CN114705768A - Sample pretreatment for detecting content of chloral hydrate in health product and detection method thereof - Google Patents

Sample pretreatment for detecting content of chloral hydrate in health product and detection method thereof Download PDF

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CN114705768A
CN114705768A CN202210212601.3A CN202210212601A CN114705768A CN 114705768 A CN114705768 A CN 114705768A CN 202210212601 A CN202210212601 A CN 202210212601A CN 114705768 A CN114705768 A CN 114705768A
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chloral hydrate
solution
detecting
health
sample
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陈正毅
袁锟婷
田池
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Guilin Medical University
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Guilin Medical University
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/30—Control of physical parameters of the fluid carrier of temperature
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/74—Optical detectors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/86—Signal analysis
    • G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information

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Abstract

The invention relates to sample pretreatment for detecting the content of chloral hydrate in a health-care product and a detection method thereof. The invention relates to a sample pretreatment method for detecting the content of chloral hydrate in a health-care product, which comprises the following steps: taking a proper amount of sample, adding acid liquor, heating, and decomposing chloral hydrate to generate HCl and HCOOH gas; and absorbing the generated gas by using water to obtain the solution to be detected. The method utilizes chloral hydrate which can generate decomposition reaction under the acidic and heating conditions and generate hydrogen chloride and formic acid with strong volatility, then combines a gas-liquid separation technology to separate the hydrogen chloride and the formic acid, and adopts an analysis technology for detecting chloride ions or formic acid to determine the content of the chloral hydrate in the health-care product. Compared with the prior art, the sample pretreatment and detection method for detecting the content of chloral hydrate in the health-care product can effectively eliminate matrix interference, shorten the time required by the sample pretreatment, greatly simplify the detection and analysis process of chloral hydrate in the health-care product, and have the advantages of simple steps and high sensitivity.

Description

Sample pretreatment for detecting content of chloral hydrate in health product and detection method thereof
Technical Field
The invention relates to the technical field of pharmaceutical analysis, in particular to a sample pretreatment and detection method for detecting the content of chloral hydrate in a health-care product.
Background
Chloral hydrate, chemical name 2, 2, 2-trichloro-1, 1-ethanediol, english name chlorine hydrate, CAS number 302-17-0.
When the chloral hydrate is used as a therapeutic drug, the hypnotic effect is mild, the Rapid Eye Movement Sleep (REMS) is not shortened, and no obvious after-effect exists; if the health care product is added to improve the efficacy, the health care product is illegal. The health product has regulating effect, and is a special food for treating diseases. The national food safety law stipulates that drugs cannot be illegally added into the health care product.
The detection technology of chloral hydrate reported at present is mostly directed at bulk drugs, such as "method for determining chloral hydrate by high performance liquid chromatography" (CN 110161156A) and "method for detecting related substances in chloral hydrate" (CN 109406690A). However, because the health-care product has complex components and contains a large amount of auxiliary agents such as saccharides, amino acids and the like, the existing technology for detecting chloral hydrate cannot be directly used for detecting chloral hydrate in the health-care product. Therefore, in the process of detection of illegal addition of drugs, a sample pretreatment process is often required, for example, methods such as solid phase extraction, solid phase micro-extraction, microwave extraction, supercritical fluid extraction and the like are adopted to separate, purify and further detect chloral hydrate, and the sample pretreatment methods are long in time consumption, complex and complicated in treatment process and not beneficial to rapid detection of drugs.
Therefore, a sample pretreatment technology and a detection method for illegally added chloral hydrate content in health care products, which are simple to develop, quick and accurate, are urgently needed, technical references are provided for national food safety standards related to chloral hydrate, safety monitoring of food and medicine in the market is facilitated, the market is standardized, and health of people is guaranteed.
Disclosure of Invention
Based on the above, the invention aims to provide a sample pretreatment method for detecting the content of chloral hydrate in a health-care product, which overcomes the problem of separating an object to be detected from a complex system of the health-care product, has the advantages of simple steps, easy operation, high sensitivity and quick detection, and is suitable for quickly analyzing the chloral hydrate in the health-care product.
