CN116953131A - A kind of detection method of glyceraldehyde and its application - Google Patents

Abstract

The invention discloses a method for detecting glyceraldehyde, a degradation impurity of fructose and glycerol. Based on the ultraviolet absorption characteristics of the product after the reaction of glyceraldehyde and a suitable derivatization reagent, the present invention establishes a simple and universal derivatization HPLV-UV method to measure glyceraldehyde produced by the degradation of glycerol or fructose. The detection method uses 2,4-dinitrophenylhydrazine or a similar type of derivatization reagent to derivatize glyceraldehyde in the test solution and the reference solution to form a product that absorbs in the ultraviolet region. Compared with glyceraldehyde, the product has reduced polarity and enhanced retention on the chromatographic column, enabling quantitative analysis of glyceraldehyde. After methodological verification, it was proved that the method has good specificity and sensitivity.

Description

A kind of detection method of glyceraldehyde and its application

Technical field

The invention belongs to the field of drug analysis and detection, and specifically relates to a derivatization method for determining glyceraldehyde, and in particular to a derivatization HPLC-UV method for detecting glyceraldehyde in injections and other dosage forms of medicines containing glycerol and/or fructose. method.

Background technique

Glycerin is widely used as an excipient in medicines; in addition, glycerol and fructose are carbohydrate compounds and are relatively safe for the human body. High-concentration solutions are often used clinically as dehydration drugs, such as glycerol-fructose sodium chloride injection. According to domestic injection production conditions and relevant regulatory requirements, high-temperature sterilization is often used in the production process of injections. The inventor found that both glycerol and fructose can be degraded to glyceraldehyde under high temperature and other conditions, resulting in the possibility that related medicines may contain glyceraldehyde impurities.

Impurities in drugs refer to all other chemical substances other than the target compound that are introduced or produced during the production, storage or use of the drug. Impurities in drugs directly affect the efficacy of the drug and may cause other adverse side effects and must be controlled.

In addition, the structure of glyceraldehyde contains a terminal aldehyde group and cannot form a hemiacetal ring structure. This structure is a mutagenic warning structure, which means that glyceraldehyde may be genetically toxic. Based on the above information, it is very necessary to establish a highly specific and sensitive glyceraldehyde detection method.

High performance liquid chromatography (HPLC) is one of the most commonly used instruments in pharmaceutical analysis, and ultraviolet detector (UV) is the most commonly used detector in high performance liquid chromatography. Due to its simple structure and lack of chromophore, glyceraldehyde has weak UV absorption; it is also highly polar and has weak retention on reversed-phase chromatography columns, making it difficult to detect using conventional HPLC-UV instruments. In pharmaceuticals, since they contain many components such as main components, main component degradation impurities, and excipients, the detection matrix is more complex, which increases the difficulty of glyceraldehyde detection. In pharmaceutical products containing glycerol and/or fructose, the detection method of glyceraldehyde has not been reported.

Contents of the invention

In view of the shortcomings of the existing technology, the present invention discloses a method for detecting glyceraldehyde, which can be used for medicines containing glycerin and/or fructose, such as glycerol injection, fructose injection, glycerol-fructose injection, and glycerol-fructose chloride. Detection of glyceraldehyde in sodium injection and other drugs.

The detection method described in the present invention uses an external standard method for quantification, and uses a glyceraldehyde reference substance to detect the glyceraldehyde in the test sample. It mainly includes derivatization pre-treatment and high-performance liquid chromatography detection. Through the detection of derivatization products, qualitative or quantitative detection of glyceraldehyde is achieved.

The specific technical solutions of the present invention are as follows:

The derivatization pretreatment method has the following steps:

(1) Prepare a glyceraldehyde reference substance aqueous solution, and pretreat the solution to obtain a reference substance stock solution; prepare a test sample aqueous solution as a test sample stock solution;

(2) Pipette appropriate amounts of the test product stock solution and the reference product stock solution into their respective reaction systems, add derivatization reagents, add pH adjusters and reaction solvents, and react at 40°C to 70°C for 1 to 3 hours to obtain Test solution and reference solution.

