CN112345651B - Method for determining content of halogenated acid in chloral hydrate or preparation thereof - Google Patents
Method for determining content of halogenated acid in chloral hydrate or preparation thereof Download PDFInfo
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Abstract
The application relates to a method for detecting the content of impurities in a medicine, in particular to a method for determining the content of chloral hydrate or halogenated acid in a preparation thereof by gas chromatography.
Description
Technical Field
The application relates to a method for detecting the content of impurities in a medicine, in particular to a method for determining the content of chloral hydrate or halogenated acid in a preparation thereof by gas chromatography.
Background
In 2017, 10 and 27, the international cancer research institution of the world health organization publishes a primarily-finished carcinogen list, and trichloroacetic acid and dichloroacetic acid are both in a class 2B carcinogen list.
However, the existing quality standards (including the standards of Chinese pharmacopoeia, United states Pharmacopeia, European Pharmacopeia, Japanese pharmacopoeia, etc.) of chloral hydrate bulk drugs and preparations do not contain a method for measuring the content of halogenated acid in chloral hydrate preparations.
Disclosure of Invention
The invention provides a method for determining the content of halogenated acid in chloral hydrate or a preparation thereof by gas chromatography, which comprises the following steps:
the first step is as follows: optionally dissolving or diluting chloral hydrate or a preparation thereof of a sample to be tested with water, and then carrying out sample pretreatment, wherein the pretreatment comprises the following operations:
(1) adding alkali into the sample, and enabling chloral hydrate to fully react with the alkali;
(2) adding an acid to the reaction solution obtained in the operation (1);
(3) adding an organic solvent which is not miscible with water for extraction to obtain an organic phase;
(4) subjecting the halogenated acid in the organic phase to an esterification derivatization reaction;
(5) optionally adding an inorganic salt solution into the reaction solution in the operation (4) for extraction, and taking an organic phase as a test solution;
the second step is that: precisely weighing a halogenated acid reference substance, dissolving the halogenated acid reference substance in water to obtain a reference substance diluent, and performing pretreatment operation on the reference substance diluent in the first step to obtain a reference substance solution; and
the third step: detecting the reference solution and the test solution respectively by gas chromatography, and calculating the content of the halogenated acid.
In the method, the chloral hydrate or the preparation thereof is pretreated appropriately to obtain a test solution, and then the test solution is detected by gas chromatography, so that factors interfering with the detection result are avoided to the maximum extent. The method of the invention has the advantages of convenient operation, high sensitivity, high accuracy and strong specificity, and meets various requirements of quantitative detection methodology.
Drawings
FIG. 1: and (3) carrying out a map obtained in the specificity verification aiming at the trichloroacetic acid detection method.
FIG. 2: and (3) carrying out trichloroacetic acid detection on the acid degradation solution to obtain a spectrum.
FIG. 3: and (3) carrying out trichloroacetic acid detection on the alkali degradation solution to obtain a spectrum.
FIG. 4: and (3) carrying out trichloroacetic acid detection on the high-temperature degradation solution to obtain a spectrum.
FIG. 5: standard curve diagram in trichloroacetic acid detection method.
FIG. 6: the spectrum obtained in the specificity verification is carried out aiming at the dichloroacetic acid detection method.
FIG. 7: standard curve diagram in the dichloroacetic acid detection method.
FIG. 8: the spectrum obtained in the comparative example.
Detailed Description
In the gas chromatography detection of the present application, the actually detected substances are products (such as methyl trichloroacetate and methyl dichloroacetate) obtained after esterification and derivatization of halogenated acids. However, for convenience, the actually detected substances will be referred to as halogenated acids (e.g., trichloroacetic acid and dichloroacetic acid).
In some embodiments, the present invention provides a method for determining the content of haloacid in chloral hydrate or a formulation thereof by gas chromatography comprising the steps of:
the first step is as follows: optionally dissolving or diluting chloral hydrate or a preparation thereof of a sample to be tested with water, and then carrying out sample pretreatment, wherein the pretreatment comprises the following operations:
(1) adding alkali into the sample, and enabling chloral hydrate to fully react with the alkali;
(2) adding an acid to the reaction solution obtained in the operation (1);
(3) adding an organic solvent which is not miscible with water for extraction to obtain an organic phase;
(4) subjecting the halogenated acid in the organic phase to an esterification derivatization reaction;
(5) optionally adding an inorganic salt solution into the reaction solution in the operation (4) for extraction, and taking an organic phase as a test solution;
the second step is that: precisely weighing a halogenated acid reference substance, dissolving the halogenated acid reference substance in water to obtain a reference substance diluent, and performing pretreatment operation on the reference substance diluent in the first step to obtain a reference substance solution; and
the third step: detecting the reference solution and the test solution respectively by gas chromatography, and calculating the content of the halogenated acid.
The steps of the method of the invention may be performed in any order, for example the second step may be performed first and then the first step.
In a preferred embodiment, the halogenated acid is a chlorinated acid, preferably trichloroacetic acid or dichloroacetic acid.