A sample pretreatment method for detecting the content of chloral hydrate in a health-care product is characterized by comprising the following steps:
taking a proper amount of sample, adding acid liquor, heating, and decomposing chloral hydrate to generate HCl and HCOOH gas;
and absorbing the generated gas by using water to obtain the solution to be detected.
The method utilizes the chloral hydrate to generate decomposition reaction under the acidic and heating conditions and generate hydrogen chloride and formic acid with strong volatility, and then combines the gas-liquid separation technology to separate the hydrogen chloride and the formic acid. Compared with the prior art that chloral hydrate is separated from the health care product by adopting a complex sample pretreatment technology, the sample pretreatment method for detecting the chloral hydrate content in the health care product converts the detection of the chloral hydrate into the detection of chloride ions and/or formic acid, can effectively eliminate matrix interference, shortens the time required by sample pretreatment, greatly simplifies the detection and analysis process of the chloral hydrate content in the health care product, and has the advantages of simple steps and high sensitivity.
Further, the acid solution is 10-60% (v/v) of non-volatile acid solution. The acid liquor is added to provide an acidic environment for the decomposition of chloral hydrate. The non-volatile acid liquid is adopted, so that on one hand, the interference of the acid liquid volatilized by the acid liquid on the detection result can be avoided, and on the other hand, the insufficient acidity provided after the acid liquid is volatilized is avoided.
Further, the non-volatile acid liquid is one of phosphoric acid and sulfuric acid. Phosphoric acid or sulfuric acid are nonvolatile acids with good thermal stability, and provide a stable acidic environment for the decomposition of chloral hydrate.
Further, the volume ratio of the acid liquor to the health product test sample is 1: 1.
Further, the heating temperature is 100-120 ℃. Ensuring enough temperature to lead the hydrated chloral to generate decomposition reaction, and adjusting the heating time according to the amount of the added sample to ensure that the hydrated chloral is completely decomposed.
A method for detecting the content of chloral hydrate in a health-care product is characterized by comprising the following steps:
preparing a series of chloral hydrate standard solutions with concentration gradients;
respectively taking the chloral hydrate standard solutions with the same volume and the series of concentration gradients, and pretreating according to a sample pretreatment method for detecting the content of chloral hydrate in the health care product, which is disclosed by any one of claims 1-5, to obtain a series of standard curve solutions;
Taking a health product test sample with the same volume as that of the chloral hydrate standard solution for pretreatment, and carrying out pretreatment according to the sample pretreatment method for detecting the chloral hydrate content in the health product of any one of claims 1-5 to obtain a solution to be tested of the test sample;
detecting chloride ions and/or formic acid in the standard curve solution, and drawing a corresponding chloral hydrate concentration-test instrument signal standard curve according to the detection result of the standard curve solution;
and detecting chloride ions and/or formic acid in the solution to be detected of the test sample, and calculating by adopting a standard curve method to obtain the content of chloral hydrate in the test sample of the health care product.
In the prior art, chloral hydrate is separated from health care products by adopting a complex sample pretreatment technology, and then the chloral hydrate can be further detected. The method for detecting the content of chloral hydrate in the health-care product converts the detection of chloral hydrate into the detection of chloride ions and/or formic acid, detects the chloride ions and/or formic acid in the standard curve solution and the solution to be detected of the sample to be tested by using the corresponding detection technology, and then calculates the content of chloral hydrate in the sample to be tested of the health-care product by using a standard curve method. The method effectively eliminates matrix interference, shortens the time required by the pretreatment of the sample, greatly simplifies the detection and analysis process of the content of chloral hydrate in the health care product, and has simple steps and high sensitivity.
Further, the standard curve method is as follows: and fitting the corresponding chloral hydrate concentration-test instrument signal standard curve to obtain a corresponding linear regression equation, substituting the corresponding test instrument signal result of the chloride ions and/or formic acid in the solution to be tested of the test sample into the corresponding linear regression equation, and calculating to obtain the chloral hydrate content in the test sample of the health care product.
