CN113866321A

CN113866321A - Detection method of Jian' er qingjie liquid

Info

Publication number: CN113866321A
Application number: CN202111399136.0A
Authority: CN
Inventors: 张元杰; 周兰; 郝文梅; 李哲媛; 来国防; 任杰红; 范兵
Original assignee: YUNNAN INSTITUTE FOR FOOD AND DRUG CONTROL
Current assignee: YUNNAN INSTITUTE FOR FOOD AND DRUG CONTROL
Priority date: 2021-11-19
Filing date: 2021-11-19
Publication date: 2021-12-31

Abstract

The invention provides a detection method of Jian' er qing Jie liquid, which comprises the following steps: detecting the mass content of mandelonitrile in the Jian' er qing hydrolysate by adopting a high-phase liquid chromatography; the solvent in the mandelonitrile reference substance solution in the high-phase liquid chromatography detection process is glacial acetic acid acetonitrile solution or phosphoric acid water solution. The method is based on the basic consensus of poor stability of mandelonitrile, solves the problem of poor stability of mandelonitrile in common solvents, and develops a method suitable for measuring the content of mandelonitrile in the healthy-child clear solution; the real content composition of the free hydrogen cyanide and the mandelonitrile in the sample is obtained by respectively detecting the free hydrogen cyanide and the mandelonitrile, the blank of quality control of the existing Jian' er Qing Jie liquid can be filled, data with discrimination can be provided for subsequent production process optimization, safety, effectiveness and other evaluations, and meanwhile, an idea is provided for quality control of a traditional Chinese medicine preparation containing bitter almond aromatic water.

Description

Detection method of Jian' er qingjie liquid

Technical Field

The invention belongs to the technical field of Jian 'er qing jue, and particularly relates to a detection method of Jian' er qing jue.

Background

The children-invigorating and digestion-promoting liquid is a Chinese patent medicine for pediatrics, is prepared by extracting six medicinal materials such as bitter apricot seed and the like, has the effects of clearing away heat and toxic materials, eliminating phlegm and stopping cough, and eliminating stagnation and regulating the middle warmer, and is used for treating oral erosion, cough and pharyngalgia, inappetence, abdominal distension and the like. The bitter apricot kernel in the prescription is prepared by distilling and extracting aromatic water. Amygdalin in bitter apricot kernel can be hydrolyzed under the catalysis of enzyme to generate glucose and prunasin under the conditions of moisture, decoction and the like, prunasin is continuously hydrolyzed to generate glucose and mandelonitrile (also called amygdaline), and the mandelonitrile is extremely unstable and is easily decomposed to generate benzaldehyde and hydrogen cyanide which is a highly toxic substance under heat. Detecting volatile oil extracted from semen Armeniacae amarum by steam distillation with gas chromatography-mass spectrometer, wherein the volatile oil contains 5.2% mandelonitrile (mangelonetrile); it is expected that mandelonitrile and its degradation hydrogen cyanide can enter into semen Armeniacae amarum aromatic water along with steam distillation and further enter into children health promotion liquid preparation.

Through the condition analysis of the bitter almond-containing traditional Chinese medicine preparation recorded in Chinese pharmacopoeia, a water vapor distillation method is mainly used for extracting a mandelonitrile solution, a mandelonitrile content detection project is established in the preparation quality standard, and meanwhile, the mandelonitrile is extremely easy to decompose into volatile free hydrogen cyanide, so that the safety risk exists, but the free hydrogen cyanide lacks a detection method with strong specificity and high sensitivity. The detection method of mandelonitrile and free hydrogen cyanide in the children-care liquid has important significance for production, quality control and other related work as the bitter almond-containing preparation prepared from the bitter almond steam distillate.

Disclosure of Invention

In view of the above, the present invention is directed to a method for detecting a Jian 'er qingjie solution, which can better perform quality detection on the Jian' er qingjie solution.

The invention provides a detection method of Jian' er qing Jie liquid, which comprises the following steps:

detecting the mass content of mandelonitrile in the Jian' er qing hydrolysate by adopting a high-phase liquid chromatography;

the solvent in the mandelonitrile reference substance solution in the high-phase liquid chromatography detection process is glacial acetic acid acetonitrile solution or phosphoric acid water solution.

Preferably, the volume of the glacial acetic acid in the glacial acetic acid acetonitrile solution is 0.1-1% of the acetonitrile volume.

Preferably, the pH value of the phosphoric acid aqueous solution is 2.0-3.5.

Preferably, the mobile phase in the detection process of the high-phase liquid chromatography is acetonitrile-phosphoric acid solution; the volume ratio of the acetonitrile to the phosphoric acid solution is (20-25) to (75-80).

Preferably, the flow rate of a chromatographic column in the detection process of the high-phase liquid chromatography is 0.5-1.5 mL/min;

the detection wavelength is 205-210 nm;

the column temperature is 25-35 ℃;

the injection volume is 5-15 mu L.

Preferably, the method further comprises the following steps:

and detecting the mass content of the free hydrogen cyanide in the Jian' er qing hydrolysate by adopting a headspace sample injection-capillary column gas chromatography.

Preferably, the control solution in the gas chromatography detection process comprises:

cyanogen component in water analysis standard substance, sodium chloride, phosphoric acid, sodium hydroxide and water.

Preferably, the preparation method of the control solution comprises the following steps:

diluting the cyanogen component analysis standard substance in water with sodium hydroxide solution, adding sodium chloride, and acidifying with phosphoric acid solution.

Preferably, the headspace equilibrium temperature in the detection process of the gas chromatography is 40-70 ℃;

the equilibrium time is not more than 1 hour;

the quantitative loop temperature is 70-80 ℃.

Preferably, the temperature of the transmission line in the detection process of the gas chromatography is 90-110 ℃;

the temperature of a sample inlet is 180-220 ℃;

the flow dividing ratio is (3-7): 1.

the method is based on the basic consensus of poor stability of mandelonitrile, solves the problem of poor stability of mandelonitrile in common solvents, and develops a method suitable for measuring the content of mandelonitrile in the healthy-child clear solution; on the basis of the method for measuring the content of the mandelonitrile, the stability of the mandelonitrile in the process of measuring the content of the free hydrogen cyanide by a headspace sample injection gas chromatography is researched, and the mandelonitrile is ensured not to be degraded in the measuring process, so that the targeted detection of the free hydrogen cyanide is realized; the method adopts a substitute matrix standard curve method, reduces the adverse effect of the matrix of the Jian' er qingjie liquid sample on the accuracy of a detection result in the detection process, and ensures the accuracy of a free hydrogen cyanide content determination result; the real content composition of the free hydrogen cyanide and the mandelonitrile in the sample is obtained by respectively detecting the free hydrogen cyanide and the mandelonitrile, the blank of quality control of the existing Jian' er Qing Jie liquid can be filled, data with discrimination can be provided for subsequent production process optimization, safety, effectiveness and other evaluations, and meanwhile, an idea is provided for quality control of a traditional Chinese medicine preparation containing bitter almond aromatic water.

Drawings

FIG. 1 is a technical route for detecting Jian' er qing Jie liquid according to an embodiment of the present invention;

FIG. 2 is a mandelonitrile standard curve obtained in example 2 of the present invention;

FIG. 3 is a high performance liquid chromatogram of a mandelonitrile control obtained in example 2 of the present invention;

FIG. 4 is a high performance liquid chromatogram of a negative control solution lacking almond obtained in example 2 of the present invention;

FIG. 5 is a high performance liquid chromatogram of bitter apricot kernel aromatic water obtained in example 2 of the present invention;

fig. 6 is a chromatogram of mandelonitrile content in the healthy-child cleansing solution provided by manufacturing enterprise a in example 3 of the present invention;

fig. 7 is a chromatogram of mandelonitrile content in the healthy-child cleansing solution provided by manufacturing enterprise B in example 3 of the present invention;

fig. 8 is a chromatogram of mandelonitrile content in the healthy-child cleansing solution provided by manufacturing enterprise C in example 3 of the present invention;

fig. 9 is a chromatogram of mandelonitrile content in the healthy-child cleansing solution provided by manufacturing enterprise D in example 3 of the present invention;

fig. 10 is a chromatogram of mandelonitrile content in the Jian' er qing dynasty solution provided by manufacturing enterprise E in example 3 of the present invention;

fig. 11 is a chromatogram of mandelonitrile content in the healthy-child cleansing solution provided by manufacturing enterprise F in example 3 of the present invention;

fig. 12 is a chromatogram of mandelonitrile content in the healthy-child cleansing solution provided by manufacturing enterprise G in example 3 of the present invention;

FIG. 13 is a graph showing the degradation rate of mandelonitrile in the Jian' er qing dynasty solution after sealing and balancing for 1 hour at different temperatures in example 5 of the present invention;

FIG. 14 shows the peak areas of free hydrogen cyanide in solutions of different concentrations of JIANER QINGJIE in example 6;

FIG. 15 is a graph showing the effect of different amounts of NaCl on the slope of a standard curve in example 6 of the present invention;

FIG. 16 is a standard curve of 10% NaCl in place of matrix in example 7 of the present invention;

FIG. 17 is a chromatogram of a control of hydrogen cyanide in example 7 of the present invention;

FIG. 18 is a chromatogram of a negative control solution lacking almond in example 7 of the present invention;

FIG. 19 is a chromatogram of aromatic water of apricot seeds in example 7 of the present invention;