In a preferred embodiment, the base in the pretreatment operation (1) is an inorganic base, preferably sodium hydroxide, potassium hydroxide, more preferably an aqueous sodium hydroxide solution or an aqueous potassium hydroxide solution.
In a preferred embodiment, the acid in the pretreatment operation (2) is a mineral acid, preferably sulfuric acid or hydrochloric acid, more preferably sulfuric acid.
In a preferred embodiment, no further treatment, for example extraction with addition of an organic solvent such as n-hexane, is carried out between the pretreatment operations (1) and (2).
In a preferred embodiment, a sodium chloride solution, preferably a saturated sodium chloride solution, is added to the solution obtained in operation (2) before the pretreatment operation (3).
In a preferred embodiment, the organic solvent in the pretreatment operation (3) is an ether having 2 to 10 carbon atoms, preferably methyl t-butyl ether.
In a preferred embodiment, the esterification derivatization reaction in the pretreatment operation (4) is a methyl esterification derivatization reaction, which preferably uses a solution of methanol sulfate as the derivatization reagent.
In a preferred embodiment, in the pretreatment operation (5), the different inorganic salt solutions can be added in a plurality of portions. For example, a sodium chloride solution may be added to perform extraction, an organic phase may be retained, a sodium bicarbonate solution may be added thereto to perform extraction, and the obtained organic phase may be used as a test solution.
In a preferred embodiment, a capillary column using 6% cyanopropylphenyl-94% methylpolysiloxane as a stationary liquid was used in the gas chromatography.
In a preferred embodiment, the gas chromatography uses an ECD detector.
In a preferred embodiment, the gas chromatography is temperature programmed chromatography.
In a preferred embodiment, the conditions for the gas chromatography are as follows:
sample inlet temperature: 208 ℃ and 212 ℃, preferably 210 ℃;
initial column temperature: 198-202 ℃, preferably 200 ℃;
detector temperature: 248-252 ℃, preferably 250 ℃; and is
The carrier gas is nitrogen.
As will be appreciated by those skilled in the art, in chromatograms obtained according to the methods of the present application, the retention time of a haloacid chromatographic peak may vary from model to model of gas chromatograph and/or from model and batch to batch of chromatographic columns.
In a preferred embodiment, the halogenated acid content is calculated by the following formula:
wherein:
AXrepresents the peak area of the halogenated acid in the test solution;
cRrepresents the concentration of halogenated acid in the control solution, mu g/ml;
v represents the dilution volume, ml, of the sample to be tested;
ARrepresents the peak area of the halogenated acid in the control solution;
w represents the amount of chloral hydrate, g, in the sample to be tested.
Examples
The invention is further described below in connection with examples, which are not provided to limit the scope of the invention.
Example 1 detection method of trichloroacetic acid and Dichloroacetic acid
1.1 instruments and reagents
The instrument comprises the following steps: gas chromatograph, GC-2014, Shimadzu, Japan; 7890B, Agilent, USA; electronic analytical balance, CPA225D, sartorius scientific instruments ltd, etc.
Reagent: a reference substance dichloroacetic acid with the content of 98.4 percent, which is purchased from Dr. Ehrenstontorfer GmbH, a reference substance trichloroacetic acid with the content of more than or equal to 99.0 percent, which is purchased from Tokyo chemical industry Co., Ltd., methyl tert-butyl ether and methanol are used as chromatographic purities, other reagents (concentrated sulfuric acid (with the density of 1.84g/mL), anhydrous sodium sulfate, sodium chloride, sodium hydroxide, sodium bicarbonate and the like) are used as analytical purities, and water is self-made ultrapure water.
10% sulfuric acid methanol solution: adding 5ml sulfuric acid slowly into 45ml methanol under stirring, and standing to room temperature to obtain the final product.
Chloral hydrate concentrate (74.48%, w/w) (batch No. 20181022) obtained from Xinjiang Tefeng pharmaceutical products Co., Ltd under 1.2 chromatographic conditions
A chromatographic column: a capillary column (30 m.times.0.53 mm. times.3 μm) using 6% cyanopropylphenyl-94% methylpolysiloxane as a stationary liquid;
temperature rising procedure: the initial temperature is 200 ℃, the temperature is maintained for 2min, the temperature is raised to 210 ℃ at the speed of 1 ℃/min, and the temperature is maintained for 3 min;
sample inlet temperature: at 210 ℃;
ECD detector temperature: 250 ℃;
column flow rate: 1.0 ml/min;
the split ratio is as follows: 50: 1;
sample introduction amount: 1 mul;
carrier gas: nitrogen (purity > 99.999%).
1.3 solution preparation
(1) Preparation of dilution solution of sample to be tested
Precisely weighing (1.01320g) chloral hydrate concentrated solution of a sample to be measured, placing the concentrated solution in a 10mL measuring flask, adding water to dissolve and dilute the concentrated solution to a scale (10mL, which is the dilution volume of the sample to be measured), shaking up, and precisely weighing 1mL as the sample diluent to be measured.
