyltrimethylammonium bromide standard solution series with the concentrations of 10mg/L, 50mg/L, 100mg/L, 250mg/L and 500mg/L respectively.
Accurately transferring 0.1mL, 0.5mL, 1.0mL, 2.5mL and 5.0mL of the hexamethonium bromide standard stock solutions into a 100mL volumetric flask, dissolving with ultrapure water, fixing the volume to a scale, and shaking up to obtain hexamethonium bromide standard solution series with the concentrations of 1mg/L, 5mg/L, 10mg/L, 25mg/L and 50mg/L respectively.
(3) Setting of ion chromatography conditions
The chromatographic conditions used were the same as in example 1, except that the eluent was a 50mmol/L perchloric acid solution, acetonitrile and water in a 10: 5: 85-5: 20: 75, gradient elution, gradient program shown in table 2.
TABLE 2 gradient program
| Time/min | 50mmol/L perchloric acid solution (%) | Acetonitrile (%) | Water (%) |
| 0 | 10 | 5 | 85 |
| 4 | 10 | 5 | 85 |
| 4.1 | 5 | 20 | 75 |
| 14 | 5 | 20 | 75 |
| 20 | 10 | 5 | 85 |
(4) Drawing of standard curve
Sequentially injecting and analyzing the 6-bromohexyltrimethylammonium bromide standard solution series, and simultaneously recording peak areas, wherein the peak areas are 2028 mu S, 11580 mu S, 24439 mu S, 67184 mu S and 133541 mu S; the concentration-peak area standard curve of 6-bromohexyltrimethylammonium bromide was plotted, and the results are shown in Table 3.
Sequentially injecting and analyzing the hexamethonium bromide standard solution series, and simultaneously recording peak areas, wherein the peak areas are 515 mu S S, 2992 mu S S, 5454 mu S S, 14165 mu S and 29028 mu S; the concentration-peak area standard curve of hexamethonium bromide was plotted and the results are shown in Table 3.
(5) Calculation of detection limits
The concentrations of 6-bromohexyltrimethylammonium bromide and hexamethonium bromide were taken as detection limits when the signal-to-noise ratio S/N was 3, and the results are shown in table 3.
TABLE 3 Linear parameters and detection limits
| Detecting items | Linear range mg/L | Regression equation | Coefficient of correlation R2 | Detection limit mg/L |
| 6-Bromohexyltrimethylammonium bromide | 10-500 | Y＝270.59X-1492.1 | 0.9997 | 1.076 |
| Ammonium hexamethobromide | 1-50 | Y＝580.84X-140.62 | 0.9997 | 0.2327 |
Embodiment 3 a method for detecting purity of 6-bromohexyltrimethylammonium bromide by ion pair chromatography adopts an ion chromatograph to perform detection, and an external standard method single-point quantitative analysis comprises the following specific steps:
(1) Preparing 6-bromohexyltrimethylammonium bromide reference substance and sample solution
Respectively weighing about 50mg of 6-bromohexyltrimethylammonium bromide standard substance (with the purity of 98 percent) and No. 1 and No. 2 and samples in a 100mL volumetric flask, dissolving with ultrapure water, fixing the volume to a scale, and shaking up to obtain the standard substance and the sample solution.
(2) Setting of ion pair chromatographic conditions
The chromatographic conditions used were as in example 1 except for gradient elution and the gradient program as in table 2.
(3) According to the chromatographic conditions, after the baseline of the instrument is balanced, a 6-bromohexyltrimethylammonium bromide standard sample, a 1# sample and a 2# sample are taken respectively, sample introduction is carried out for 6 times in parallel, and peak areas are recorded and detailed in table 4. The chromatogram for detecting the 6-bromohexyltrimethylammonium bromide standard in this example is shown in fig. 1.
(4) The purity of 6-bromohexyltrimethylammonium bromide in the sample is calculated by the following formula.
The results are shown in Table 4.
In the formula
WiPurity of 6-bromohexyltrimethylammonium bromide in sample,%;
Aiis the sample peak area,. mu.S.s;
miis sample mass, mg;
Asstandard peak area, μ S;
msis the standard substance mass, mg;
Pspurity of standard,%;
TABLE 46 purity of bromohexyltrimethylammonium bromide
As can be seen from table 4, the purity of 6-bromohexyltrimethylammonium bromide in samples # 1 and # 2 was 95.5% and 97.9%, respectively, and the RSD was less than 0.80%, respectively, which indicates that the precision of the test in this example was good.
Example 4 method for detecting hexamethonium bromide as impurity in 6-bromohexyltrimethylammonium bromide by ion pair chromatography
The method adopts an ion chromatograph to carry out detection and external standard method standard curve quantitative analysis, and comprises the following specific steps:
(1) preparing a hexamethonium bromide standard stock solution
The standard solution of hexamethonium bromide was prepared as in example 1.
(2) Preparing ammonium hexamethobromide standard solution series
The ammonium hexamethobromide standard solution series was prepared as in example 2.
(3) Setting of ion pair chromatographic conditions
The chromatographic conditions used were as in example 1 except for gradient elution and the gradient program as in table 2.