After the above reaction, glyceraldehyde generates a hydrazone structure corresponding to the derivatization reagent:

In the step (1), the pretreatment of the reference substance stock solution is to place the prepared glyceraldehyde aqueous solution in an environment of 50 to 100°C for 0.5 to 5 hours to ensure that glyceraldehyde is fully dissolved in the water to facilitate subsequent processing. The derivatization reaction can proceed completely. Among them, the way to maintain the ambient temperature can be dry heating or wet heating. Wet heating is preferred, and water bath heating is preferred. Furthermore, the treatment temperature of the glyceraldehyde solution is preferably 60 to 70°C.

In the step (2), the derivatization reagent is a hydrazine compound containing strong UV-absorbing groups such as benzene ring and naphthalene ring, and the reaction system is an acetonitrile-water solution, and an appropriate pH regulator is added.

Furthermore, the derivatization reagent is preferably 2,4-dinitrophenylhydrazine, wherein the concentration of the derivatization reagent is 2 to 10 mg/ml.

Furthermore, an acidic pH adjuster is added to make the pH environment of the derivatization reaction acidic. Hydrochloric acid solution is preferably used. Further, 1 mol/L hydrochloric acid solution is preferably used, and 30 to 300 μl is added.

Furthermore, the temperature of the derivatization reaction is selected from 40°C to 70°C, with 50°C to 60°C being preferred; the reaction time is from 0.5 to 5 hours, with 1 hour being preferred.

The HPLC-UV detection method is a reversed-phase chromatography method. The high-performance liquid chromatograph used must have an online vacuum degasser, a dual pump or a quaternary pump, an automatic sampler, a column oven, a UV detector or Diode array detector. The chromatographic column uses octadecylsilane bonded silica gel as the filler; phosphate buffer or water is used as mobile phase A, and acetonitrile or acetonitrile-water is used as mobile phase B. The elution method is isocratic or gradient elution; the column The temperature is 25~40℃; the flow rate is 1.0~2.0ml/min, and the injection volume is 5~30μl.

Furthermore, the detection wavelength is selected from 300 to 400 nm, preferably from 360 to 380 nm, and further preferably from 365 nm.

Further, the chromatographic column was selected as GL Science InertSustain C18 column, the mobile phase was selected as water and acetonitrile, the column temperature was 30°C, the flow rate was 1.5ml/min, and the following gradient program was used for elution:

Table 1 Elution gradient table

The external standard method was used to calculate the glyceraldehyde content in the test sample. The structure of glyceraldehyde contains a chiral center, and its configuration is divided into D-type and L-type. In this method, the two configurations of glyceraldehyde peaked separately after empirical biochemistry. The content of glyceraldehyde in the test sample was calculated based on the sum of the peak areas of the two derivatives.

Beneficial effects of the invention

The present invention establishes a simple and universal derivatization HPLC-UV method for determination based on the fact that a derivatization reagent containing a chromophore and a hydrazine group in the structure can react with an aldehyde group in glyceraldehyde to generate a derivatization product with UV absorption. Glyceraldehyde content.

Methodological validation results show that glycerol, fructose and other degradation impurities of both will not interfere with the analysis, and the method has good specificity. In addition, the detection sensitivity of the method is high, the limit of quantification can reach 0.1534μg/ml; the linear relationship is good (r>0.995); the RSD of repeatability and intermediate precision are 0.44% and 1.3% respectively; the average recovery rate is 96.9-100.9% (RSD<2.0%), there is no obvious matrix interference; and the derivatization product has good stability within 24 hours. This method can be used to detect glyceraldehyde impurities in medicines containing glycerol and/or fructose, thereby further ensuring the product quality of related medicines and patient medication safety.

Description of the drawings

In Figure 1, Example 1 shows the chromatogram of the derivatized solution after forced degradation of glycerol.

In Figure 2, in Example 1, the chromatogram of the derivatized solution after fructose has been forced to degrade.

Figure 3 is the chromatogram of the derivatization solution of derivatization reagent, glyceraldehyde, glycerin and fructose under the chromatographic conditions of the present invention.

Figure 4 is the ultraviolet absorption spectrum of the glyceraldehyde derivatization product under the chromatographic conditions of the present invention.