(2) Preparation of test solution
Placing the sample diluent (1ml) to be tested in a 10ml centrifuge tube with a plug, adding 1ml of 1mol/L sodium hydroxide solution, and fully and uniformly mixing. After the reaction is completely finished (namely the solution is colorless, clear and transparent or has a transparent solution layering phenomenon, about 10min), adding 1ml of 3mol/L sulfuric acid solution, shaking up, standing for 1 min, adding 1ml of saturated sodium chloride solution, shaking up, standing for 3min, precisely adding 5ml of methyl tert-butyl ether for primary extraction, standing and layering. Methyl tert-butyl ether (4 ml) was measured precisely, placed in a 20ml headspace bottle, added with 10% methanol sulfate (4 ml), sealed, and placed in an oven at 60 ℃ for derivatization for 1.5 hours. Taking out the headspace bottle, cooling, opening the cover, adding 5ml of saturated sodium chloride solution, shaking for 1 minute, standing for 3 minutes, discarding the lower layer water and all solids by using a dropper, adding 1ml of saturated sodium bicarbonate solution, shaking uniformly, standing for 1 minute, taking the ether layer, filtering by using a filter paved with anhydrous sodium sulfate, and taking the filtrate as a sample solution.
(3) Preparation of control solutions
Trichloroacetic acid and dichloroacetic acid reference substances are precisely weighed, respectively 95.13mg and 99.99mg, dissolved by water and quantitatively diluted to prepare a solution containing 9.513 mu g of trichloroacetic acid and 9.839 mu g of dichloroacetic acid in each 1ml of the solution as reference substance diluent.
Precisely measuring 1ml of reference substance diluent, and treating the reference substance diluent according to the preparation method of the test solution to obtain the reference substance solution.
1.4 assay method
Precisely measuring 1 μ l of each of the reference solution and the sample solution, respectively injecting into a gas chromatograph, and recording chromatogram.
1.5 calculation of
Example 2 trichloroacetic acid detection method validation
SUMMARY
The trichloroacetic acid detection method was verified, and the verification results are summarized in table 1.
TABLE 1 summary of trichloroacetic acid detection method validation
DETAILED DESCRIPTIONS
2.1 instruments and reagents
2.1.1 instrumentation
TABLE 2 Instrument Equipment
| Serial number | Name of instrument | Model number | Manufacturer of the product |
| 1 | Gas chromatograph | GC-2014 | |
| 2 | Gas chromatograph | 7890B | Agilent, USA |
| 3 | Electronic balance | CPA225D | Sadoris scientific instruments Ltd |
| 4 | Electronic balance | BSA224S | Sadoris |
| 5 | Electric heating constant temperature water bath | DZKW-S-4 | Beijing City Guangming medical |
| 6 | Electric heating blowing dry box | GZX-9246MBE | Medical equipment of Shanghai Boxun practice company Limited |
2.1.2 reagents
Trichloroacetic acid reference substance (batch number: YBMXD-TJ), content 99.0%, Tokyo Kaisha;
chloral hydrate concentrate (74.48%, w/w) (batch No. 20170322), Texacon pharmaceuticals, Inc.;
methyl tert-butyl ether and methanol were chromatographically pure, the remaining reagents (such as sodium chloride, anhydrous sodium sulfate, sodium bicarbonate, concentrated sulfuric acid (density of 1.84g/mL), sodium hydroxide and hydrochloric acid) were analytically pure, and water was purified water.
2.2 chromatographic conditions
A chromatographic column: 6% cyanopropylphenyl-94% methylpolysiloxane as stationary phase (DB-624 UI);
temperature rising procedure: the initial temperature is 200 ℃, the temperature is maintained for 2min, the temperature is raised to 210 ℃ at the speed of 1 ℃/min, and the temperature is maintained for 3 min;
sample inlet temperature: at 210 ℃;
ECD detector temperature: 250 ℃;
column flow rate: 1.0 ml/min;
the split ratio is as follows: 50: 1;
sample introduction amount: 1 mul;
carrier gas: nitrogen (purity > 99.999%).
2.3 solution preparation
2.3.1 preparation of dilution of sample to be tested
Taking about 1g of chloral hydrate concentrated solution of a sample to be measured, precisely weighing, placing in a 10mL measuring flask, adding water to dissolve and dilute to a scale (10mL, which is the dilution volume of the sample to be measured), shaking up, and precisely weighing 1mL as the sample diluent to be measured.
2.3.2 preparation of test solutions
Placing a sample diluent (1ml) to be detected in a 10ml centrifuge tube with a plug, adding 1ml of 1mol/L sodium hydroxide solution, shaking up, standing for 10 minutes, adding 1ml of 3mol/L sulfuric acid solution, shaking up, standing for 1 minute, adding 1ml of saturated sodium chloride solution, shaking up, standing for 3 minutes, precisely adding 5ml of methyl tert-butyl ether for primary extraction, standing and layering. The methyl tert-butyl ether layer (4 ml) was precisely weighed, placed in a 20ml headspace bottle, added with 4ml of 10% methanol sulfate solution, sealed, and placed in an oven (i.e., the above-mentioned electrothermal forced air drying oven) at 60 ℃ for derivatization for 1.5 hours. Taking out the headspace bottle, cooling, opening the cover, adding 5ml of saturated sodium chloride solution, shaking for 1 minute, standing for 3 minutes, discarding the lower layer water and all solids by using a dropper, adding 1ml of saturated sodium bicarbonate solution, shaking uniformly, standing for 1 minute, taking the ether layer, filtering by using a filter paved with anhydrous sodium sulfate, and taking the filtrate as a sample solution.