FIG. 20 is a chromatogram of the detection limit of free hydrogen cyanide in example 7 of the present invention;

FIG. 21 shows the results of the Standard addition method (lot 06119017) in example 8 of the present invention;

FIG. 22 shows the result of the Standard addition method (lot 190930) in example 8 of the present invention;

fig. 23 is a chromatogram for measuring the content of free hydrogen cyanide in the Jian' er qing dynasty solution provided by the manufacturing enterprise a in example 9 of the present invention;

fig. 24 is a chromatogram for measuring the content of free hydrogen cyanide in the Jian' er qing dynasty solution provided by the manufacturing enterprise B in example 9 of the present invention;

fig. 25 is a chromatogram for measuring the content of free hydrogen cyanide in the Jian' er qing dynasty solution provided by manufacturing enterprise C in example 9 of the present invention;

fig. 26 is a chromatogram for measuring the content of free hydrogen cyanide in the Jian' er qing dynasty solution provided by manufacturing enterprise D in example 9 of the present invention;

fig. 27 is a chromatogram for measuring the content of free hydrogen cyanide in the Jian' er qing dynasty solution provided by manufacturing enterprise E in example 9 of the present invention;

fig. 28 is a chromatogram for measuring the content of free hydrogen cyanide in the healthy baby hydrolysate provided by manufacturing enterprise F in example 9 of the present invention;

fig. 29 is a chromatogram for measuring the content of free hydrogen cyanide in the Jian' er qing dynasty solution provided by manufacturing enterprise G in example 9 of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings 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.

determining the mass content of mandelonitrile in the Jian' er qing hydrolysate by adopting a high-phase liquid chromatography;

According to the invention, researches show that a mandelonitrile reference substance solution is prepared by using common solvents such as methanol, ethanol, acetonitrile, water and the like, and is respectively detected by high performance liquid chromatography sample injection, and the change rate of the peak area proves that the mandelonitrile is extremely unstable in the solvent, which is a key reason that the mandelonitrile is lack of a mature detection method at present.

In the invention, the volume of glacial acetic acid in the glacial acetic acid acetonitrile solution is preferably 0.1-1% of the acetonitrile volume, more preferably 0.3-1%, more preferably 0.5-1%, more preferably 0.8-1%, and most preferably 1%.

In the invention, the pH value of the phosphoric acid aqueous solution is preferably 2.0-3.5, more preferably 2.5-3, and most preferably 2.5.

According to the invention, through research, acetonitrile solutions containing glacial acetic acid with different concentrations and phosphoric acid aqueous solutions with different pH values are used for preparing mandelonitrile reference substance solutions, the mandelonitrile reference substance solutions are respectively subjected to sample injection detection by high performance liquid chromatography, and through peak area change rate, the mandelonitrile reference substance solutions prepared by using 0.1-1.0% (v/v) glacial acetic acid acetonitrile solutions or phosphoric acid aqueous solutions with pH values of 2.0-3.5 can be kept stable within 12 hours, so that the content determination of mandelonitrile becomes possible, and meanwhile, a thought is provided for stability research in the storage process of mandelonitrile.

The invention has no special limitation on the chromatograph and the chromatographic column used in the high performance liquid chromatography detection process. In the invention, the chromatographic conditions in the high performance liquid chromatography detection process are preferably performed by using Shimadzu LC-2040 high performance liquid chromatograph; an Agilent TC-C18 high performance liquid chromatography column (4.6mm multiplied by 250mm, 5 μm) is adopted; the mobile phase is acetonitrile-phosphoric acid solution; the phosphoric acid solution is preferably a phosphoric acid aqueous solution, and the volume concentration of the phosphoric acid solution is preferably 0.05-0.15%, more preferably 0.08-0.12%, and most preferably 0.1%; the volume ratio of the acetonitrile to the phosphoric acid solution is preferably (20-25): 75-80), more preferably (21-24): 76-79, more preferably (22-23): 77-78, and most preferably 23: 77; the flow rate of the chromatographic column is preferably 0.5-1.5 mL/min, more preferably 0.8-1.2 mL/min, and most preferably 1 mL/min; the detection wavelength is preferably 205-210 nm, more preferably 206-209 nm, more preferably 207-208 nm, and most preferably 207 nm; the column temperature is preferably 25-35 ℃, more preferably 28-32 ℃, and most preferably 30 ℃; the injection volume is preferably 5-15 μ L, more preferably 8-12 μ L, and most preferably 10 μ L.

The method for detecting the Jian' er qingjie liquid provided by the invention preferably further comprises the following steps:

In the present invention, hydrogen cyanide has a boiling point of about 25.6 ℃ at normal atmospheric pressure, and can be detected using gas chromatography; sucrose and other substances which are difficult to gasify exist in the Jian' er qing Jie liquid, and the mode of headspace sampling after heating balance is preferably selected for detection; the mandelonitrile is degraded by heating to generate hydrogen cyanide, key parameters such as equilibrium temperature, time and the like need to be set, so that the degradation of the mandelonitrile in the detection process is avoided, and the determination of the free hydrogen cyanide is ensured.

In the present invention, the control solution in the gas chromatography detection process preferably includes:

In the present invention, the sodium chloride, phosphoric acid and sodium hydroxide are preferably superior pure; the water is preferably ultrapure water.

In the invention, in order to avoid the use of virulent sodium (potassium) cyanide, a traceable 'standard substance for analyzing cyanogen components in water' is selected, and the matrix of the standard substance is 0.1mol/L sodium hydroxide; in this matrix, cyanogen is present in the form of cyanate, and needs to be acidified to volatile hydrogen cyanide for gas chromatography detection.

In the present invention, the preparation method of the control solution preferably includes:

In the present invention, the sodium hydroxide solution is preferably an aqueous sodium hydroxide solution; the concentration of the sodium hydroxide solution is preferably 0.08-0.12 mol/L, more preferably 0.09-0.11 mol/L, and most preferably 0.1 mol/L.

In the invention, the content of cyanate in the diluted standard substance for analyzing the cyanogen component in water is preferably 20-30 mug/mL, more preferably 23-27 mug/mL, and most preferably 25 mug/mL.

In the present invention, the phosphoric acid solution is preferably an aqueous phosphoric acid solution; the volume concentration of the phosphoric acid solution is preferably 5-12%, more preferably 6-11%, more preferably 7-10%, and most preferably 8-9%.

In the invention, the pH value of the acidified water cyanogen component analysis standard substance (control solution) is preferably 2.1-6.4, more preferably 2.5-6, more preferably 3-5.5, more preferably 3.5-5, and most preferably 4-4.5.

In order to reduce the influence of the matrix enhancement effect on the content detection result, the following method can be selected: diluting to reduce matrix enhancement effect brought by the heat-clearing and digestion liquid for children; measured using standard addition methods; and preparing a reference substance solution and preparing a blank sample matrix standard curve by using the blank Jian' er clear solution sample matrix without free hydrogen cyanide so as to match the matrix environment of the hydrogen cyanide in the reference substance solution and the sample solution. The methods have the defects of high sensitivity requirement on detection means, complicated operation process, difficult preparation of blank sample matrixes and the like. The invention establishes a method for preparing a substitute matrix reference substance solution by using sodium chloride and further directly measuring the content of free hydrogen cyanide in the healthy infant clear solution by using a substitute matrix standard curve.

In the invention, the mass content of the sodium chloride in the reference solution is preferably 5-15%, more preferably 8-12%, and most preferably 10%.

In the present invention, the test solution in the gas chromatography detection process preferably includes:

jian' er Qing Jie liquid and phosphoric acid solution.

In the present invention, the phosphoric acid solution is preferably an aqueous phosphoric acid solution; the volume concentration of the phosphoric acid solution is preferably 8-12%, more preferably 9-11%, and most preferably 10%.

In the present invention, the volume ratio of the healthy infant digestion solution to the phosphoric acid solution is preferably 10: (0.8 to 0.12), more preferably 10: (0.9 to 0.11), most preferably 10: 1.

the invention has no special limitation on the chromatograph in the detection process of the gas chromatography; in the invention, an Agilent 7890B chromatograph is preferably adopted in the gas chromatography detection process, and an Agilent 7697A headspace automatic sample injector is provided.

In the invention, the headspace equilibrium temperature in the detection process of the gas chromatography is preferably 40-70 ℃, more preferably 45-60 ℃, and most preferably 50 ℃; the balancing time is preferably not more than 1 hour, more preferably 20-40 min, more preferably 25-35 min, and most preferably 30 min; the quantitative ring temperature is preferably 70-80 ℃, more preferably 73-77 ℃, and most preferably 75 ℃; the temperature of the transmission line is preferably 90-110 ℃, more preferably 95-105 ℃, and most preferably 100 ℃; the injection port temperature is preferably 180-220 ℃, more preferably 190-210 ℃, and most preferably 200 ℃; the preferred split ratio is (3-7): 1, more preferably (4-6): 1, most preferably 5: 1; preferably a bonded polystyrene-divinylbenzene capillary column (Agilent PLOT Q, 30 m.times.0.32 mm. times.40 μm); preferably, the temperature of the column oven is programmed, and the temperature raising method preferably comprises the following steps:

keeping the initial temperature, raising the temperature to the final temperature, and keeping the temperature.

In the present invention, the time for maintaining the initial temperature is preferably 0.8 to 1.2min, more preferably 0.9 to 1.1min, and most preferably 1 min.