Further, detecting chloride ions and formic acid in the standard curve solution and the test sample solution to be tested by adopting ion chromatography, and drawing a chloral hydrate standard solution concentration-chloride ion peak area standard curve and a chloral hydrate standard solution concentration-formic acid peak area standard curve. The ratio of the area of the formic acid peak to the area of the chloride ion chromatographic peak obtained by detecting the standard curve solution by adopting an ion chromatography is in a certain range, and the certain range can be regarded as a proportionality constant range (the concentration of the chloral hydrate standard solution can be adjusted according to actual requirements, so that the proportionality constant range meeting the requirements is obtained). When the ion chromatographic profile of the solution to be tested of the test sample is consistent with the ion chromatographic profile of the solution with the standard curve, and the ratio of the area of the formic acid peak to the area of the chlorine ion chromatographic peak falls within the range of the proportionality constant of the chloral hydrate standard solution with a certain concentration, indicating that chloral hydrate is added into the health care product. And calculating to obtain the content of chloral hydrate in the health-care product by combining a standard curve method, and carrying out quantitative analysis on the content of chloral hydrate added in the health-care product.
Further, detecting formic acid in the standard curve solution and the test sample solution to be tested by using a hydrophilic interaction chromatography, and drawing a concentration-formic acid peak area standard curve of the chloral hydrate standard solution. Under normal conditions, the health-care product does not contain formic acid, so when formic acid is contained in the liquid to be detected obtained by the pretreatment of the health-care product, chloral hydrate can be primarily determined to be contained in the health-care product. And then calculating the content of chloral hydrate in the health-care product by combining a standard curve method, and carrying out quantitative analysis on the chloral hydrate added in the health-care product.
Further, detecting chloride ions in the standard curve solution and the solution to be detected of the sample by adopting a silver nitrate-ethylene glycol fluorescence resonance light scattering method, and drawing a concentration-scattered light intensity standard curve of the chloral hydrate standard solution; or detecting chloride ions in the standard curve solution and the solution to be detected of the sample to be tested by adopting a silver nitrate turbidimetric method, and drawing a concentration-absorbance standard curve of the chloral hydrate standard solution. The method can only detect chloride ions and can carry out quantitative analysis on chloral hydrate added in the health care product.
For a better understanding and practice, the invention is described in detail below with reference to examples and the accompanying drawings.
Drawings
FIG. 1 is a chemical equation of thermal decomposition of chloral hydrate under acidic conditions.
FIG. 2 is a graph of an ion chromatogram and a linear regression fit provided in example 2, wherein a is a graph of a linear regression fit of the concentration of chloral hydrate standard solution to the area of formic acid peak; FIG. b is an ion chromatogram of formic acid and chloride ions; figure c is a linear regression fit of chloral hydrate standard solution concentration-peak area of chloride ion peak.
FIG. 3 is an ion chromatogram of a test sample solution provided in example 2.
Fig. 4 is a HILIC chromatogram and a linear regression fit chart provided in example 3, in which a is a linear regression fit chart of concentration of chloral hydrate standard solution and formic acid peak area, and b is a HILIC chromatogram of formic acid.
FIG. 5 is a HILIC chromatogram of a test sample provided in example 3.
FIG. 6 is a linear regression fit of chloral hydrate standard solution concentration versus scattered light intensity provided in example 4.
FIG. 7 is a linear regression fit of the concentration-absorbance of chloral hydrate standard solution provided in example 5.
Fig. 8 is an HPLC spectrum of chloral hydrate standard solution and health product provided in comparative example 1.
Detailed Description
In order to further explain the present invention, this embodiment takes a health product containing a chloral hydrate drug illegally added as an example, and details a sample pretreatment for detecting the content of chloral hydrate in the health product and a detection method thereof provided by the present application are described. However, it should be understood by those skilled in the art that the specific examples are illustrative of the concept of the present invention and are not to be construed as limiting the alternative embodiments of the present invention, and that the determination of the drug content based on the actual health product can be performed by those skilled in the art using the method of the present invention as well.
The invention provides a sample pretreatment method for detecting the content of chloral hydrate in a health-care product, which comprises the following steps:
taking a proper amount of sample, adding acid liquor, heating, and decomposing chloral hydrate to generate HCl and HCOOH gas; and absorbing the generated gas by water to obtain a solution to be tested.
Preferably, the acid liquor is 10-60% (v/v) of a non-volatile acid liquor.
Preferably, the non-volatile acid liquid is one of phosphoric acid and sulfuric acid.