2.3.3 preparation of reference stock solutions
Taking 100mg of trichloroacetic acid as a reference substance, precisely weighing, placing in a 100ml measuring flask, adding water to dissolve, diluting to scale, and shaking uniformly to obtain a reference substance stock solution.
2.3.4 preparation of control solutions
Precisely measuring 1ml of the reference stock solution, placing in a 100ml measuring flask, adding water to dilute to scale, and shaking up to obtain the reference diluent. Precisely measuring 1ml of reference substance diluent, and treating the reference substance diluent according to the preparation method of the test solution to obtain the reference substance solution.
2.3.5 preparation of blank solution
Precisely measuring 1ml of methanol, and carrying out the same treatment on the methanol as the diluent of the sample to be detected to obtain a blank solution.
2.3.6 preparation of adjuvant solution
Weighing all auxiliary materials except chloral hydrate according to the composition of the chloral hydrate concentrated solution, and stirring for dissolving to obtain the chloral hydrate.
2.3.7 preparation of negative solutions
Taking the adjuvant solution, and preparing a negative solution according to the preparation method of the test solution.
2.4 System applicability
The control solution was taken and measured 6 times by using the gas chromatograph 7890B under the above-mentioned chromatographic conditions, and the results are shown in table 3.
TABLE 3 System suitability test results
| Serial number | Retention time (min) | Peak area | Number of theoretical plate | Symmetry factor |
| 1 | 9.663 | 8920 | 60472 | 0.98 |
| 2 | 9.663 | 8941 | 59366 | 0.98 |
| 3 | 9.669 | 9016 | 57381 | 1.01 |
| 4 | 9.661 | 9022 | 61534 | 0.98 |
| 5 | 9.659 | 8924 | 60420 | 1.00 |
| 6 | 9.666 | 9151 | 58381 | 1.00 |
| Mean value of | 9.664 | 8996 | 59592 | 0.99 |
| RSD(％) | 0.04 | 0.98 | / | / |
Note: and/represents no relevant information.
The result shows that the peak area RSD of 6 times of tests is less than 5.0%, the retention time RSD is less than 1.0%, the number of theoretical plates is more than 20000, and the symmetry factor is less than 1.5, which indicates that the system has good applicability.
2.5 specificity
2.5.1 solvent, adjuvant interference test
The blank solution, the control solution, the negative solution and the test solution were taken and measured by gas chromatograph 7890B according to the above chromatographic conditions, and the results are shown in FIG. 1.
As can be seen from FIG. 1, the solvent and excipients did not interfere with the detection.
2.5.2 forced degradation test
The forced degradation test is to prepare a degradation sample solution under the conditions of acid, alkali, oxidation and high temperature, measure by using a gas chromatograph 7890B according to the chromatographic conditions, and examine the impurity separation and detection capability of the method.
2.5.2.1 acid degradation test
Taking about 1g of chloral hydrate concentrated solution, precisely weighing, placing in a 10ml measuring flask, adding 2ml of 1mol/L hydrochloric acid solution, plugging, carrying out water bath at 90 ℃ for 2h, cooling, adding 2ml of 1mol/L sodium hydroxide solution, adding water to dilute to scale, and shaking up. Precisely measuring 1ml of acid degradation sample, treating according to the preparation method of the test solution to obtain acid degradation solution, and measuring with gas chromatograph 7890B according to the above chromatographic conditions, with the result shown in FIG. 2.
The results show that the main peak is well separated from the adjacent impurity peaks under acid degradation conditions.
2.5.2.2 alkali degradation test
Precisely weighing about 1g of chloral hydrate concentrated solution, placing the concentrated solution into a 10ml measuring flask, adding 1ml of 0.5mol/L sodium hydroxide solution, sealing for 5min, immediately adding 1ml of 0.5mol/L hydrochloric acid solution, adding water to dilute to scale, and shaking up. Precisely measuring 1ml of alkali degradation sample, treating according to the preparation method of the test solution to obtain alkali degradation solution, and measuring with gas chromatograph 7890B according to the above chromatographic conditions, with the result shown in FIG. 3.
The results show that the main peak is well separated from the adjacent impurity peaks under alkaline degradation conditions.
2.5.2.3 high temperature degradation test
Precisely weighing about 1g of chloral hydrate concentrated solution, placing in a 10ml measuring flask with a plug, placing in a water bath at 90 ℃, heating for 5h in a dark place, cooling, adding water to dilute to a scale, and shaking up. Precisely measuring 1ml of high temperature degradation sample, processing the sample solution according to the preparation method of the sample solution to obtain high temperature degradation solution, and measuring by using a gas chromatograph 7890B according to the chromatographic conditions, wherein the result is shown in figure 4.