In the invention, the initial temperature is preferably 38-42 ℃, more preferably 39-41 ℃ and most preferably 40 ℃.

In the invention, the temperature rise speed is preferably 20-30 ℃/min, more preferably 22-28 ℃/min, and most preferably 24-26 ℃/min.

In the invention, the final temperature is preferably 260-280 ℃, more preferably 265-275 ℃ and most preferably 270 ℃.

In the invention, the heat preservation time is preferably 5-9 min, more preferably 6-8 min, and most preferably 7 min.

In the invention, the detector in the detection process of the headspace sample injection gas chromatography is preferably an FID detector; the detection temperature is preferably 290-310 ℃, more preferably 295-305 ℃, and most preferably 300 ℃.

In the invention, the content of the free hydrogen cyanide in the test sample is preferably calculated according to an external standard curve method in the detection process of the gas chromatography.

The method uses traceable water cyanogen component analysis standard substance, selects low-concentration phosphoric acid solution as an acidification reagent through pH detection, and the addition amount is one tenth of the volume of a matrix, thereby realizing the acidification of the matrix of the standard substance and reducing the risk of heat loss of hydrogen cyanide; evaluating by using a high performance liquid chromatography, under the same acidification condition as a standard substance, selecting a proper headspace heating balance temperature to ensure that the mandelonitrile is not degraded into hydrogen cyanide in the determination process so as to realize the detection of free hydrogen cyanide; evaluating the influence of a blank sample matrix of the Jian' er clear solution on the final equilibrium concentration of hydrogen cyanide in the headspace heating balance process, finding that the matrix effect is obvious, preparing a blank sample matrix reference solution to ensure the accuracy of a content detection result, selecting sodium chloride to match the sample matrix, and further optimizing the headspace heating balance time; under the selected headspace heating equilibrium temperature and time, 10% sodium chloride is used for replacing the blank sample matrix of the Jianer qingjie liquid to prepare a reference substance solution, and a substitute matrix standard curve method which is simple and convenient to operate and accurate in result is established.

The virulent substance hydrogen cyanide exists in the almond aromatic water, the infant-care solution and other preparations containing the almond aromatic water in a free state and in a mandelonitrile form, is an important substance basis for exerting the cough-relieving effect and is also an important factor influencing the medication safety. In the self-made bitter almond aromatic water and the infant-care and digestion-promoting liquid produced by different enterprises, the total amount of hydrogen cyanide and the proportions of different existing forms of hydrogen cyanide are greatly different, and even if the total amount of hydrogen cyanide is similar, the proportions of different existing forms of hydrogen cyanide are also greatly different. The content of hydrogen cyanide in the free state is directly related to the safety and effectiveness of the drug. Mandelonitrile is the main component of the "mandelonitrile solution" of the antitussive drug, and according to the related reports of animal experiments, the metabolic pathway in vivo can be degraded to generate benzaldehyde and hydrogen cyanide, but a secondary metabolic pathway without generating hydrogen cyanide also exists. It can be seen that the same amount of free hydrocyanic acid as that of mandelonitrile is not necessarily the same in pharmacological activity and safety. Meanwhile, the pharmacological activity and safety of the mixture of free hydrogen cyanide and mandelonitrile with the same total amount of hydrogen cyanide are not necessarily the same. The invention can obtain the real content composition of the free hydrogen cyanide and the mandelonitrile in bitter almond aromatic water preparations such as bitter almond aromatic water, healthy liquid and the like through respectively and quantitatively detecting the free hydrogen cyanide and the mandelonitrile, and provides data with discrimination for the optimization of the production process, the safety, the effectiveness evaluation, the stability investigation and the like.

In the invention, headspace sampling refers to that a solid or liquid sample is prepared into a sample solution, then the sample solution is placed in a closed small bottle, the sample solution is heated in a heating chamber with constant temperature control until the concentration of volatile components in the sample solution reaches the balance between liquid and gas, and then a certain volume of headspace air is automatically sucked by a sample injector and injected into a gas chromatograph.

In the present invention, the matrix refers to all components of the specimen except for the analyte.

In the invention, the matrix effect means that when headspace sampling is adopted, a test sample and a reference sample are in incompletely the same matrix, so that the difference of the balance coefficients of volatile components in the test sample and the reference sample between liquid state and gas state is caused, and the accuracy of content detection is influenced; the standard addition method can eliminate the influence of matrix effect caused by the difference between the matrix of the test solution and the matrix of the reference solution, so the accuracy of the quantitative method can be generally verified by adopting the standard addition method.

In the present invention, the aromatic water refers to a nearly saturated or saturated aqueous solution of medicinal materials containing volatile components prepared by steam distillation.

In the invention, mandelonitrile is maleonitrile, CAS No. 532-28-5, alias phenylethanonitrile, alpha-hydroxybenzonitrile and mandelonitrile.

In the present invention, hydrogen cyanide is hydrogen cyanide, CAS number 74-90-8, a highly toxic substance, also known as hydrocyanic acid.

In the prior art, the content of total hydrogen cyanide is detected by adopting a titration method after distillation, the sample consumption is large, the treatment process is complicated, and the collected target hydrogen cyanide is a highly volatile and toxic substance and has certain potential safety hazard; the detection result is equivalent to the content of total hydrogen cyanide, the real content distribution situation of the free hydrogen cyanide in the sample cannot be objectively reflected, and the difference of potential toxicity risks cannot be reflected because the sample with the same detection result obtained by using the technology cannot reflect the real composition of the mandelonitrile and the free hydrogen cyanide, considering that the GHS Classification of the mandelonitrile (the global unified chemical Classification and Labeling System, GHS for short) is HC301 (swallow toxicity) and the GHS Classification of the hydrogen cyanide is HC300 (swallow lethal).

In the prior art, the content of hydrogen cyanide is detected by using a special nitrogen and phosphorus detector instead of a general flame ionization detector, and special requirements are configured on instruments; the problem that the final equilibrium concentration of hydrogen cyanide is greatly influenced by a complex sample matrix in the headspace heating balance process, so that the accuracy of a content detection result is influenced, is not found and solved; the degradation condition of mandelonitrile in the headspace heating detection process cannot be evaluated, and the content of free hydrogen cyanide in the detection result is difficult to ensure.

The prior art detects the content of mandelonitrile, has poor stability, is easy to degrade spontaneously and difficult to detect accurately, and does not research the stability of the mandelonitrile in a solvent; the sample matrix is relatively simple.

The flow of the method for detecting the Jian' er qingjie liquid in the embodiment of the invention is shown in figure 1, and the invention solves the problem that a mandelonitrile reference substance is rapidly degraded in a conventional solvent at room temperature and is difficult to detect; on the basis, a technical scheme with strong specificity and high sensitivity is provided for accurately detecting the content of the mandelonitrile in the Jian' er qing dynasty liquid by using a high performance liquid chromatography; the problem that mandelonitrile in a sample is endogenously converted into hydrogen cyanide is avoided, and the targeted detection of free hydrogen cyanide of highly toxic substances in the Jian' er qingjie liquid is really realized; the influence of matrix effect in the process of detecting the content of the free hydrogen cyanide by headspace sample injection gas chromatography is reduced, and a technical scheme with simple operation, strong specificity and high sensitivity is provided for accurately detecting the content of the free hydrogen cyanide in the healthy baby hydrolysate; the method has the advantages that the real contents of the mandelonitrile and the free hydrogen cyanide can be determined through accurate quantitative detection of the mandelonitrile and the free hydrogen cyanide in the healthy-child clear solution, the defect that the prior art can only detect the total amount of the hydrogen cyanide is overcome, and a real and objective basis is provided for the problems of effectiveness, safety and stability evaluation, package tightness, production process optimization and the like of bitter-almond aromatic water preparations such as the bitter-almond aromatic water and the healthy-child clear solution.

EXAMPLE 1 stability of mandelonitrile in solvent

Detecting a mandelonitrile solution by adopting high performance liquid chromatography:

chromatographic conditions are as follows: shimadzu LC-2040 high performance liquid chromatograph, Agilent TC-C18 high performance liquid chromatography column (4.6mm × 250mm, 5 μm); the mobile phase is acetonitrile-0.1% phosphoric acid solution (23:77), and the flow rate of the chromatographic column is 1 mL/min; the detection wavelength is 207 nm; the column temperature is 30 ℃; sample introduction volume: 10 μ L.

Mandelonitrile control (Shanghai Allan Biotechnology science and technology Co., Ltd., code M158766, batch number B2019085, designation purity 97.067%).

The method comprises the following steps: taking a proper amount of mandelonitrile reference substances, respectively dissolving the mandelonitrile reference substances by using methanol, ethanol, acetonitrile, water, a phosphoric acid aqueous solution with the pH value of 1.5-6.0 and a glacial acetic acid acetonitrile solution of 0.1-1.0% (v/v) to prepare a solution containing about 200 mu g of mandelonitrile per 1mL, sealing, and standing at the constant temperature of 20 ℃; sampling and detecting every 30 minutes from 0 minute, recording peak areas, and calculating the degradation rate according to the peak areas; the results of the measurements are shown in tables 1 to 3.