Preferably, the volume ratio of the acid liquor to the health product test sample is 1: 1.
Preferably, the heating temperature is 100-.
The chloral hydrate can be subjected to decomposition reaction under the acidic and heating conditions to generate hydrogen chloride and formic acid with strong volatility, and then the hydrogen chloride and the formic acid are separated by combining a gas-liquid separation technology. Compared with the prior art, the sample pretreatment method for detecting the content of chloral hydrate in the health care product effectively eliminates matrix interference, shortens the time required by sample pretreatment, greatly simplifies the detection and analysis process of chloral hydrate in the health care product, and has the advantages of simple steps and high sensitivity.
The invention also provides a method for detecting the content of chloral hydrate in the health-care product, which comprises the following steps:
Preparing a series of chloral hydrate standard solutions with concentration gradients;
respectively taking equal volumes of the chloral hydrate standard solutions with the concentration gradients, and pretreating according to a sample pretreatment method for detecting the content of chloral hydrate in the health care product in any one of claims 1-5 to obtain a series of standard curve solutions;
taking a health product test sample with the same volume as that of the chloral hydrate standard solution for pretreatment, and carrying out pretreatment according to the sample pretreatment method for detecting the chloral hydrate content in the health product of any one of claims 1-5 to obtain a solution to be tested of the test sample;
detecting chloride ions and/or formic acid in the standard curve solution, and drawing a corresponding chloral hydrate concentration-test instrument signal standard curve according to the detection result of the standard curve solution;
and detecting chloride ions and/or formic acid in the solution to be detected of the test sample, and calculating by adopting a standard curve method to obtain the content of chloral hydrate in the test sample of the health care product.
Preferably, the standard curve method is as follows: and fitting the corresponding chloral hydrate concentration-test instrument signal standard curve to obtain a corresponding linear regression equation, substituting the corresponding test instrument signal result of the chloride ions and/or formic acid in the solution to be tested of the test sample into the corresponding linear regression equation, and calculating to obtain the chloral hydrate content in the test sample of the health care product.
Preferably, the standard curve solution and the test sample solution are subjected to chloride ion and formic acid detection by ion chromatography, and a chloral hydrate standard solution concentration-chloride ion peak area standard curve and a chloral hydrate standard solution concentration-formic acid peak area standard curve are drawn.
Preferably, the formic acid in the standard curve solution and the test sample solution is detected by hydrophilic interaction chromatography, and a standard curve of the concentration of the chloral hydrate standard solution and the area of the formic acid peak is drawn.
Preferably, detecting chloride ions in the standard curve solution and the solution to be detected of the sample to be tested by adopting a silver nitrate-ethylene glycol fluorescence resonance light scattering method, and drawing a chloral hydrate standard solution concentration-scattered light intensity standard curve; or detecting chloride ions in the standard curve solution and the solution to be detected of the sample to be tested by adopting a silver nitrate turbidimetric method, and drawing a concentration-absorbance standard curve of the chloral hydrate standard solution.
The specific detection technique can be selected according to actual situations, including but not limited to the detection technique sections listed in the present invention.
Example 1
Pretreatment of a sample:
the invention adopts the miniaturized heat-assisted sample pretreatment device disclosed in the patent No. CN 105910881B to pretreat the sample, and the device can realize the integration of the acidolysis, heating and reabsorption process of the decomposition products of the chloral hydrate. The device comprises a carrier gas system, a heating cavity, a gasification pool and a receiving pipe; the gasification pool is arranged in the heating cavity and used for placing samples, the heating cavity is a cavity, an air inlet and an air outlet are arranged at two ends of the cavity, and a sample inlet is arranged above the cavity and in a position corresponding to the gasification pool; the carrier gas system is connected with the heating cavity through a gas inlet; the receiving pipe is connected with the heating cavity through the air outlet, and a solution for absorbing the gasified sample is arranged in the receiving pipe.
The method for pretreating the sample comprises the following steps:
a quantity of sample was taken and mixed with 50% (v/v) phosphoric acid at a ratio of 1: mixing at a volume ratio of 1 to obtain a mixed sample solution.