Figure 5 Linear relationship diagram between glyceraldehyde concentration and peak area.

Specific implementation methods

The terms used in the present invention generally have the meanings commonly understood by those of ordinary skill in the art, unless otherwise stated.

The present invention will be described in further detail below with reference to specific examples and data. It should be understood that these examples are only for illustrating the present invention and do not limit the scope of the present invention in any way.

In the following examples, various procedures and methods not described in detail are conventional methods well known in the art.

1.1 Instruments

The instruments used in this method are shown in the table below:

Table 2 Instrument information

name manufacturer model Electronic analytical balance (0.01mg) Sartorius BT125D Electronic analytical balance (0.1mg) Sartorius BSA124S Electronic analytical balance (0.001mg) Sartorius MSE3.6P-0CE-DM Electronic analytical balance (0.01mg) Sartorius BT25S High performance liquid chromatography Shimadzu LC-2030C 3D, DAD detector, quaternary pump High performance liquid chromatography Shimadzu LC-2030C 3D, UV detector, quaternary pump High performance liquid chromatography Agilent Agilent 1260UV detector, quaternary pump Drug strong light irradiation test chamber Chongqing Yongsheng SHH-300GD-2 Vertical pressure steam sterilizer Shanghai Boxun YXQ-LS-50SⅡ Digital display constant temperature water bath Changzhou Guohua HH-4 Ultrapure water instrument Sichuan Youpu UPR-Ⅱ-5T Ultrasonic cleaner Kunshan Hechuang KH-250B

1.2 Reagents

The reagents used in the present invention are shown in the following table:

Table 3 Reagent information table

1.3 Preparation of solution

(1) Solvent: Measure 60ml acetonitrile and add 40ml water, shake well, and it is ready.

(2) 1mol/L hydrochloric acid: Take 90ml of hydrochloric acid, add an appropriate amount of water to make it 1000ml, shake well, and it is ready.

(3) Derivatization reagent: Take about 250 mg of 2,4-dinitrophenylhydrazine, weigh it accurately, place it in a 50 ml measuring bottle, add acetonitrile and sonicate to dissolve and dilute to the mark, shake well, and get (about 5 mg/ml ).

(4) Glyceraldehyde stock solution: Take about 10 mg of glyceraldehyde reference substance, weigh it accurately, put it in a 100 ml measuring bottle, add water to dilute to the mark, shake well, place it in a 50°C water bath for 1 hour, take it out, and cool it to get (about 0.1 mg/ml).

1.4 Derivatization process

Precisely measure 2 ml of the sample to be tested, put it into a 20 ml measuring bottle, add 2 ml of 2,4-dinitrophenylhydrazine solution and 50 μl of 1 mol/L hydrochloric acid solution, add solvent to dilute to the mark, shake well, and immediately place it in a 50°C water bath for reaction for 60 seconds minutes, cool it in an ice bath, take it out, filter it, and use it as the test solution.

Precisely measure 2 ml of glyceraldehyde stock solution, place it in a 20 ml measuring bottle, add 2 ml of 2,4-dinitrophenylhydrazine solution and 50 μl of 1 mol/L hydrochloric acid solution, add solvent to dilute to the mark, shake well, and immediately place in a 50°C water bath for reaction 60 minutes, cool in an ice bath, filter, and use as reference solution.

Precisely measure 2 ml of water, place it in a 20 ml measuring flask, add 2 ml of 2,4-dinitrophenylhydrazine solution and 50 μl of 1 mol/L hydrochloric acid solution, add solvent to dilute to the mark, shake well, and immediately place in a 50°C water bath for reaction for 60 minutes. Cool in an ice bath, filter, and use as a blank solution.