TABLE 1 degradation rates of mandelonitrile in different solvents

TABLE 2 degradation rate of mandelonitrile in phosphoric acid aqueous solutions of different pH

TABLE 3 degradation rate of mandelonitrile in glacial acetic acid acetonitrile solutions of different concentrations

Solvent(s)	Time of standing	Rate of degradation
			0.10% glacial acetic acid acetonitrile	12 hours	0.7％
0.25% glacial acetic acid acetonitrile	12 hours	0.6％
			0.50% glacial acetic acid acetonitrile	12 hours	1.3％
0.75% glacial acetic acid acetonitrile	12 hours	0.9％
			1.0% glacial acetic acid acetonitrile	12 hours	0.1％

Mandelonitrile is extremely unstable in methanol, ethanol and water, can be degraded for 13-53% in 30 minutes, and the degradation rate can reach 49-69% in 2 hours; in acetonitrile, mandelonitrile is relatively stable, the degradation rate is about 7% within 2 hours, and the content determination of mandelonitrile is difficult to complete due to the continuous degradation of the mandelonitrile reference substance; the mandelonitrile reference substance solution prepared by 0.1-1.0% (v/v) of glacial acetic acid acetonitrile solution or phosphoric acid aqueous solution with the pH value of 2.0-3.5 is kept stable within 12 hours, and the degradation rate is lower than 2.0%; selecting phosphoric acid aqueous solution with pH of 2.5 to prepare mandelonitrile reference substance, establishing high performance liquid chromatography, and determining mandelonitrile content in the Jian' er clear solution.

Example 2

Detection of mandelonitrile in Jian' er qing Jie liquid by high performance liquid chromatography

The instrument comprises the following steps: shimadzu LC-2040 high performance liquid chromatograph.

Preparation of control solutions: taking a mandelonitrile reference substance (Shanghai Aladdin Biotechnology, Inc., number M158766, lot number B2019085, mark purity 97.067%), precisely weighing, adding phosphoric acid aqueous solution with pH value of 2.5, diluting step by step, and preparing into reference substance solutions with series concentrations of 1.033 μ g, 5.164 μ g, 10.328 μ g, 24.432 μ g, 48.864 μ g, 103.279 μ g, 147.493 μ g, 198.599 μ g, 244.318 μ g and 294.987 μ g for each 1mL respectively to obtain the final product.

Preparation of a test solution: collecting appropriate amount of JIANER QINGJIE liquid, filtering with 0.45 μm microporous membrane, and collecting the filtrate.

Preparation of negative control solution: according to the recipe preparation method carried by the variety of 'Jianer Qing Jie' in the tenth volume of Chinese medicinal prescription preparation from the drug Standard of the Ministry of health, a negative control solution without bitter almond aromatic water is prepared, and is filtered by a 0.45 mu m microporous membrane, and the subsequent filtrate is taken, thus obtaining the drug.

Preparing bitter almond aromatic water: taking 50g of bitter almond medicinal material, distilling and extracting 50mL of aromatic water, adding phosphoric acid aqueous solution with the pH value of 2.5 to dilute to 1000mL, shaking up, taking a proper amount, filtering by using a 0.45 mu m microporous membrane, and taking the subsequent filtrate to obtain the bitter almond health care beverage.

Chromatographic conditions are as follows: agilent TC-C18 high performance liquid chromatography column (4.6 mm. times.250 mm, 5 μm); the mobile phase is acetonitrile-0.1% phosphoric acid water solution (23:77), and the flow rate of the chromatographic column is 1 mL/min; the detection wavelength is 207 nm; the column temperature is 30 ℃; sample introduction volume: 10 μ L.

(1) Linear relation

Taking the mandelonitrile series concentration reference substance solution, carrying out sample injection measurement according to the conditions and recording the peak area, wherein the result is shown in a table 4; taking the peak area value as the ordinate and the concentration (μ g/mL) as the abscissa, a standard curve is drawn, as shown in FIG. 2, and the regression equation is: y 33498.87068x-1060.85339, R0.9999; the result shows that the peak area and the concentration of mandelonitrile have a good linear relationship in the range of 1.033-294.987 mu g/mL.

TABLE 4 mandelonitrile content determination-Linear relationship examination

Mandelonitrile concentration μ g/mL	Peak area
		1.033	31478
5.164	173238
		10.328	348943
24.432	780467
		48.864	1635278
103.279	3502224
		147.493	4933171
198.599	6644051
		244.318	8234144
294.987	9834829

(2) Specificity

Taking the negative control solution and bitter almond aromatic water, carrying out sample injection measurement according to the conditions, and recording a chromatogram; as shown in fig. 3 to 5, the negative control solution has no spectrum interference at the same retention time as the chromatographic peak of mandelonitrile; the chromatogram of the bitter almond aromatic water shows a chromatographic peak with the same retention time as that of a mandelonitrile reference substance.

(3) Precision of the instrument

The above-mentioned control solution having mandelonitrile concentration of 48.864 μ g/mL was sampled and measured 6 times in succession under the above-mentioned conditions, and a chromatogram was recorded, and the peak area RSD was 0.1% (n ═ 6), as shown in Table 5, the instrument precision was good.

TABLE 5 measurement of mandelonitrile content-investigation of instrument precision

(4) Stability of

Sampling JIAN' ER QINGJIE liquid sample (lot number 191136) at 0h, 2h, 4h, 6h, 8h, 10h, and 12h respectively, and determining; the relative deviation from the peak area at 0h was 0.31% to 0.53%, and the RSD was 0.2% (n ═ 7), and the test solution remained substantially stable for 12 hours, as shown in table 6.

TABLE 6 measurement of mandelonitrile content-investigation of instrument precision

(5) Repeatability of

Taking a proper amount of the Jian' er clear solution sample (batch No. 08220006), preparing 6 parts of sample solution in parallel, injecting and measuring according to the conditions, recording a chromatogram, substituting peak areas into the standard curve, and calculating to obtain the average mandelonitrile concentration of 154.578 mu g/mL and RSD of 0.1% (n is 6) as shown in Table 7.

TABLE 7 mandelonitrile content determination-repeatability

(6) Accuracy test

Taking 5mL of a Jian ' er clear solution sample (batch No. 08220006), placing the sample in a 10mL measuring flask, respectively adding 367.204 mu g/mL of reference substance solution (prepared by phosphoric acid solution with pH value of 2.5) 1mL, 2mL and 3mL of reference substance solution according to three levels of 50%, 100% and 150% of mandelonitrile content measured under the term of ' repeatability ', adding phosphoric acid solution with pH value of 2.5 to a constant volume to scale, and shaking up; recovery was calculated as measured by the method at 3 parts per level, resulting in an average recovery of 97.4% and an RSD of 0.6% (n-9), which met the requirements of the quantitative analysis, as shown in table 8.

TABLE 8 determination of mandelonitrile content-accuracy test

(7) Quantitative limit and detection limit

Taking 0.35mL of a control solution with the mandelonitrile concentration of 2.538 mu g/mL, placing the control solution into a 10mL measuring flask, adding the almond lacking negative control solution to the measuring flask, fixing the volume to a scale, shaking up, and measuring by the method, wherein the signal-to-noise ratio is 2.93, so the detection limit of the method is 0.102 mu g/mL.

Taking 1mL of a control solution with mandelonitrile concentration of 2.538 mu g/mL, placing the control solution in a 10mL measuring flask, adding the almond lacking negative control solution to a constant volume to scale, and shaking up; 6 samples were prepared in parallel and measured by the method, and the results were calculated to give 6 samples with an average signal to noise ratio of 10.9, an average recovery of 92.4% and an RSD of 2.7% (n-6), with a certain precision and accuracy, as shown in table 9, and thus the limit of quantitation by the method was 0.254 μ g/mL.

TABLE 9 mandelonitrile content determination-quantitation limits

(8) Durable mobile phase composition

The effect of volume ratio of three mobile phase compositions of acetonitrile-0.1% phosphoric acid (21:79), acetonitrile-0.1% phosphoric acid (23:77), and acetonitrile-0.1% phosphoric acid (25:75) was examined using an Agilent TC-C18 column (4.6 mm. times.250 mm, 5 μm); as a result, under each condition, the separation degree of the mandelonitrile chromatographic peak and the adjacent interference peak is greater than 1.5, and the RSD of the content determination result of the same batch of the Jian' er clear solution sample (batch No. 08220006) is 1.4% (n ═ 6), as shown in table 10, it meets the verification requirement of the analysis method.

TABLE 10 mandelonitrile assay-durability-mobile phase composition

(9) Durability-column flow

Using Agilent TC-C18 chromatographic column (4.6mm multiplied by 250mm, 5 μm), the influence of three chromatographic column flow rates of 0.8mL/min, 1.0mL/min and 1.2mL/min when acetonitrile-0.1% phosphoric acid (23:77) is used as a mobile phase is respectively examined; as a result, under each condition, the separation degree of the mandelonitrile chromatographic peak and the adjacent interference peak is greater than 1.5, and the RSD of the content determination result of the same batch of the Jian' er clear solution sample (batch No. 08220006) is 0.1% (n ═ 6), as shown in table 11, it meets the verification requirement of the analysis method.

TABLE 11 determination of mandelonitrile content-durability-flow on chromatography column

(10) Durability-column temperature of chromatographic column

The influence of the column temperatures of three types of columns, namely, 25 ℃, 30 ℃ and 35 ℃, when acetonitrile-0.1% phosphoric acid (23:77) is used as a mobile phase, was examined using an Agilent TC-C18 column (4.6 mm. times.250 mm, 5 μm); as a result, under each condition, the separation degree of the mandelonitrile chromatographic peak and the adjacent interference peak is greater than 1.5, and the RSD of the content determination result of the same batch of the Jian' er clear solution sample (batch No. 08220006) is 0.3% (n ═ 6), as shown in table 12, it meets the verification requirement of the analysis method.