(4) Drawing of standard curve
The standard curve for hexamethonium bromide was plotted as in example 2.
(5) Preparing 6-bromohexyl trimethyl ammonium bromide sample solution
Weighing about 150mg (W) of 6-bromohexyltrimethylammonium bromide No. 1 and No. 2 samples, adding ultrapure water to 100mL (V), injecting for analysis, and recording peak areas (Y, MuS). In this example, chromatograms for detecting hexamethonium bromide as an impurity in 6-bromohexyltrimethylammonium bromide 1# and 2# samples are shown in fig. 2 and 3.
(6) The amount of hexamethonium bromide was calculated according to the following formula, and the results are shown in Table 5.
In the formula
X is the mass concentration of ammonium hexamethobromide, mg/L;
Y is the peak area of hexamethonium bromide,. mu.S.s;
m is the mass percentage of ammonium hexamethobromide;
v is the volume of the sample solution with constant volume, mL;
w is the mass of the sample, mg;
TABLE 56 content of hexamethylamine bromide impurity in bromohexyltrimethylammonium bromide
As can be seen from Table 5, the purity of the hexamine bromide in the sample No. 1 and the sample No. 2 is 0.66% and 0.26%, respectively, and the RSD is less than 1.10%, and the test precision is good.
Example 5 spiking recovery experiment
Respectively carrying out a 6-bromohexyltrimethylammonium bromide and hexamethonium bromide tri-level labeling test on a 6-bromohexyltrimethylammonium bromide 1# sample, detecting by adopting an ion chromatograph, and carrying out quantitative analysis on a standard curve by an external standard method, wherein the method comprises the following specific steps:
(1) preparing standard stock solution of 6-bromohexyltrimethylammonium bromide and hexamethonium bromide
Standard stock solutions of 6-bromohexyltrimethylammonium bromide and hexamethonium bromide were prepared as in example 1.
(2) Standard solution series of 6-bromohexyltrimethylammonium bromide and hexamethonium bromide are prepared
Standard solutions of 6-bromohexyltrimethylammonium bromide and hexamethonium bromide were prepared as in example 2.
(3) Setting of ion chromatography conditions
The chromatographic conditions used were as in example 1 except for gradient elution and the gradient program as in table 2.
(4) Drawing of Standard Curve
Standard curves for 6-bromohexyltrimethylammonium bromide and hexamethonium bromide were plotted as in example 2.
(5) Preparing 6-bromohexyltrimethylammonium bromide and hexamethonium bromide standard-adding solution
150.37mg of 6-bromohexyltrimethylammonium bromide 1# sample is precisely weighed and placed in a 10mL volumetric flask, dissolved by ultrapure water, and the volume is determined to the scale, so that 1# sample mother liquor is prepared. According to the contents of 6-bromohexyltrimethylammonium bromide and hexamethonium bromide in the sample No. 1 in tables 4 and 5, which are 95.5% and 0.66%, respectively, the mass concentrations of the 6-bromohexyltrimethylammonium bromide and the hexamethonium bromide in the mother liquor are 14360mg/L and 99.24mg/L, respectively.
Preparing a 6-bromohexyltrimethylammonium bromide standard solution: accurately transferring 0.1mL, 0.1mL and 0.1mL of No. 1 sample mother liquor into a 10mL volumetric flask, respectively adding 1.0mL, 1.5mL and 2.0mL of 6-bromohexyltrimethylammonium bromide standard stock solution (1000mg/L), diluting with ultrapure water to a constant volume to a scale mark, shaking uniformly, carrying out sample injection analysis according to example 2, simultaneously recording peak areas, and calculating the standard recovery rate.
Preparing a hexamethonium bromide standard solution, accurately transferring 1mL, 1mL and 1mL1# sample mother solution into a 10mL volumetric flask, respectively adding 50 muL, 100 muL and 150 muL of hexamethonium bromide standard stock solution (1000mg/L) into the volumetric flask, diluting the stock solution with ultrapure water to a constant volume to a scale mark, shaking uniformly, injecting a sample for analysis according to example 2, simultaneously recording the peak area, and calculating the standard recovery rate, wherein the result is shown in Table 6.
TABLE 6 results of recovery test with addition of standard
As can be seen from Table 6, the standard recovery rate of 6-bromohexyltrimethylammonium bromide is between 98.6% and 103.3%, and the standard recovery rate of hexamethonium bromide is between 95.8% and 99.1%, so that the standard recovery rate of a common sample is between 90% and 110%, and the method is accurate and reliable in measurement result.
Comparative example 1
The ion chromatograph is adopted for detection, and the method specifically comprises the following steps:
(1) preparing standard stock solution of 6-bromohexyltrimethylammonium bromide and hexamethonium bromide
Standard stock solutions of 6-bromohexyltrimethylammonium bromide and hexamethonium bromide were prepared as in example 1.
(2) Preparing a mixed standard solution of 6-bromohexyltrimethylammonium bromide and hexamethonium bromide
A standard solution of 6-bromohexyltrimethylammonium bromide and hexamethonium bromide was prepared as in example 1.