1.5 Chromatographic conditions

Chromatographic column: Octadecyl bonded silica gel is used as filler (GL Sciences Inc Inert Sustain C18 column, 4.6×150mm, 5μm or a column with equivalent performance);

Mobile phase A: water;

Mobile phase B: acetonitrile;

Flow rate: 1.5ml/min;

Column temperature: 30℃;

Detection wavelength: 365nm;

Injection volume: 10μl;

Elute according to the gradient in the following table:

Table 4 Elution gradient table

Example 1 Degradation of glycerol and fructose to produce glyceraldehyde

Take glycerol and fructose to prepare glycerol stock solution and fructose stock solution respectively, and conduct forced degradation experiments, including strong acidic damage, strong alkaline damage, high temperature damage, oxidative damage and light damage. The specific conditions are shown in the table below.

Table 5 forced degradation method

Take each of the above-destructed solutions and conduct derivatization respectively, and detect the glyceraldehyde content in each solution using the external standard method. The experimental results are shown in the table below.

Table 6 Glyceraldehyde produced by the degradation of glycerol and fructose

The results in the above table show that the glyceraldehyde content of glycerol increases significantly under oxidative conditions; fructose generates more glyceraldehyde under high temperature and alkali destruction conditions; in addition, fructose generates a small amount of glyceraldehyde under strong acid conditions.

Example 2 Effect of pretreatment of glyceraldehyde stock solution on derivatization effect

Glyceraldehyde stock solutions were prepared separately and processed and derivatized under different conditions. The results are shown in the table below:

Table 7 Derivatization Conditions Investigation 1

Note: The derivatization level is the ratio of the sum of the peak areas of the above solutions to the sum of the peak areas of the glyceraldehyde solution with basically the same concentration after complete derivatization.

From the results in the above table, it can be seen that if the glyceraldehyde stock solution is derivatized for 5 hours at room temperature without special treatment, the degree of derivatization reaction is only 43%, and it takes 5 hours at 50°C for the reaction to be basically complete. After the glyceraldehyde stock solution is treated, even if it is placed under low temperature conditions, as long as the time is long enough, the derivatization efficiency can be greatly increased.

Based on the above results, the same glyceraldehyde solution was used to examine the effect of storage time at 50°C on the derivatization level. The results are shown in the table below. It shows that the completion of the subsequent derivatization reaction can be ensured by placing the glyceraldehyde stock solution at 50°C for 0.5 to 5 hours.

Table 8 Derivatization condition investigation 2

Example 3 Selection of Derivatization Temperature and Time

The glyceraldehyde solution and the test solution were derivatized at different temperatures for 1 hour respectively. The results showed that the derivatization level of the glyceraldehyde solution and the test solution reached more than 90% within 1 hour at a temperature of 40 to 70°C. There was no significant difference in the results of the derivatization reaction of glyceraldehyde solution at 50°C for 1 to 5 hours.

Table 9 Derivatization Conditions Investigation 3

Note: The test solution in the above table refers to the test solution prepared with glycerol-fructose sodium chloride injection.

Example 4 Methodology Verification and Application

4.1 Exclusiveness

Precisely measure 2 ml of water, place it in a 20 ml measuring flask, add 2 ml of 2,4-dinitrophenylhydrazine solution and 50 μl of 1 mol/L hydrochloric acid solution, add solvent to dilute to the mark, shake well, and immediately place in a 50°C water bath for reaction for 60 minutes. Cool in an ice bath, filter, and inject sample for analysis.

Take about 10 mg of the glyceraldehyde reference substance, weigh it accurately, put it in a 100 ml measuring flask, add water to dilute to the mark, shake well, put it in a 50°C water bath for 1 hour, take it out, cool it, accurately measure 2 ml, put it in a 20 ml measuring flask, add 2 , 2 ml of 4-dinitrophenylhydrazine solution and 50 μl of 1 mol/L hydrochloric acid solution, add solvent to dilute to volume, shake well, immediately place in a 50°C water bath to react for 60 minutes, cool in an ice bath, filter, and inject sample for analysis.

Take appropriate amounts of glycerol and fructose, add water to prepare a 100 mg/ml glycerol solution and a 50 mg/ml fructose solution respectively. Precisely measure 2 ml of each, place it in a 20 ml measuring bottle, add 2 ml of 2,4-dinitrophenylhydrazine solution and Dilute 50 μl of 1 mol/L hydrochloric acid solution to the mark with solvent, shake well, and immediately place in a 50°C water bath to react for 60 minutes. Place in an ice bath to cool, take it out, filter, and inject a sample for analysis.