TABLE 12 determination of mandelonitrile content-durability-column temperature investigation of the chromatography

(11) Selection of chromatography columns

When the same batch of samples of the healthy product clear liquid were subjected to the chromatographic separation measurement using a TC-C18 column (4.6 mm. times.250 mm, 5 μm), an Inertsil ODS-3 column (4.6 mm. times.250 mm, 5 μm), a Zorbax SB-C18 column (4.6 mm. times.250 mm, 5 μm), a Syncronics C18 column (4.6 mm. times.250 mm, 5 μm), and an Inertsustain C18 column (4.6 mm. times.250 mm, 5 μm), respectively, as the mobile phases selected as described above, the degrees of separation of the mandelonitrile chromatographic peak from the adjacent interfering peak were each greater than 1.5, and the RSD (Table 5) was 0.9% (n ═ 10), as shown in Table 13, and thus there was no particular requirement for the columns.

TABLE 13 mandelonitrile content determination-durability-different brand model chromatography column investigation

Example 3 detection of mandelonitrile content in Jian' er qing Jie liquid sample and self-made bitter apricot seed aromatic water of different enterprises

Detecting mandelonitrile in the Jian' er qing Jie liquid and bitter almond aromatic water by high performance liquid chromatography

Detection conditions are as follows: agilent TC-C18 high performance liquid chromatography column (4.6 mm. times.250 mm, 5 μm); the mobile phase is acetonitrile-0.1% phosphoric acid water solution (23:77), and the flow rate of the chromatographic column is 1 mL/min; the detection wavelength is 207 nm; the column temperature is 30 ℃; sample introduction volume: 10 μ L.

The determination method comprises the following steps: and calculating the content of mandelonitrile in the sample according to an external standard curve method.

The detection results are shown in table 14, table 15, and fig. 6 to 12.

TABLE 14 content of mandelonitrile in Jian' er Qing Jie liquid of different manufacturers

TABLE 15 content of mandelonitrile in homemade bitter almond aromatic water

Bitter apricot kernel aromatic water	Mandelonitrile mu g/mL	Corresponding to mu g/mL of hydrogen cyanide
			1	6.942	1.409
2	14.952	3.035
			3	5.797	1.177
4	14.104	2.863
			5	12.557	2.549

Example 4

Taking a proper amount of cyanogen component analysis standard substances in water, diluting the cyanogen component analysis standard substances with 0.1mol/L sodium hydroxide solution to prepare a reference substance solution with cyanogen radical of 20 mu g/mL, dividing 10mL, respectively adding 5% phosphoric acid solution, 6% phosphoric acid solution, 7% phosphoric acid solution, 8% phosphoric acid solution, 9% phosphoric acid solution, 10% phosphoric acid solution, 11% phosphoric acid solution and 12% phosphoric acid solution, and acidifying 1 mL; the pH value after acidification is about 2.1-6.4; precisely measuring 2mL of the solution respectively, placing the solution into 10mL headspace bottles, sealing, and carrying out headspace sample injection detection under the same conditions.

The detection conditions are as follows: detecting the pH value by using a Mettler toledo Delta 320pH meter; an Agilent 7890B chromatograph equipped with an Agilent 7697A headspace automatic sample injector to determine peak area; equilibrating at 60 deg.C for 45 min, introducing sample at headspace with inlet temperature of 200 deg.C and split ratio of 5:1, heating with bonded polystyrene-divinylbenzene capillary column (Agilent PLOT Q, 30m × 0.32mm × 40 μm) at initial temperature of 40 deg.C for 1min, at 24 deg.C/min to 270 deg.C for 7 min; FID detector, temperature 300 ℃.

As shown in table 16, the peak area RSD was 1.1% (n: 8), and the acidification of the control substrate was achieved with 5% to 12% phosphoric acid solution at a volume ratio of 1:10 to the substrate, and 10% phosphoric acid solution was selected as the acidifying agent in one tenth of the volume of the substrate in view of the convenience of the operation.

TABLE 16 determination of the content of free Hydrogen cyanide-acidification of phosphoric acid solutions of different concentrations

Example 5

Detecting the content of mandelonitrile by high performance liquid chromatography

Taking 6 pairs of stock solutions of the Jian' er clear solution sample (batch No. 08220006) and acidified sample solutions (hereinafter referred to as acidified samples) obtained by using 10% phosphoric acid according to the method in the embodiment 4, detecting the content of the mandelonitrile by using a high performance liquid chromatography, wherein the detection structure is shown in table 17, the relative deviation is less than 0.9%, and the P is more than 0.05 through paired t test, and the result has no significant difference, which indicates that the addition of the acidifying reagent does not cause the transient degradation of the mandelonitrile in the sample.

TABLE 17 detection results of mandelonitrile content in stock solutions and acidified samples of samples

The content of the sample stock solution is mu g/mL	Acidified sample content mu g/mL	Relative mean deviation
			151.903	152.286	0.25％
150.785	151.099	0.21％
			152.418	153.752	0.87％
152.515	153.774	0.82％
			152.032	151.915	0.08％
152.076	152.002	0.05％

Taking the stock solution and the acidified sample of the Jian 'er qingjie solution sample (batch No. 08220006), respectively carrying out sealing and balancing treatment at 40 ℃, 50 ℃, 60 ℃, 70 ℃ and 80 ℃ for 1 hour, and detecting the content of mandelonitrile by using a high performance liquid chromatography according to the method, wherein the degradation rate of the acidified sample is obviously lower than that of the stock solution of the sample, and the acidification can increase the stability of the Jian' er qingjie solution at high temperature; when the headspace equilibrium time is not more than 1 hour at 40-70 ℃, the degradation rate of mandelonitrile in the healthy care clear solution acidified by 10% phosphoric acid solution is less than 0.9%, as shown in table 18 and fig. 14, the detection of free hydrogen cyanide can be realized, and the lower headspace equilibrium temperature is selected to be 50 ℃ on the premise of meeting the sensitivity of a flame ionization detector.

TABLE 18 degradation rate of mandelonitrile in the hermetically balanced 1-hour Jian' er clear solution at different temperatures

Temperature of	Sample stock solution degradation rate	Degradation rate of acidified sample
			40℃	0.85％	-0.05％
50℃	1.88％	0.33％
			60℃	3.16％	0.24％
70℃	11.41％	0.81％
			80℃	24.48％	1.58％

Example 6

Detection of free hydrogen cyanide in Jian' er qing hydrolysate by headspace sample injection gas chromatography

The instrument comprises the following steps: agilent 7890B chromatograph equipped with Agilent 7697A headspace autosampler.

Standard substance: standard substance for analyzing cyanogen component in water (national institute of metrology science, GBW (E)080115, lot No. 20045, labeled amount 50.0. mu.g/mL (in cyanate group)).

Chromatographic conditions are as follows: the temperature of a sample inlet is 200 ℃, and the split ratio is 5: 1; using bonded polystyrene-divinylbenzene capillary column (Agilent PLOT Q, 30m × 0.32mm × 40 μm), column oven programmed temperature (initial temperature 40 deg.C, hold for 1min, temperature to 270 deg.C at 24 deg.C/min, hold for 7 min); FID detector, temperature 300 ℃.

Taking a proper amount of a cyanogen component analysis standard substance in water, adding 0.1mol/L sodium hydroxide for dilution, preparing a reference substance solution containing 2.0 mu g/mL, 5.0 mu g/mL and 10 mu g/mL of cyanogen radical respectively per 1mL, dividing into 2mL, placing in a 10mL headspace bottle, adding 0.2mL of 10% phosphoric acid solution, carrying out headspace sample injection detection, and drawing a water matrix standard curve.

Taking a proper amount of a cyanogen component analysis standard substance in water, adding 0.1mol/L sodium hydroxide for dilution, preparing a reference substance solution containing 2.0 mu g/mL, 5.0 mu g/mL and 10 mu g/mL of cyanogen radicals respectively per 1mL, dividing into 2mL, placing in a 10mL headspace bottle which is added with 0.1g of sodium chloride in advance, adding 0.2mL of 10% phosphoric acid solution, carrying out headspace sample injection detection, and drawing a 5% sodium chloride matrix standard curve.

Taking 1mL of the Jian' er clear solution samples (batch No. 05519106) respectively, placing the samples into 10mL headspace bottles which are pre-added with 0.2mL of 10% phosphoric acid solution, respectively adding water and 1mL of reference substance solution containing 2.0 mu g/mL, 5.0 mu g/mL and 10 mu g/mL of cyanide radical, sealing, carrying out headspace sample injection detection according to the detection method, deducting the peak area of the water-added samples, and drawing a 50% sample matrix standard curve.

And carrying out univariate variance analysis on the slopes of the water matrix standard curve and the 50% sample matrix standard curve, wherein the result P value is 0.04 and less than 0.05, and the difference of the slopes of the curves is obvious, which indicates that a non-negligible matrix effect exists and the accuracy of a detection result is influenced.