The results show that the main peak is well separated from the adjacent impurity peaks under high temperature conditions.
2.5.3 conclusions of the specificity test
The results of the special tests show that the solvent and the auxiliary materials do not interfere with the determination of trichloroacetic acid, and chromatographic peak separation degrees in acid, alkali and high-temperature strong degradation samples are good without interfering the determination. Thus, the method specificity is good.
2.6 detection and quantitation limits
Taking a reference substance diluent, diluting with water to prepare solutions for measuring the detection limit and the quantification limit with different concentrations, precisely measuring 1ml of the solution for measuring the detection limit and the quantification limit, and treating the solution for measuring the detection limit and the quantification limit according to the preparation method of the test solution to obtain the reference substance solution. According to the chromatographic conditions, a gas chromatograph GC-2014 is used for measuring, and S/N is more than or equal to 3 as a detection limit and S/N is more than or equal to 10 as a quantification limit. The results are shown in Table 4.
TABLE 4 detection Limit and quantitation Limit test results
Note: and/represents no relevant information.
The result shows that the trichloroacetic acid detection limit concentration is 0.011 mu g/ml; the limit concentration of quantification is 0.218 mu g/ml, and the RSD of the main peak area tested under 6 times of limit concentration of quantification is less than 5.0%.
2.7 Linear sum Range
Trichloroacetic acid control stock solutions were quantitatively diluted with purified water to give solutions having concentrations of 1.038, 10.38, 20.76, 41.51, and 62.27 μ g/ml, respectively, which were used as solutions for a series of standard curves, and the results were determined according to the methods of the present application, respectively, and are shown in Table 5. The concentration was plotted on the abscissa (x) and the peak area was plotted on the ordinate (y) to obtain FIG. 5.
TABLE 5 measurement results
| Serial number | Concentration (μ g/ml) | Peak area |
| 1 | 1.038 | 1780 |
| 2 | 10.38 | 18600 |
| 3 | 20.76 | 38423 |
| 4 | 41.51 | 76933 |
| 5 | 62.27 | 119802 |
The results show that the linear regression equation of trichloroacetic acid is that y is 1922.5x-1164.9, r is 0.9997 and is more than 0.999, and the trichloroacetic acid has good linearity between 1.038 mug/ml and 62.27 mug/ml.
2.8 precision
Repeatability: taking the chloral hydrate concentrated solution, preparing 6 parts of test solution for determination.
Intermediate precision: the same sample, different personnel with different instruments and equipment, on different dates according to the law determination.
The results are shown in Table 6.
TABLE 6 results of precision test
The result shows that the RSD of the trichloroacetic acid content measured by 6 parts of sample is respectively 2.24 percent and 1.05 percent, the RSD of 12 parts of sample by different personnel and equipment is 1.87 percent, and the method precision meets the requirement.
2.9 accuracy
Taking 25ml of reference substance diluent, placing the reference substance diluent in a 50ml measuring flask, adding water to dilute the reference substance diluent to a scale, and shaking up the reference substance diluent to obtain a reference substance solution for low-concentration recovery rate; taking the reference substance diluent as a reference substance solution for medium concentration recovery rate; taking 10ml of the reference substance stock solution, placing the reference substance stock solution in a 25ml measuring flask, adding water to dilute to the scale, taking 2ml, placing the reference substance stock solution in a 50ml measuring flask, adding water to dilute to the scale, shaking up to obtain the reference substance solution for high-concentration recovery rate.
Precisely measuring 0.5ml of a sample diluent to be measured (wherein the content of trichloroacetic acid is additionally measured according to the method of the application), placing the sample diluent into a 10ml centrifuge tube with a plug, adding 0.5ml of a control solution for low, medium and high concentration recovery rates, preparing three solutions with different concentrations of recovery rates according to the preparation method of the sample solution, and measuring according to the method to calculate the recovery rates. The above operation was repeated 3 times, and the results are shown in Table 7.
TABLE 7 accuracy test results
The result shows that the single recovery rate and the average recovery rate of each concentration are 90.27-98.63%, the RSD of the recovery rate is less than 5.0%, and the method has good accuracy.
2.10 solution stability
Taking the test solution, standing at room temperature, sampling at 0, 2, 4, 6, 8, and 12h, and determining according to the method. The results are shown in Table 8.
TABLE 8 test article solution stability investigation results
The result shows that the content of the test result at each time point has no obvious change compared with the result of 0h, and the RSD is less than 5.0 percent, which shows that the test solution is stable after being placed at room temperature for 12 h.
2.11 durability
The trichloroacetic acid content of the test sample solution was measured at different injection port temperatures, column temperatures and detector temperatures (see Table 9), and the results are shown in Table 10.