(3) Setting of ion chromatography conditions
The chromatographic conditions used were the same as in example 1, except that the column was a polymer column packed with a polystyrene-divinylbenzene matrix, and the gradient procedure is as shown in Table 2.
(4) The mixed standard solution of 6-bromohexyltrimethylammonium bromide and hexamethonium bromide is detected by an ion chromatograph, and a spectrogram is shown in figure 4.
As can be seen from FIG. 4, the weakly retained component, hexamethonium bromide, peaks at 4.098min with symmetrical peak shapes, but the strongly retained component, 6-bromohexyltrimethylammonium bromide, was not eluted.
(5) Adjusting the acetonitrile ratio in the eluate, and the spectrogram is shown in FIG. 5.
The eluent was 50mmol/L perchloric acid solution, acetonitrile and water at 10: 5: 85-5: 50: 45, and gradient elution. As can be seen from FIG. 5, the strong retention component, 6-bromohexyltrimethylammonium bromide, peaked at 16.698 min. The use of this type of column is not recommended in view of the fact that acetonitrile content in the eluent is up to 50% which has an effect on the lifetime of the suppressor.
While there have been shown and described what are at present considered the fundamental principles and essential features of the invention and its advantages, it will be apparent to those skilled in the art that the invention is not limited to the details of the foregoing exemplary embodiments, but is capable of other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (9)
1. A method for measuring related substances in 6-bromohexyltrimethylammonium bromide by using an ion pair chromatography is characterized in that the method for measuring 6-bromohexyltrimethylammonium bromide and impurity hexamethonium bromide by using the ion pair chromatography comprises the following steps:
(1) preparation of Standard solution
Weighing 6-bromohexyltrimethylammonium bromide and hexamethonium bromide standard substances, respectively dissolving with ultrapure water to a constant volume, and shaking up to obtain standard substance solutions of the 6-bromohexyltrimethylammonium bromide and the hexamethonium bromide;
(2) preparation of sample solution
Weighing a 6-bromohexyltrimethylammonium bromide sample, dissolving the 6-bromohexyltrimethylammonium bromide sample with ultrapure water to a constant volume, and shaking up to obtain a sample solution;
(3) determination of standard and sample solutions
According to certain chromatographic conditions, carrying out sample injection analysis on a standard substance and a sample solution by using an ion chromatograph, and simultaneously recording peak areas;
The chromatographic conditions are as follows: the chromatographic column is a reversed phase C18 column; the eluent of the chromatographic column is a mixed solution consisting of perchloric acid solution, acetonitrile and water; the temperature of the chromatographic column is 25-45 ℃; the detector is a bipolar pulse conductance detector, and the temperature of the detector is 30-45 ℃; the suppressor is a cation suppressor, the current of the suppressor is 15-60 mA, the regenerative liquid of the suppressor is 30mmol/L sodium hydroxide, and the flow rate is 1.0-2.0 mL/min;
(4) calculating the content of the substance to be detected
Calculating to obtain the contents of 6-bromohexyltrimethylammonium bromide and hexamethonium bromide;
the eluent is prepared by mixing 50mmol/L perchloric acid solution, acetonitrile and water in a volume ratio of 10: 5: 85-5: 20: 75 in proportion.
2. The method for determining related substances in 6-bromohexyltrimethylammonium bromide according to claim 1, wherein the elution solution is gradient elution, 0-4.1min, and the volume ratio of 50mmol/L perchloric acid solution to acetonitrile to water is 10: 5: 85 parts by weight; 4.1-14min, wherein the volume ratio of 50mmol/L perchloric acid solution to acetonitrile to water is 5: 20: 75; 14-20min, wherein the volume ratio of 50mmol/L perchloric acid solution to acetonitrile to water is 5: 20: and 75, regulating to 10: 5: 85.
3. The method for determining related substances in 6-bromohexyltrimethylammonium bromide according to claim 2, wherein the flow rate of the leacheate is 1.0 mL/L.
4. The method for determining related substances in 6-bromohexyltrimethylammonium bromide according to claim 1, wherein the temperature of the chromatographic column is 30 ℃.
5. The method for detecting related substances in 6-bromohexyltrimethylammonium bromide according to claim 1, wherein the temperature of the detector is 32 ℃.
6. The method for detecting related substances in 6-bromohexyltrimethylammonium bromide according to claim 1, wherein the suppressor current is 30 mA.
7. The method for determining related substances in 6-bromohexyltrimethylammonium bromide according to claim 1, wherein the flow rate of the suppressor regenerant is 1.7 mL/min.
8. The method for determining related substances in 6-bromohexyltrimethylammonium bromide according to claim 1, wherein the sample volumes of the standard solution and the sample solution are 5 μ L.
9. The method for determining related substances in 6-bromohexyltrimethylammonium bromide according to claim 1, wherein the filler of the chromatographic column is octadecylsilane chemically bonded silica with 5 μm; the chromatographic column has an inner diameter of 4mm and a length of 250 mm.
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