Carrying out univariate variance analysis on the slopes of the standard curve of the 50% sample matrix and the standard curve of the 5% sodium chloride matrix, wherein the P value is 0.35 and is more than 0.05, the difference of the slopes of the curves is not obvious, and the sample matrix effect is similar to that of the 10% sodium chloride matrix; as shown in table 19.

TABLE 19 Standard curves for water matrix, 50% sample matrix and 5% sodium chloride matrix

According to the parameter optimization result, taking a proper amount of cyanogen component analysis standard substances in water, adding 0.1mol/L sodium hydroxide for dilution, preparing a reference substance solution with cyanogen radical of 25 mug/mL per 1mL, dividing into 2mL, placing in a 10mL headspace bottle with 0.2g of sodium chloride added in advance, adding 0.2mL of 10% phosphoric acid solution, respectively balancing at 50 ℃ for 15min, 20min, 25min, 30min and 35min, then detecting headspace sample injection, as shown in Table 20, after the result of 30min, the hydrogen cyanide peak area is not obviously increased, and therefore the headspace balance time is determined to be 30 min.

TABLE 20 headspace balance time study

Equilibration time min	Peak area
			15	138.8
20	139.7
		25	139.2
30	141.3
		35	141.6

0.5mL, 1.0mL and 2.0mL of the Jian 'er qing dynasty liquid samples (lot No. 05519106) were respectively taken, and placed in 10mL headspace bottles, water was added to make 2mL, then 10% phosphoric acid solution was added to make 0.2mL, and sealed to prepare sample solutions containing 25%, 50% and 100% Jian' er qing dynasty liquid, and the headspace sample injection detection peak areas were determined according to the above-mentioned detection method, as shown in Table 21.

TABLE 21 Peak areas of free Hydrogen cyanide in samples of Jian' er Qing-Jie solutions with different percentages

The percentage content of Jianer qingjie liquid	Actually measured peak area	Theoretical peak area (calculated as 100% recovery)
			25％	12.5	12.5
50％	27.1	25
			100％	58.6	50

Taking the percentage content of the matrix of the Jian' er qing dynasty liquid as the abscissa and the peak area as the ordinate, and performing linear relation fitting, as shown in fig. 14; the result shows that the percentage content of the healthy-infant clear solution has good linear relation with the actually-measured peak area, the correlation coefficient is 0.9998, but the slope of the correlation coefficient is obviously higher than that of a curve fitted by the percentage content of the healthy-infant clear solution and the theoretical peak area; the matrix of the Jian' er qing Jie liquid has a matrix enhancing effect on the determination of hydrogen cyanide, and the matrix effect is directly and linearly related to the content of the matrix in a certain range.

Respectively taking 1mL of blank sample matrixes of the Jianer qingjie liquid without hydrogen cyanide, placing the blank sample matrixes into 10mL of headspace bottles in which 0.2mL of 10% phosphoric acid solution is added in advance, respectively adding 1mL of reference substance solution containing 4.0 mu g/mL, 10.0 mu g/mL, 20 mu g/mL and 40 mu g/mL of cyanide radical, sealing, balancing the headspace at 50 ℃ for 30min, carrying out sample injection detection according to the method, and drawing a 50% blank sample matrix standard curve.

Taking a proper amount of a cyanogen component analysis standard substance in water, adding 0.1mol/L sodium hydroxide for dilution to prepare a reference substance solution containing 2.0 mu g/mL, 5.0 mu g/mL, 10 mu g/mL and 20 mu g/mL of cyanogen acid radicals respectively per 1mL, dividing into 2mL, putting the 2mL into a 10mL headspace bottle which is added with 0.1g of sodium chloride in advance, adding 0.2mL of 10% phosphoric acid solution, sealing, balancing the headspace at 50 ℃ for 30min, carrying out sample injection detection according to the method, and drawing a 5% sodium chloride matrix standard curve.

Taking a proper amount of a cyanogen component analysis standard substance in water, adding 0.1mol/L sodium hydroxide for dilution to prepare a reference substance solution containing 2.0 mu g/mL, 5.0 mu g/mL, 10 mu g/mL and 20 mu g/mL of cyanogen acid radicals respectively per 1mL, dividing into 2mL, putting the 2mL into a 10mL headspace bottle which is added with 0.2g of sodium chloride in advance, adding 0.2mL of 10% phosphoric acid solution, sealing, balancing the headspace at 50 ℃ for 30min, carrying out sample injection detection according to the method, and drawing a 10% sodium chloride matrix standard curve.

Taking a proper amount of a cyanogen component analysis standard substance in water, adding 0.1mol/L sodium hydroxide for dilution to prepare a reference substance solution containing 2.0 mu g/mL, 5.0 mu g/mL, 10 mu g/mL and 20 mu g/mL of cyanogen acid radicals respectively per 1mL, dividing into 2mL, putting the 2mL into a 10mL headspace bottle which is added with 0.3g of sodium chloride in advance, adding 0.2mL of 10% phosphoric acid solution, sealing, balancing the headspace at 50 ℃ for 30min, carrying out sample injection detection according to the method, and drawing a 15% sodium chloride matrix standard curve.

Taking a proper amount of a cyanogen component analysis standard substance in water, adding 0.1mol/L sodium hydroxide for dilution, preparing a reference substance solution containing 2.0 mu g/mL, 5.0 mu g/mL, 10 mu g/mL and 20 mu g/mL of cyanogen acid radicals respectively per 1mL, dividing into 2mL, placing in a 10mL headspace bottle, adding 0.2mL of 10% phosphoric acid solution, sealing, balancing for 30min at 50 ℃ headspace, carrying out sample injection detection according to the method, and drawing a water matrix standard curve.

Performing linear model one-factor analysis of variance on the slopes of the matrix standard curves of the 50% blank samples and the slopes of the matrix standard curves of the water, the 5%, the 10% and the 15% sodium chloride respectively, wherein the slopes of the matrix standard curves of the water, the 10% and the 15% sodium chloride are significantly different from the slopes of the matrix standard curves of the 50% blank samples as shown in table 22; the slope and intercept of the 5% sodium chloride matrix standard curve and the 50% blank sample matrix standard curve have no significant difference.

TABLE 22 surrogate matrix Standard Curve

Fitting a linear relation by taking the sodium chloride content (5%, 10%, 15%) as an abscissa and the slope of the corresponding standard curve as an ordinate, as shown in fig. 15; the result linear equation is that y is 7.297x +4.468, the correlation coefficient R is 0.9989, and the result shows that the influence of the sodium chloride content in the matrix on the slope of the standard curve is in a linear relation in a certain range, so that the simulation of the matrix of the blank sample can be realized by adjusting the sodium chloride content, the matrix is used for preparing a substitute matrix reference solution, and the content of the free hydrogen cyanide in the sample is directly measured; for the children-care clear solution, the substitute matrix is 10% sodium chloride.

Example 7

The method for detecting the content of free hydrogen cyanide in the Jian' er qing Jie liquid by replacing the matrix standard curve method comprises the following steps:

Reagent and reagent testing: a standard substance for analyzing cyanogen components in water (China institute of metrology science, GBW (E)080115, batch No. 20045, and a mark amount of 50.0 mu g/mL (in terms of cyanate)); sodium chloride, phosphoric acid and sodium hydroxide are super-pure, and water is ultrapure water.

Preparation of surrogate matrix control solution: taking a proper amount of a cyanogen component analysis standard substance in water, adding 0.1mol/L sodium hydroxide for dilution, gradually diluting to prepare a series of concentration reference substance solutions containing 0.5 mu g, 1.0 mu g, 2.0 mu g, 5.0 mu g, 10.0 mu g, 15.0 mu g and 20.0 mu g of cyanide radicals in each 1mL, dividing into 2mL according to the molecular weight, putting the samples into 10mL headspace bottles containing 0.2g of sodium chloride in advance (even if the final content of the sodium chloride is 10%), adding 0.2mL of 10% phosphoric acid solution, and sealing to obtain the product, wherein the concentrations of the reference substance solutions are respectively 0.519 mu g, 1.039 mu g, 2.077 mu g, 5.194 mu g, 10.387 mu g, 15.581 mu g and 20.775 mu g in each 1 mL.

Preparation of a test solution: taking 2mL of the Jian ' er qingjie liquid sample, putting the Jian ' er qingjie liquid sample into a 10mL headspace bottle, adding 0.2mL of 10% phosphoric acid solution, and sealing to obtain the Jian ' er qingjie liquid.

Preparation of bitter almond lacking negative control solution: preparing a negative control solution without bitter almond aromatic water according to a prescription preparation method carried in the tenth volume of 'Jianer Qing Jie' variety of a Chinese medicinal prescription preparation from drug standards of the ministry of health, taking 2mL, placing in a 10mL headspace bottle, adding 0.2mL of 10% phosphoric acid solution, and sealing to obtain the medicinal composition.

Preparing bitter almond aromatic water: taking 50g of bitter almond medicinal material, distilling and extracting 50mL of aromatic water, adding phosphoric acid aqueous solution with the pH value of 2.5 to dilute to 1000mL, shaking up, taking 2mL, putting into a 10mL headspace bottle which is added with 0.2g of sodium chloride in advance (even if the final content of the sodium chloride is 10%), adding 0.2mL of water, and sealing to obtain the bitter almond beverage.