Adding 800 mu L of the mixed sample liquid into a gasification cell in a gas membrane separation sample pretreatment device, adjusting the heating voltage (7.0V) to ensure that the heating temperature is within the range of 100-120 ℃, using air as carrier gas, ensuring the flow rate to be 300mL/min, and containing 5.0mL of water in a receiving tube; stopping heating after 30min, transferring the absorption liquid, and fixing the volume to 10.0mL to obtain the solution to be detected.
Example 2
Detecting chloride ions and formic acid in the solution to be detected by adopting an ion chromatography, and calculating the concentration of chloral hydrate in the health care product test sample by using a linear regression equation of the formic acid and the chloride ions.
Preparation of standard curve solution: preparing chloral hydrate standard solutions with concentration gradients of 5.0, 10.0, 20.0, 30.0, 40.0 and 50.0 mu g/mL series. The chloral hydrate standard solutions with the series of concentration gradients are subjected to sample pretreatment according to the steps in the example 1, so that corresponding standard curve solutions are obtained.
Pretreatment of a health product sample: the sample pretreatment is carried out on the health product test sample according to the steps in the embodiment 1, and the absorption liquid is diluted by 40 times to obtain the corresponding test sample solution.
Sample detection: detection analysis was performed using a Thermo Scientific Integraon RFIC ion chromatograph with a 4.0mm (i.d). times.250 mm (L) column of Thermo Scientific Dionex lonpact AS19, column temperature set at 30 deg.C, mobile phase 20mmol/L KOH, and flow rate 1.000 mL/min.
Referring to FIG. 2, wherein, the graph a is a linear regression fit graph of chloral hydrate standard solution concentration-formic acid peak area; FIG. b is an ion chromatogram of formic acid and chloride ions; and the graph c is a linear regression fitting graph of the concentration of the chloral hydrate standard solution and the peak area of the chloride ion. In the range of 5.0-50.0 mug/mL chloral hydrate, the retention time t of the standard curve solutionR4.68min (formic acid peak) and tRThe peak area at 5.44min (chloride ion peak) gradually increases along with the increase of the concentration of the chloral hydrate standard solution, the linear regression equations of formic acid and chloride ion are y 52172x and y 8909x respectively, wherein x represents the concentration of the chloral hydrate standard solution, y represents the peak area, and the correlation coefficients are R20.9999 and R2The detection limit is 0.1 mu g/mL and 0.5 mu g/mL respectively which is 0.9998, and the ion chromatography detection of the ultra-trace chloral hydrate can be satisfied. When the concentration of the chloral hydrate is 5.0-50.0 mug/mL, the ratio of the formic acid peak area to the chloride ion peak area of an ion chromatogram is 5.76-5.96.
Please refer to fig. 3, which is an ion chromatogram of the solution to be tested of the sample, the profile of which is consistent with chloral hydrate, the peak areas of the detected formic acid and chloride ion are 2671206 and 456141, respectively, the ratio thereof is 5.85, and falls within the range of 5.76-5.96, indicating that the sample contains chloral hydrate; calculating by using a linear regression equation of formic acid and chloride ions to obtain the concentration of chloral hydrate in the health care product test sample diluted by 40 times, wherein the concentration of the chloral hydrate is 51.2 mu g/mL; multiplying by the dilution factor of 40 to obtain the chloral hydrate concentration of 2.05mg/mL in the health care product test sample.
Example 3
Detecting formic acid by hydrophilic interaction chromatography (HILIC, also called reversed phase chromatography), and calculating the concentration of chloral hydrate in the sample of the health product by using a linear regression equation of the formic acid.
Preparation of standard curve solution: preparing chloral hydrate standard solutions with concentration gradients of 5.0, 10.0, 20.0, 40.0, 60.0, 80.0 and 100.0 mu g/mL. The chloral hydrate standard solutions with the series of concentration gradients are subjected to sample pretreatment according to the steps in the example 1, so that corresponding standard curve solutions are obtained.
Pretreatment of a health product sample: the health product sample is pretreated according to the steps in the embodiment 1, and the absorption liquid is diluted by 40 times to obtain the corresponding test sample solution.