TABLE 9 parameters for variation of chromatographic conditions
| Chromatographic conditions | Original conditions | Examination conditions |
| Sample inlet temperature (. degree. C.) | 210 | 208 and 212 |
| Initial column temperature (. degree. C.) | 200 | 198 and 202 |
| Detector temperature (. degree.C.) | 250 | 248 and 252 |
TABLE 10 durability test results
Note: only the initial column temperature was different and the subsequent temperature program was the same.
The result shows that under the conditions of the sample inlet temperature, the column temperature and the detector temperature, the measured content has no obvious change, and the durability meets the requirement.
Example 3 verification of Dichloroacetic acid detection method
SUMMARY
The dichloroacetic acid detection method was verified, and the verification results are summarized in table 11.
TABLE 11 summary of verification of dichloroacetic acid detection method
DETAILED DESCRIPTIONS
3.1 instruments and reagents
3.1.1 instrumentation
Watch 12 instrumentation
| Serial number | Name of instrument | Model number | Manufacturer of the product |
| 1 | Gas chromatograph | 7890B | Agilent, |
| 2 | Electronic balance | CPA225D | Sadoris scientific instruments Ltd |
| 3 | Electronic balance | BSA224S | Sadoris scientific instruments Ltd |
| 4 | Electric heating blowing dry box | GZX-9246MBE | Medical equipment of Shanghai Boxun practice company Limited |
3.1.2 reagents
Chloral hydrate concentrate (74.48%, w/w) (batch numbers: 20181101, 2181102), Nanjing Tefeng pharmaceutical Co., Ltd;
dichloroacetic acid control (batch No. G149179), content 98.4%, dr. ehrenstorfer GmbH;
the methyl tert-butyl ether and methanol are chromatographically pure, the other reagents (such as sodium chloride, anhydrous sodium sulfate, sodium bicarbonate, concentrated sulfuric acid (density of 1.84g/mL) and sodium hydroxide) are analytically pure, and the water is purified water.
3.2 chromatographic conditions
A chromatographic column: 6% cyanopropylphenyl-94% methylpolysiloxane as stationary phase (DB-624);
temperature rising procedure: the initial temperature is 200 ℃, the temperature is maintained for 2min, the temperature is raised to 210 ℃ at the speed of 1 ℃/min, and the temperature is maintained for 3 min;
sample inlet temperature: at 210 ℃;
ECD detector temperature: 250 ℃;
column flow rate: 1.0 ml/min;
the split ratio is as follows: 50: 1;
sample introduction amount: 1 mul;
carrier gas: nitrogen (purity > 99.999%).
3.3 solution preparation
3.3.1 preparation of dilution of sample to be tested
Taking about 1g of chloral hydrate concentrated solution of a sample to be measured, precisely weighing, placing in a 10mL measuring flask, adding water to dissolve and dilute to a scale (10mL, which is the dilution volume of the sample to be measured), shaking up, and precisely weighing 1mL as the sample diluent to be measured.
3.3.2 preparation of test solutions
Placing a sample diluent (1ml) to be tested in a 10ml centrifuge tube with a plug, adding 1ml of 1mol/L sodium hydroxide solution, shaking up, standing for 10 minutes, adding 1ml of 3mol/L sulfuric acid solution, shaking up, standing for 1 minute, adding 1ml of saturated sodium chloride solution, shaking up, standing for 3 minutes, precisely adding 5ml of methyl tert-butyl ether (the volume of the organic solvent used in the primary extraction) for primary extraction, standing and layering. The methyl tert-butyl ether layer (4 ml) was precisely weighed, placed in a 20ml headspace bottle, added with 4ml of 10% methanol sulfate solution, sealed, and placed in an oven (i.e., the above-mentioned electrothermal forced air drying oven) at 60 ℃ for derivatization for 1.5 hours. Taking out the headspace bottle, cooling, opening the cover, adding 5ml of saturated sodium chloride solution, shaking for 1 minute, standing for 3 minutes, discarding the lower layer water and all solids by using a dropper, adding 1ml of saturated sodium bicarbonate solution, shaking uniformly, standing for 1 minute, taking the ether layer, filtering by using a filter paved with anhydrous sodium sulfate, and taking the filtrate as a sample solution.
3.3.3 preparation of reference stock solutions
Taking 100mg of dichloroacetic acid as a reference substance, precisely weighing, placing in a 100ml measuring flask, adding water to dissolve, diluting to scale, and shaking uniformly to obtain a reference substance stock solution.
3.3.4 preparation of control solutions
Precisely measuring 1ml of the reference stock solution, placing in a 100ml measuring flask, adding water to dilute to scale, and shaking up to obtain the reference diluent. Precisely measuring 1ml of reference substance diluent, and treating the reference substance diluent according to the preparation method of the test solution to obtain the reference substance solution.
3.3.5 preparation of blank solution
Precisely measuring 1ml of methanol, and carrying out the same treatment on the methanol as the diluent of the sample to be detected to obtain a blank solution.
3.3.6 preparation of adjuvant solution
Weighing all auxiliary materials except chloral hydrate according to the composition of the chloral hydrate concentrated solution, and stirring for dissolving to obtain the chloral hydrate.