Chromatographic conditions are as follows: the headspace equilibrium temperature is 50 ℃, the equilibrium time is 30min, the quantitative loop temperature is 75 ℃, and the transmission line temperature is 100 ℃; the temperature of a sample inlet is 200 ℃, and the split ratio is 5: 1; using bonded polystyrene-divinylbenzene capillary column (Agilent PLOT Q, 30m × 0.32mm × 40 μm), column oven programmed temperature (initial temperature 40 deg.C, hold for 1min, temperature to 270 deg.C at 24 deg.C/min, hold for 7 min); the FID temperature was 300 ℃.

Detection method: detecting by headspace sampling-capillary column gas chromatography, and calculating the content of free hydrogen cyanide in the sample according to external standard curve method.

Methodology investigation:

(1) drawing of standard curve of alternative substrate

Taking the substitute matrix reference substance solution, carrying out sample injection detection according to the conditions and recording the peak area, wherein the peak area is shown in a table 23; the peak area values are plotted as ordinate and the concentration of hydrogen cyanide (. mu.g/mL) as abscissa to plot a standard curve of the surrogate matrix, as shown in FIG. 16, with the regression equation of y 4.96615x-0.13984, R²0.9999; the result shows that the peak area and the concentration of the hydrogen cyanide present a good linear relationship within the range of 1.033-294.987 mu g/mL.

TABLE 23 alternative matrix Hydrogen cyanide Standard Curve

Concentration of hydrogen cyanide μ g/mL	Peak area
		0.519	2.50
1.039	5.05
		2.077	10.10
5.194	25.95
		10.387	51.20
15.581	76.75
		20.775	103.45

(2) Specificity

Sampling the above semen Armeniacae amarum negative control solution and semen Armeniacae amarum aromatic water, detecting according to the above conditions, recording chromatogram, as shown in FIG. 17 and FIG. 18, wherein the semen Armeniacae amarum negative control solution has no spectrum interference at the same position as the retention time of hydrogen cyanide chromatographic peak; the chromatogram of the aromatic water of bitter apricot seeds shows the chromatographic peak with the same retention time as that of the hydrogen cyanide reference product, as shown in FIG. 19.

(3) Precision of the instrument

The above-mentioned control solution having a hydrogen cyanide concentration of 15.581 μ g/mL was subjected to continuous sample injection and detection 5 times under the above-mentioned conditions, and a chromatogram was recorded, and the peak area RSD value was 1.2% (n ═ 5), as shown in table 24, and the instrument precision was good.

TABLE 24 precision of the instrument

(4) Repeatability of

Taking a proper amount of the Jian' er clear solution sample (batch No. 06119017), preparing 6 parts of sample solution in parallel, carrying out sample injection detection according to the conditions, recording a chromatogram, substituting peak areas into the standard curve of the substitute matrix, and calculating to obtain the average hydrogen cyanide content of 10.482 mu g/mL and RSD of 1.0% (n is 6) as shown in Table 25.

Table 25 repeatability (batch 06119017)

Another suitable amount of Jian' er qing dynasty liquid sample (batch 190930) was taken, 6 portions of sample solution were prepared in parallel, the sample injection detection was performed according to the above conditions, the chromatogram was recorded, the peak area was substituted into the above-mentioned substitute matrix standard curve, the calculation resulted in an average hydrogen cyanide content of 3.508 μ g/mL, the RSD was 2.9% (n ═ 6), as shown in table 26.

Table 26 repeatability (batch 190930)

(5) Accuracy test

Dividing 1mL of the same batch of Jian' er clear solution sample (batch No. 06119017, hydrogen cyanide content is 10.482 mug/mL), putting the sample into a 10mL headspace bottle which is added with 0.1g of sodium chloride in advance, adding 0.2mL of 10% phosphoric acid solution, respectively adding 1mL of control solution containing 5.194 mug/mL, 10.387 mug/mL and 20.775 mug/mL of hydrogen cyanide according to three levels of low, medium and high, and sealing; 3 parts of the feed is injected into each level according to the conditions, and the recovery rate is calculated; the average recovery was 96.7% and the RSD was 2.6% (n-9), and the method accuracy met the quantitative analysis requirements as shown in table 27.

TABLE 27 accuracy test (batch No. 06119017)

Taking 1mL of the same Jian' er clear solution sample (batch No. 190930, hydrogen cyanide content is 3.418 mug/mL), putting the sample into a 10mL headspace bottle which is added with 0.1g of sodium chloride in advance, adding 0.2mL of 10% phosphoric acid solution, adding 1mL of control solution containing 2.077 mug/mL, 5.194 mug/mL and 10.387 mug/mL of hydrogen cyanide according to three levels of low, medium and high, and sealing; 3 parts of the feed is injected into each level according to the conditions, and the recovery rate is calculated; the average recovery was 97.7% and the RSD was 2.4% (n-9), and the accuracy of the method was satisfactory for quantitative analysis as shown in table 28.

TABLE 28 accuracy test (batch 190930)

(6) Detection limit

2mL of blank sample (batch No. 191201) without hydrogen cyanide is taken and placed in a 10mL headspace bottle, 0.02mL of reference substance solution containing 5.194 mu g of hydrogen cyanide per 1mL is added, 0.18mL of 10% phosphoric acid solution is added, sealing is carried out, and the sample injection detection is carried out according to the conditions; the signal-to-noise ratio was 3.6 and the detection limit was 0.052. mu.g/mL, as shown in FIG. 20.

(7) Limit of quantification

Taking 6 parts of blank samples (batch No. 191201) without hydrogen cyanide, dividing each part into 2mL of blank samples, placing the blank samples into 10mL of headspace bottles, adding 0.04mL of reference substance solution containing 10.387 mu g of hydrogen cyanide into each 1mL of blank samples, adding 0.16mL of 10% phosphoric acid solution, sealing, and carrying out sample injection detection according to the conditions; the average recovery was 90.9%, and RSD was 6.7% (n ═ 6), as shown in table 29; the result shows that the method has certain precision and accuracy near the quantitative limit of 0.208 mug/m, and meets the relevant regulation of the quantitative limit.

TABLE 29 limit of quantitation of hydrogen cyanide

(8) Stability of

Taking 2mL of Jian' er qingjie liquid sample (batch number 06119017), putting into a 10mL headspace bottle, adding 0.2mL of 10% phosphoric acid solution, sealing, preparing 3 parts in parallel at the same time, and injecting samples and detecting according to the conditions for 0h, 6h and 12h respectively; the relative deviation of the peak areas with 0h is 1.1% (6h) and 1.7% (12h), and the sample solution is basically stable within 12 h.

Example 8

For the verification of the quantitative method in the headspace sampling process, the following description is given in 0861 "method for measuring residual solvent" in the four ministry of general rules of the design, 2020 of the Chinese pharmacopoeia, "when headspace sampling is used, the sample and the reference are in an incompletely identical matrix, so the matrix effect in the gas-liquid equilibrium process (the influence of the composition difference of the sample solution and the reference solution on the headspace liquid equilibrium) should be considered; because the standard addition method can eliminate the influence of matrix effect caused by different matrixes of the test solution and the reference solution, the accuracy of the quantitative method is usually verified by adopting the standard addition method; when the results of the standard addition method are inconsistent with those of other setting methods, the results of the standard addition method are used as the standard "; although the results show that the alternative matrix standard curve method provided by the invention meets the basic requirements of the verification of the analysis method, the difference between the measurement result and the true value cannot be reflected; thus, the assay results were again validated using standard addition methods:

Reagent and reagent testing: a standard substance for analyzing cyanogen components in water (China institute of metrology science, GBW (E)080115, batch No. 20045, and a mark amount of 50.0 mu g/mL (in terms of cyanate)); phosphoric acid is super pure, and water is ultrapure water.

Chromatographic conditions are as follows: the headspace equilibrium temperature is 50 ℃, the equilibrium time is 30min, the quantitative loop temperature is 75 ℃, and the transmission line temperature is 100 ℃; the temperature of a sample inlet is 200 ℃, and the split ratio is 5: 1; using bonded polystyrene-divinylbenzene capillary column (Agilent PLOT Q, 30m × 0.32mm × 40 μm), column oven programmed temperature (initial temperature 40 deg.C, hold for 1min, temperature to 270 deg.C at 24 deg.C/min, hold for 7 min); FID Detector 300 ℃.

Preparation of a test solution:

the same batch of Jian' er qing dynasty liquid samples (the samples used for the repeatability and recovery rate test, batch No. 06119017, hydrogen cyanide content 10.482 mug/mL) are respectively taken for 1mL, respectively placed in 10mL headspace bottles, added with 0.2mL of 10% phosphoric acid solution, and then respectively added with 1mL of water and reference substance solutions containing 5.194 mug/mL, 10.387 mug/mL and 20.775 mug/mL of hydrogen cyanide, quickly sealed, and subjected to sample injection detection according to the conditions.

The same batch of Jian' er qing dynasty liquid samples (the samples used in the repeatability and recovery rate test, batch No. 190930, hydrogen cyanide content of 3.418 mug/mL) are respectively taken for 1mL, respectively placed in 10mL headspace bottles, added with 0.2mL of 10% phosphoric acid solution, and then respectively added with 1mL of water, control solution containing 2.077 mug/mL, 5.194 mug/mL and 10.387 mug/mL of hydrogen cyanide, quickly sealed, and subjected to sample injection detection according to the conditions.