Sample detection: the detection and analysis were carried out by liquid chromatography of Shimadzu corporation, Japan, in which the separation column was a 4.0mm (i.d). times.250 mm (L) Shishengtang PC HILIC S5 column, the column temperature was set at 40 ℃ and the mobile phase was NH at pH 3.05 mmol/L4H2PO4Aqueous solution (water phase) and acetonitrile (organic phase), wherein the chromatographic gradient adopts 10 percent of water phase-90 percent of organic phase for isocratic elution, the flow rate is 0.800mL/min, and the detection wavelength is 206 nm.
Referring to fig. 4, wherein, a is a linear regression fit graph of concentration of chloral hydrate standard solution-formic acid peak area, and b is a HILIC chromatogram of formic acid; in the range of 5.0-100.0 mu g/mL of chloral hydrate standard solution concentration, the retention time t of the standard curve solution isRThe peak area at 3.75min (formic acid peak) is gradually increased along with the increase of the concentration of the chloral hydrate standard solution, and the linear regression equation is that y is 10176.3x, wherein x represents the concentration of the chloral hydrate standard solution, y represents the peak area, and the correlation coefficient R is20.9991, the detection limit is 0.2 mug/mL respectively, which indicates that the HILIC liquid chromatography detection of ultra trace chloral hydrate can be satisfied.
Referring to fig. 5, a HILIC chromatogram of a test solution of a test sample, the profile of which is consistent with chloral hydrate, and the peak area of the peak of the formic acid is 529200; calculating the concentration of the chloral hydrate in the health care product test sample diluted by 40 times to be 52.0 mu g/mL through a linear regression equation of formic acid; multiplying by the dilution factor of 40 to obtain the chloral hydrate concentration of 2.08 mg/mL in the health care product test sample.
Example 4
Detecting chloride ions by adopting a silver nitrate-ethylene glycol fluorescence resonance light scattering method, and calculating the concentration of chloral hydrate in the health care product test sample through a linear regression equation of the chloride ions.
Preparation of standard curve solution: preparing chloral hydrate standard solutions with concentration gradients of 5.0, 10.0, 20.0, 40.0 and 80.0 mu g/mL series. The chloral hydrate standard solutions with the series of concentration gradients are subjected to sample pretreatment according to the steps in the example 1 to obtain corresponding standard curve solutions.
Pretreatment of a health product test sample: the sample pretreatment is carried out on the health product sample according to the steps in the embodiment 1, and the absorption liquid is diluted by 40 times to obtain the corresponding sample to be tested.
Sample detection: respectively taking 5.0mL of solution to be detected, sequentially adding 1.0mL of 1mol/L nitric acid, 1.0mL of ethylene glycol and 1.0mL of 1mol/L silver nitrate, diluting to a constant volume of 10.0mL, uniformly oscillating, standing for 15min, and detecting and analyzing by using a fluorescence instrument of Shimadzu corporation, wherein the excitation wavelength and the emission wavelength are set to be 515nm, the width of the excitation wavelength slit is set to be 3.0nm, the width of the emission wavelength slit is set to be 1.0nm, and the sensitivity level is set to be High.
Referring to fig. 6, which is a linear regression fit of chloral hydrate standard solution concentration versus scattered light intensity; in the range of the chloral hydrate concentration of 5.0-80.0 mug/mL, the scattered light intensity of the standard curve solution under 515nm exciting light gradually increases along with the increase of the chloral hydrate standard solution concentration, and the linear regression equation is that y is 10.65x +16.78, wherein x represents the chloral hydrate standard solution concentration, y represents the scattered light intensity at 515nm, and the correlation coefficient R is 20.9959, the detection limit is 1.0 mug/mL respectively, which indicates that the detection of ultra trace chloral hydrate can be satisfied.
By this method, the scattering intensity of the test sample liquid was measured to be 575.91; calculating by a linear regression equation to obtain the concentration of chloral hydrate in the health care product test sample diluted by 40 times to be 52.5 mu g/mL; multiplying by the dilution factor of 40 to obtain the chloral hydrate concentration of 2.10mg/mL in the health care product test sample.
Example 5
And detecting chloride ions by using a silver nitrate turbidimetric method, and calculating the concentration of chloral hydrate in the health care product test sample by using a linear regression equation of the chloride ions.