The chromatogram is shown in Figure 3; the ultraviolet absorption chromatogram of the glyceraldehyde derivative is shown in Figure 4. In the above chromatogram, the double peaks produced after derivatization of glyceraldehyde are completely separated from the derivatization reagent peak and other chromatographic peaks introduced by glycerol and fructose. Glycerin, fructose and other impurities do not interfere with the determination of glyceraldehyde, indicating the specificity of the method. good.

4.2 Linear

Prepare a glyceraldehyde stock solution of approximately 0.2 mg/ml, pipet appropriate amounts, and dilute with water to prepare a series of glyceraldehyde solutions of 0.01, 0.025, 0.05, 0.08, 0.10, 0.12, and 0.15 mg/ml. Derivatize them according to the law. Inject. Taking the concentration of glyceraldehyde as the abscissa and the sum of the peak areas of the derivatized products as the ordinate, use the least squares method to make a linear regression equation for the sum of the peak areas of its derivatives (A) with the concentration of glyceraldehyde solution C (μg/ml). Results: The linear relationship between glyceraldehyde and glyceraldehyde was good in the range of 0.01 to 0.15 mg/ml, and the correlation coefficient was r>0.995. The results are shown in Figure 5.

4.3 Sensitivity

The glyceraldehyde stock solution was diluted step by step, derivatized according to the law and sampled for analysis. The peak height of the chromatographic peak of the glyceraldehyde derivative and the baseline noise near the peak were measured. The signal-to-noise ratio (S/N) was not less than The detection limit is calculated at 3:1; the quantification limit is calculated based on the signal-to-noise ratio (S/N) being no less than 10:1. The solution with the quantification limit concentration is continuously injected 6 times, and the solution with the detection limit concentration is continuously injected 3 times.

The results showed that the detection limit of glyceraldehyde was 0.0377 μg/ml, and the quantitation limit was 0.2622 μg/ml.

4.4 Precision test

Prepare the reference solution, the test product solution and the test product spiked solution respectively, calculate the glyceraldehyde content in the test product spiked solution according to the external standard method, and examine the recovery rate of glyceraldehyde added to the test product spiked solution. And calculate the relative standard deviation of each 6 measurement results. Two people conducted the detection on different days using different chromatographs.

As a result, the recovery rate of 6 copies of Person A was between 99.3% and 100.9%, and the RSD was 0.6%; the recovery rate of 6 copies of Person B was between 100.5% and 103.8%, and the RSD was 1.2%; the recovery rate of 12 copies of Person B was between 100.5% and 103.8%. The RSD is 1.3%. It shows that the precision of this method is good.

4.5 Accuracy

Prepare glyceraldehyde solutions of 0.025, 0.05, 0.1 and 0.12mg/ml respectively, add them to the test product and derivatize according to the law to prepare test product spiked solutions with different concentration levels, combined with the background amount in the test solution measured in parallel , to examine the recovery rate of glyceraldehyde, and examine 3 copies of each concentration in parallel.

Recovery rate = (measured amount in the test sample spiked solution - background amount in the test sample solution)/added amount.

The experimental results show that the recovery rate of glyceraldehyde is between 96.9% and 100.9%, the average recovery rate of 12 solutions is 99.3%, and the RSD is 1.6%. The accuracy of this method is good.

4.6 Solution stability

Take the glyceraldehyde stock solution, derivatize it according to the law to prepare the reference solution, place it in the autosampler for different times and repeat the injection, and examine the stability of the reference solution based on the changes in the derivatized peak area.

Results: Within 24 hours at room temperature, the peak area of glyceraldehyde derivatives changed by less than ±0.5%, and the RSD of the peak area was 0.15%. The stability of glyceraldehyde derivatives was good.

Example 5

Precisely measure 2 ml of water, place it in a 20 ml measuring flask, add 2 ml of 2,4-dinitrophenylhydrazine solution and 50 μl of 1 mol/L hydrochloric acid solution, add solvent to dilute to the mark, shake well, and immediately place in a 50°C water bath for reaction for 60 minutes. Cool in an ice bath, filter, and use as a blank solution.