3.3.7 negative solution
Taking the adjuvant solution, and preparing a negative solution according to the preparation method of the test solution.
3.4 System applicability
The control solution was taken and measured 6 times by using the gas chromatograph 7890B under the above-mentioned chromatographic conditions, and the results are shown in table 13.
TABLE 13 System suitability test results
| Serial number | Retention time (min) | Peak area | Number of theoretical plate | Symmetry factor |
| 1 | 6.720 | 5458.3 | 64030 | 0.97 |
| 2 | 6.718 | 5394.0 | 66670 | 0.97 |
| 3 | 6.717 | 5255.1 | 67553 | 0.97 |
| 4 | 6.719 | 5469.6 | 64876 | 0.96 |
| 5 | 6.719 | 5463.2 | 64908 | 0.97 |
| 6 | 6.720 | 5500.4 | 64045 | 0.97 |
| Mean value of | 6.719 | 5423.4 | 65347 | 0.97 |
| RSD(％) | 0.02 | 1.65 | 2.21 | 0.42 |
The result shows that the peak area RSD of 6 times of tests is less than 5.0%, the retention time RSD is less than 1.0%, the number of theoretical plates is more than 20000, and the symmetry factor is less than 1.5, which indicates that the system has good applicability.
3.5 specificity
3.5.1 solvent, adjuvant interference test
The blank solution, the control solution, the negative solution and the test solution were taken and measured by gas chromatograph 7890B under the above chromatographic conditions, and the results are shown in FIG. 6.
As can be seen from fig. 6, the solvent, excipients did not interfere with the detection.
3.6 detection and quantitation limits
Taking a reference substance diluent, diluting with water to prepare solutions for measuring the detection limit and the quantification limit with different concentrations, precisely measuring 1ml of the solution for measuring the detection limit and the quantification limit, and treating the solution for measuring the detection limit and the quantification limit according to the preparation method of the test solution to obtain the reference substance solution. According to the above chromatographic conditions, the detection limit was S/N.gtoreq.3 and the quantitative limit was S/N.gtoreq.i 0, respectively, as measured by gas chromatograph 7890B. The results are shown in Table 14.
TABLE 14 detection Limit and quantitation Limit test results
Note: and/represents no relevant information.
The result shows that the detection limit of the dichloroacetic acid is 0.050 mu g/ml; the limit of quantitation is 0.202 mug/ml, and the RSD of the main peak area tested under 6 times of limit concentrations is less than 6.0%.
3.7 Linearity and Range
The stock solutions of dichloroacetic acid control were quantitatively diluted with purified water to obtain solutions of 0.2022, 0.5055, 1.011, 5.055, 10.11 and 20.22. mu.g/ml, which were used as solutions for series of standard curves and were measured according to the methods of the present application, and the measurement results are shown in Table 15. Linear regression analysis was performed with the concentration as abscissa (x) and the peak area as ordinate (y), to obtain fig. 7.
Table 15 measurement results
| Serial number | Concentration (μ g/ml) | Peak area |
| 1 | 0.2022 | 311.7 |
| 2 | 0.5055 | 496.9 |
| 3 | 1.011 | 929.5 |
| 4 | 5.055 | 3866.8 |
| 5 | 10.11 | 7459.4 |
| 6 | 20.22 | 15314.2 |
The result shows that the linear regression equation of the dichloroacetic acid is that y is 746.88x +111.52 and r is 0.999, and the dichloroacetic acid has good linearity in 0.2022-20.22 mu g/m.
3.8 repeatability
The aqueous chloral hydrate concentrated solution was used to prepare 6 parts of test sample solution for measurement, and the measurement results are shown in Table 16.
TABLE 16 results of the repeatability tests
The result shows that the RSD of the content of 6 parts of sample dichloroacetic acid is 3.86 percent, which indicates that the repeatability of the method meets the requirement.
3.9 accuracy
Taking about 1g of chloral hydrate concentrated solution, and preparing the sample diluent to be detected according to the preparation method of the sample diluent to be detected (wherein the content of trichloroacetic acid is determined according to the method of the application).
Precisely measuring 0.5ml of the sample diluent to be measured, placing the sample diluent in a 10ml centrifuge tube with a plug, adding 0.5ml of the reference substance diluent, preparing a recovery rate solution according to the preparation method of the test solution, measuring the sample according to the method, and calculating the recovery rate. The above operation was repeated 6 times, and the results are shown in Table 17.
TABLE 17 accuracy test results
The result shows that the single recovery rate and the average recovery rate are 104.81-116.19%, the RSD of the recovery rate is less than 5.0%, and the method has good accuracy.
Comparative example
1.1 instruments and reagents
The instrument comprises the following steps: gas chromatograph, GC-2014, Shimadzu, Japan; electronic analytical balance, CPA225D, sartorius scientific instruments ltd, etc.
The reagents are as described in example 1.