Preparation of standard curve solution: preparing chloral hydrate standard solutions with concentration gradients of 1.0, 2.0, 4.0, 6.0, 8.0 and 10.0mg/mL series. The chloral hydrate standard solutions with the series of concentration gradients are subjected to sample pretreatment according to the steps in the example 1, so that corresponding standard curve solutions are obtained.
Pretreatment of a health product sample: the sample pretreatment is carried out on the health product sample according to the steps in the embodiment 1, and the corresponding sample solution to be tested is obtained.
Sample detection: respectively taking 5.0mL of solution to be detected, sequentially adding 1.0mL of 1mol/L nitric acid, 1.0mL of ethylene glycol and 1.0mL of 1mol/L silver nitrate, diluting to a constant volume of 10.0mL, uniformly oscillating, standing for 15min, and detecting and analyzing by using an ultraviolet spectrophotometer of Shimadzu corporation, wherein the detection wavelength is set to 515 nm.
Please refer to fig. 7, which is a linear regression fitting graph of concentration-absorbance of chloral hydrate standard solution; in the range of 1.0-10.0mg/mL of the chloral hydrate standard solution concentration, the absorbance of the standard curve solution at 515nm gradually increases along with the increase of the chloral hydrate standard solution concentration, and the linear regression equation is that y is 0.03854x-0.0094, wherein x represents the concentration of the chloral hydrate standard solution, y represents the absorbance at 515nm, and the correlation coefficient R is2The detection limit is 0.9861, and is 0.2mg/mL respectively, which indicates that the detection of chloral hydrate with higher content can be satisfied. In addition, the method can be visually compared to determine concentration based on turbidity.
By this method, the absorbance of the test sample solution was measured to be 0.075 (cloudiness visible to the naked eye); and calculating by a linear regression method to obtain the concentration of the chloral hydrate in the health care product test sample to be 2.2 mg/mL.
Comparative example 1
The content of chloral hydrate in the health care product is tested by a test method disclosed in a comparison document 'method for measuring chloral hydrate by high performance liquid phase' (CN 110161156A).
The concentration of the chloral hydrate standard substance is 1.0mg/mL
The pretreatment method of the health care product comprises the following steps: diluting by 10 times, passing through a 0.22 μm membrane, and performing machine-based detection.
The HPLC detection conditions are as follows:
The chromatographic column was Shim-pack GIST C185 μm 4.6X 250mm, the detection wavelength was 210nm, the flow rate was 1.0 mL/min, the column temperature was 40 ℃ and the sample injection amount was 20 μ L.
The mobile phase A is water, the mobile phase B is acetonitrile, gradient elution is adopted, and the reaction time is 0-17 min: 10% B-10% B, 17-25 min: 10% B-90% B, 25-35 min: 90% B-90% B (gradient elution is used to completely wash out impurities in the sample and ensure no residual material in the column after analysis).
FIG. 8 is HPLC chromatogram of chloral hydrate standard and health product, and it can be seen from FIG. 8 that retention time of chloral hydrate standard is tR15.14min (t in the comparison file)RThe chromatographic method in this application is not significantly different from the control file) at 15.135 min. It has an interference peak (t) near the sample peakR14.73min), the quantification of chloral hydrate was greatly influenced.
In addition, a great number of impurity peaks (0-5min and 22-35min) can be seen from a chromatogram, and the direct detection of chloral hydrate in the health care product by adopting the method for many times is likely to damage a chromatographic column, so that the high performance liquid chromatography of the comparison document cannot be directly used for detecting chloral hydrate in the health care product.
In the embodiment, only one health-care product is compared, and more impurities which interfere with the detection of chloral hydrate may exist in the matrix of other health-care products.
The method provided by the invention can effectively eliminate the interference of impurities in the health care product, can combine with a quick detection technology to shorten the detection time, has the potential of being applied to field detection, and can quickly screen illegal addition of chloral hydrate in the health care product.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is specific and detailed, but not to be construed as limiting the scope of the invention. It should be noted that, to those skilled in the art, changes and modifications may be made without departing from the spirit of the present invention, and it is intended that the present invention encompass such changes and modifications.