Prepare 0.1 mg/ml glyceraldehyde stock solution according to the law, accurately measure 2 ml, place it in a 20 ml measuring bottle, add 2 ml of 2,4-dinitrophenylhydrazine solution and 50 μl of 1 mol/L hydrochloric acid solution, add solvent to dilute to the mark, and shake Mix well, immediately put it in a 50°C water bath for 60 minutes, cool it in an ice bath, filter it, use it as a reference solution, and inject samples for analysis according to the law.

Precisely measure 2 ml of the sample to be tested, put it into a 20 ml measuring bottle, add 2 ml of 2,4-dinitrophenylhydrazine solution and 50 μl of 1 mol/L hydrochloric acid solution, add solvent to dilute to the mark, shake well, and immediately place it in a 50°C water bath for reaction for 60 seconds minutes, put it in an ice bath to cool, take it out, filter it, use it as the test solution, and inject samples for analysis according to the law.

The content of glyceraldehyde was calculated based on the peak area according to the external standard method. Take one batch of samples from each of two manufacturers and two specifications of glycerol-fructose sodium chloride injection for measurement.

Table 10 Detection results of glyceraldehyde in 4 batches of glycerol-fructose sodium chloride injection by the method of the present invention

sample Manufacturer specifications 1-250ml Manufacturer specifications 1-500ml Manufacturer 2-250ml specifications Manufacturer 2-500ml specifications Test results 34.50μg/ml 34.05μg/ml 17.09μg/ml 18.82μg/ml

Claims (10)

1. A method for determining glyceraldehyde produced by the degradation of glycerol or fructose using a derivatized HPLC-UV method, which is characterized by comprising the following steps: (1) Prepare a glyceraldehyde aqueous solution and pretreat the solution to obtain a reference substance stock solution; prepare a test sample aqueous solution as a test sample stock solution; (2) Pipette appropriate amounts of the test product stock solution and the reference product stock solution into their respective reaction systems, add derivatization reagents, add pH adjusters and reaction solvents, and react at 40°C to 70°C for 1 to 3 hours to obtain Test solution and reference solution; (3) Use the HPLC-UV method to detect the relevant derivatization products of the reference solution and the test solution, and perform calculations with the external standard method. 2. The method according to claim 1, characterized in that the test sample is a liquid medicine containing glycerin and/or fructose. 3. The method according to claim 2, characterized in that the liquid medicine containing glycerol and/or fructose is glycerin injection, fructose injection, glycerol-fructose injection or glycerol-fructose sodium chloride injection. 4. The method according to claim 1, characterized in that the pretreatment is: placing the glyceraldehyde aqueous solution at 50-100°C for 0.5-5 hours. 5. The method according to claim 1, characterized in that the derivatization reagent is a compound containing a strong chromophore and a hydrazine group. 6. The method according to claim 5, characterized in that the derivatization reagent is 2,4-dinitrophenylhydrazine. 7. The method according to claim 5, characterized in that the step (2) uses acetonitrile-water as the reaction solvent at 50-60°C for 1 hour. 8. The method according to claim 1, characterized in that in the step (3), in the HPLC-UV method, the chromatographic column uses octadecylsilane bonded silica gel as a filling agent; phosphate buffer Or water is used as mobile phase A, and acetonitrile or acetonitrile-water is used as mobile phase B. The elution method is isocratic or gradient elution; the column temperature is 25~40°C; the flow rate is 1.0~2.0ml/min. 9. The method according to claim 8, characterized in that the HPLC-UV method has an ultraviolet detection wavelength of 300-400 nm. 10. The method according to claim 8, characterized in that the method of gradient elution is as follows: From 0 to 20 minutes, the volume percentage of mobile phase A dropped from 87% to 70%; From 20 to 21 minutes, the volume percentage of mobile phase A dropped from 70% to 20%; 21 to 25 minutes, keep the volume percentage of mobile phase A at 20%; From 25 to 26 minutes, the volume percentage of mobile phase A increased from 20% to 87%; For 27 to 35 minutes, keep the volume percentage of mobile phase A at 87%.