1.2 preparation of dilution of sample to be tested
Taking about 1g of chloral hydrate concentrated solution of a sample to be measured, precisely weighing, placing in a 10mL measuring flask, adding water to dissolve and dilute to a scale (10mL, which is the dilution volume of the sample to be measured), shaking up, and precisely weighing 1mL as the sample diluent to be measured.
1.3 preparation of test solutions
Placing a sample diluent (1ml) to be detected in a 10ml centrifuge tube with a plug, adding 1ml of purified water, shaking up, standing for 10 minutes, adding 1ml of 3mol/L sulfuric acid solution, shaking up, standing for 1 minute, adding 1ml of saturated sodium chloride solution, shaking up, standing for 3 minutes, precisely adding 5ml of methyl tert-butyl ether for primary extraction, and standing for layering. The methyl tert-butyl ether layer (4 ml) was precisely weighed, placed in a 20ml headspace bottle, added with 4ml of 10% methanol sulfate solution, sealed, and placed in an oven at 60 ℃ for derivatization for 1.5 hours. Taking out the headspace bottle, cooling, opening the cover, adding 5ml of saturated sodium chloride solution, shaking for 1 minute, standing for 3 minutes, discarding the lower layer water and all solids by using a dropper, adding 1ml of saturated sodium bicarbonate solution, shaking uniformly, standing for 1 minute, taking the ether layer, filtering by using a filter paved with anhydrous sodium sulfate, and taking the filtrate as a sample solution.
1.4 gas chromatography detection
The sample solution was analyzed by gas chromatography under the chromatographic conditions in example 1, and the chromatogram was shown in FIG. 8.
As can be seen from FIG. 8, the peaks of the dichloroacetic acid and trichloroacetic acid are included in the other peaks. This indicates that if the sample to be tested is not pretreated with alkali, the impurities in the sample to be tested cannot be quantitatively detected.
Various modifications of the invention in addition to those described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference, including all patents, patent applications, journal articles, books, and any other publications, cited in this application is hereby incorporated by reference in its entirety.
Claims (14)
1. A method for determining the content of chloral hydrate or of halogenated acids in its formulations by gas chromatography, comprising the following steps:
the first step is as follows: optionally dissolving or diluting chloral hydrate or a preparation thereof of a sample to be tested with water, and then carrying out sample pretreatment, wherein the pretreatment comprises the following operations:
(1) adding alkali into the sample, and enabling chloral hydrate to fully react with the alkali, wherein the alkali is inorganic alkali;
(2) adding an acid into the reaction liquid obtained in the operation (1), wherein the acid is an inorganic acid;
(3) adding an organic solvent which is immiscible with water for extraction to obtain an organic phase, wherein the organic solvent is ether containing 2-10 carbon atoms;
(4) carrying out esterification derivatization reaction on halogenated acid in an organic phase, wherein the esterification derivatization reaction is methyl esterification derivatization reaction;
(5) optionally adding an inorganic salt solution into the reaction solution in the operation (4) for extraction, and taking an organic phase as a test solution;
the second step is that: precisely weighing a halogenated acid reference substance, dissolving the halogenated acid reference substance in water to obtain a reference substance diluent, and performing pretreatment operation on the reference substance diluent in the first step to obtain a reference substance solution; and
the third step: respectively detecting the reference solution and the test solution by gas chromatography, and calculating the content of the halogenated acid;
wherein the halogenated acid is trichloroacetic acid or dichloroacetic acid.
2. The method of claim 1, wherein the alkali in the pretreatment operation (1) is sodium hydroxide or potassium hydroxide.
3. The process of claim 1, wherein the base in the pretreatment operation (1) is an aqueous sodium hydroxide solution or an aqueous potassium hydroxide solution.
4. A process according to any one of claims 1 to 3, wherein the acid in the pretreatment operation (2) is sulfuric acid or hydrochloric acid.
5. The process of claim 4 wherein the acid in the pretreatment operation (2) is sulfuric acid.
6. The process of any of claims 1-3, wherein the organic solvent in the pre-treatment operation (3) is methyl tert-butyl ether.
7. The method of any one of claims 1 to 3, wherein the esterification derivatization reaction in the pretreatment operation (4) is a methyl esterification derivatization reaction, and a methanol sulfate solution is used as the derivatization reagent.
8. The method of any one of claims 1-3, wherein a capillary column with 6% cyanopropylphenyl-94% methylpolysiloxane as a stationary liquid is used in the gas chromatography.
9. The method of any one of claims 1-3, wherein the gas chromatography uses an ECD detector.
10. The method of any one of claims 1-3, wherein the gas chromatography is temperature programmed chromatography.
11. The method of any one of claims 1-3, wherein the conditions of the gas chromatography are as follows:
sample inlet temperature: 208-212 ℃;
initial column temperature: 198-202 ℃;
detector temperature: 248-252 ℃; and is
The carrier gas is nitrogen.
12. The method of claim 11, wherein the injection port temperature is 210 ℃.
13. The process of claim 11, wherein the initial column temperature is 200 ℃.
14. The method of claim 11, wherein the detector temperature is 250 ℃.
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