Claims (10)

1. A sample pretreatment method for detecting the content of chloral hydrate in a health-care product is characterized by comprising the following steps:
taking a proper amount of sample, adding acid liquor, heating, and decomposing chloral hydrate to generate HCl and HCOOH gas;
and absorbing the generated gas by using water to obtain the solution to be detected.
2. The sample pretreatment method for detecting the content of chloral hydrate in a health product according to claim 1, characterized in that: the acid liquid is 10-60% (v/v) of non-volatile acid liquid.
3. The sample pretreatment method for detecting the content of chloral hydrate in a health product according to claim 2, characterized in that: the non-volatile acid liquid is one of phosphoric acid or sulfuric acid.
4. The sample pretreatment method for detecting the content of chloral hydrate in a health product according to claim 2, characterized in that: the volume ratio of the acid solution to the sample is 1: 1.
5. The sample pretreatment method for detecting the content of chloral hydrate in a health product according to claim 1, characterized in that: the heating temperature is 100-120 ℃.
6. A method for detecting the content of chloral hydrate in a health-care product is characterized by comprising the following steps:
preparing a series of chloral hydrate standard solutions with concentration gradients;
respectively taking the chloral hydrate standard solutions with the same volume and the series of concentration gradients, and pretreating according to a sample pretreatment method for detecting the content of chloral hydrate in the health care product of any one of claims 1 to 5 to obtain a series of standard curve solutions;
taking a health-care product test sample with the same volume as that of the chloral hydrate standard solution for pretreatment, and carrying out pretreatment according to the sample pretreatment method for detecting the chloral hydrate content in the health-care product of any one of claims 1 to 5 to obtain a solution to be detected of the test sample;
detecting chloride ions and/or formic acid in the standard curve solution, and drawing a corresponding chloral hydrate concentration-test instrument signal standard curve according to the detection result of the standard curve solution;
And detecting chloride ions and/or formic acid in the solution to be detected of the test sample, and calculating by adopting a standard curve method to obtain the content of chloral hydrate in the test sample of the health care product.
7. The method for detecting the content of chloral hydrate in a health product according to claim 6, wherein the method comprises the following steps: the standard curve method comprises the following steps: and fitting the corresponding chloral hydrate concentration-test instrument signal standard curve to obtain a corresponding linear regression equation, substituting the corresponding test instrument signal result of the chloride ions and/or formic acid in the solution to be tested of the test sample into the corresponding linear regression equation, and calculating to obtain the chloral hydrate content in the test sample of the health care product.
8. The method for detecting the content of chloral hydrate in a health product according to claim 6, wherein the method comprises the following steps: and detecting chloride ions and formic acid in the standard curve solution and the solution to be detected of the sample by adopting ion chromatography, and drawing a chloral hydrate standard solution concentration-chloride ion peak area standard curve and a chloral hydrate standard solution concentration-formic acid peak area standard curve.
9. The method for detecting the content of chloral hydrate in a health product according to claim 6, wherein the method comprises the following steps: and detecting formic acid in the standard curve solution and the solution to be detected of the sample by using a hydrophilic interaction chromatography, and drawing a standard curve of the concentration of the chloral hydrate standard solution and the area of the peak of the formic acid.
10. The method for detecting the content of chloral hydrate in a health product according to claim 6, wherein the method comprises the following steps: detecting chloride ions in the standard curve solution and the solution to be detected of the sample by using a silver nitrate-ethylene glycol fluorescence resonance light scattering method, and drawing a concentration-scattered light intensity standard curve of the chloral hydrate standard solution; or detecting the chloride ions in the standard curve solution and the solution to be detected of the sample by using a silver nitrate turbidimetric method, and drawing a concentration-absorbance standard curve of the chloral hydrate standard solution.
CN202210212601.3A 2022-03-04 2022-03-04 Sample pretreatment for detecting content of chloral hydrate in health product and detection method thereof Pending CN114705768A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115436541A (en) * 2022-09-29 2022-12-06 成都施贝康生物医药科技有限公司 Content detection method of chloral hydrate

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115436541A (en) * 2022-09-29 2022-12-06 成都施贝康生物医药科技有限公司 Content detection method of chloral hydrate
CN115436541B (en) * 2022-09-29 2024-04-26 成都施贝康生物医药科技有限公司 Method for detecting content of chloral hydrate

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