The calculation method comprises the following steps:

the peak area was plotted against the amount of the added control, the fitted line was extended to intersect the extension line of the content axis, the distance between this intersection point and the origin was equivalent to the content of hydrogen cyanide in the amount (1mL) of the sample solution taken, and the results of the measurement are shown in FIG. 21, FIG. 22 and Table 30.

TABLE 30 comparison of results from the Standard addition method and the Standard Curve method

From the results, the determination results of the water matrix standard curve method are higher than those of the standard addition method and the alternative matrix standard curve method, the existence of the matrix enhancement effect is verified again, and the relative deviations are respectively 12.4% and 20.2%, which indicates that the method is insufficient in the aspect of quantitative accuracy. Compared with the standard addition method, the relative deviation of the result measured by the substitute matrix standard curve method provided by the invention is only 1.3 percent and 6.7 percent respectively, the absolute deviation is less than 0.25 mug/mL, the result of the substitute matrix standard curve method is accurate, and the method is simple, convenient and feasible.

In the process of the methodology repeatability investigation, the average peak areas directly measured by sampling the two batches of the Jian' er clear solution with the batch numbers 190930 and 06119017 are 17.3 and 51.9 in sequence; in the subsequent recovery rate investigation, sodium chloride with the final concentration of 5% is added into each sample with the standard concentration to compensate matrix dilution caused by halving the sampling amount of the healthy infant clear solution, so that direct reading calculation of a standard curve method for substituting 10% sodium chloride for the matrix is facilitated. The difference is that in each standard addition concentration sample of the standard addition method, 50% of sample matrix is always kept, sodium chloride is not added, a water matrix standard curve (y is 4.6849x-0.5351, calculated by cyanate radical) is used to calculate the recovery rate of each standard addition concentration, the result of recovery rate investigation by using the water matrix standard curve method under the conventional thought can be simulated, the recovery rate of each standard addition concentration is between 86.6% and 103.7%, as shown in table 31, the recovery rate does not exceed the recovery rate limit regulation of 80% to 115% in the "four-part general rule 9101" analytical method verification guiding principle "of the year 2020 edition of Chinese pharmacopoeia, and the objective fact that the measurement result has a larger difference with the" true value "of the standard addition method cannot be reflected by the recovery rate investigation only.

TABLE 31 recovery of each concentration by standard addition method (calculation of standard curve for water base)

See methodology verification, repeatability investigation section for details.

Further analysis, in the above methodology repeatability investigation process, the average peak areas directly measured by sampling two batches of Jian' er qing dynasty solutions of the batches 190930 and 06119017 are 17.3 and 51.9 in sequence, and since they are 100% of sample matrix, they are substituted into 10% sodium chloride substituted matrix standard curve (y is 5.1779x-0.2207, calculated by cyanate), so that the "true original amount" of hydrogen cyanide in the sample can be calculated, and the "true original amount" and the "added amount" of the reference substance are prepared by standard addition method, and finally exist in 50% of sample matrix, so that they are substituted into 50% blank sample matrix standard curve (y is 4.8632x-0.4845, calculated by cyanate), the theoretical total peak area corresponding to the "true original amount" and the "added amount" can be calculated, and theoretically, after the above numerical value is transferred, the conventional thought can be predicted, the result of the 'measured recovery rate' directly calculated by using a water matrix standard curve method. The relative average deviation between the predicted recovery rate and the "actually measured recovery rate" is 0.6% -3.3% by comparison, and the results are consistent as shown in table 32; fully indicates that the sodium chloride with different contents can be used as a substitute of a blank sample matrix, reflects the influence of a matrix effect in the determination process of the content of the free hydrogen cyanide in the healthy infant digestion solution, replaces a matrix standard curve method, and is completely feasible.

TABLE 32 recovery of each concentration by Standard addition method (Multi-stage Standard Curve calculation prediction)

Example 9

Detecting the content of free hydrogen cyanide in the Jian' er qing Jie liquid by gas chromatography

Preparing bitter almond aromatic water: taking 50g of bitter almond medicinal material, distilling and extracting 50mL of aromatic water, adding phosphoric acid aqueous solution with the pH value of 2.5 to dilute to 1000mL, shaking up, precisely measuring 2mL, placing in a 10mL headspace bottle which is added with 0.2g of sodium chloride in advance (even if the final content of the sodium chloride is 10%), adding 0.2mL of 10% phosphoric acid solution, and sealing to obtain the bitter almond beverage.

Preparation of a test solution: precisely measuring 2mL of the Jianer qingjie liquid sample, putting the sample into a 10mL headspace bottle, adding 0.2mL of 10% phosphoric acid solution, and sealing to obtain the Jianer qingjie liquid.

The determination method comprises the following steps: detecting by headspace sampling-capillary column gas chromatography, and calculating the content of free hydrogen cyanide in the sample according to external standard curve method.

The results of the measurements are shown in tables 33, 34, 3, 36 and fig. 23 to 29.

TABLE 33 content of free hydrogen cyanide in Jian' er Qing Jie liquor samples from different manufacturers

TABLE 34 content of free hydrogen cyanide in homemade bitter apricot kernel aromatic water

Bitter apricot kernel aromatic water	Mu g/mL of free hydrogen cyanide
		1	4.378
2	4.982
		3	10.217
4	4.438
		5	4.8

TABLE 35 content of hydrogen cyanide in Jian' er Qing Jie liquor from different enterprises

TABLE 36 content of hydrogen cyanide in homemade bitter apricot kernel aromatic water

The key point of the method is that a mandelonitrile reference substance is prepared by using 0.1-1.0% (v/v) glacial acetic acid acetonitrile solution or phosphoric acid water solution with the pH value of 2.0-3.5, so that a stable mandelonitrile solution can be obtained (theoretically, the glacial acetic acid acetonitrile and acid in the water solution are replaced, and the aim of stabilizing the mandelonitrile reference substance solution can also be achieved); sodium chloride with a final concentration of 10% is used as a substitute matrix to prepare a substitute matrix control solution (theoretically, potassium chloride and other similar water-soluble neutral salts can also achieve the purpose); the total hydrogen cyanide content was evaluated using the free hydrogen cyanide plus mandelonitrile composition. The invention solves the technical problems of poor stability of mandelonitrile solution, matrix enhancement effect in the process of detecting hydrogen cyanide by headspace sample introduction gas chromatography and the like; the fact that the highly toxic substance hydrogen cyanide exists in preparations containing bitter almond aromatic water such as bitter almond aromatic water, a health-care liquid for clearing away children in a free state and mandelonitrile form is respected, and the determination result is the direct and objective reflection of the contents of the bitter almond aromatic water and the bitter almond aromatic water in the production process and the storage process; the pharmacological activity and safety of free hydrogen cyanide and mandelonitrile based on the same equivalent weight of hydrogen cyanide are not necessarily the same; the fact that the pharmacological activity and safety of the mixture of free hydrogen cyanide and mandelonitrile with the same total amount of hydrogen cyanide are not necessarily the same makes up the deficiency of the total hydrogen cyanide titration method; through the respective detection of the free hydrogen cyanide and the mandelonitrile, the real content composition of the free hydrogen cyanide and the mandelonitrile in bitter almond aromatic water preparations such as bitter almond aromatic water, a healthy infant clear solution and the like can be obtained, and data with discrimination can be provided for the optimization of the production process, the safety, the effectiveness evaluation, the stability investigation and the like.

While the invention has been described and illustrated with reference to specific embodiments thereof, such description and illustration are not intended to limit the invention. It will be clearly understood by those skilled in the art that various changes in form and details may be made therein without departing from the true spirit and scope of the invention as defined by the appended claims, to adapt a particular situation, material, composition of matter, substance, method or process to the objective, spirit and scope of this application. All such modifications are intended to be within the scope of the claims appended hereto. Although the methods disclosed herein have been described with reference to particular operations performed in a particular order, it should be understood that these operations may be combined, sub-divided, or reordered to form equivalent methods without departing from the teachings of the present disclosure. Accordingly, unless specifically indicated herein, the order and grouping of the operations is not a limitation of the present application.

Claims

1. A detection method of Jian' er qing jie liquid comprises the following steps:

2. The method according to claim 1, wherein the volume of glacial acetic acid in the glacial acetic acid acetonitrile solution is 0.1-1% of the acetonitrile volume.

3. The method according to claim 1, wherein the pH of the aqueous phosphoric acid solution is 2.0 to 3.5.

4. The method according to claim 1, wherein the mobile phase in the high-phase liquid chromatography detection process is acetonitrile-phosphoric acid solution; the volume ratio of the acetonitrile to the phosphoric acid solution is (20-25) to (75-80).

5. The method according to claim 1, wherein the flow rate of the chromatographic column in the detection process of the high-phase liquid chromatography is 0.5-1.5 mL/min;

the detection wavelength is 205-210 nm;

the column temperature is 25-35 ℃;

the injection volume is 5-15 mu L.

6. The method of claim 1, further comprising:

7. The method of claim 6, wherein the gas chromatography assay process control solution comprises:

8. The method of claim 7, wherein the control solution is prepared by a method comprising:

9. The method according to claim 6, wherein the headspace equilibrium temperature during the detection by gas chromatography is 40-70 ℃;

the equilibrium time is not more than 1 hour;

the quantitative loop temperature is 70-80 ℃.

10. The method of claim 6, wherein the temperature of the transmission line during the detection process of the gas chromatography is 90-110 ℃;

the temperature of a sample inlet is 180-220 ℃;

the flow dividing ratio is (3-7): 1.