Brand-new formaldehyde content liquid chromatography detection method and application

Abstract

The invention relates to the technical field of formaldehyde detection, and discloses a brand-new formaldehyde content liquid chromatography detection method which comprises the following steps: step 1, preparing a kit solution; step 2, adding the kit solution into a to-be-detected sample or putting the kit solution in a detection environment, and adding a solvent for dilution to obtain a test solution; 3, precisely measuring the kit solution, and adding a solvent for dilution to obtain a derivative blank solution; step 4, respectively sucking the same amount of the test solution and the derivative blank solution, and injecting into high performance liquid chromatography for quantitative detection; according to the method, the use of toxic reagents is reduced, the operation is simple, the specificity is high, the sensitivity is high, and the durability and repeatability are good; the method can be applied to detection of formaldehyde in raw material medicines, auxiliary materials, solvents, preparations, foods and environments.

Description

Technical Field

[0001] This invention relates to the field of formaldehyde detection technology, specifically to a novel liquid chromatography method for formaldehyde content detection and its application. Background Technology

[0002] Formaldehyde is a highly toxic organic compound that can damage cell proteins, trigger allergic reactions, and cause DNA and chromosome damage, reducing cell viability and accelerating cell death, posing a significant potential threat to human health. On October 27, 2017, formaldehyde was listed as a Group 1 carcinogen by the World Health Organization. Formaldehyde can be found in pharmaceuticals, food, and the environment. It readily undergoes nucleophilic addition reactions with active ingredients containing secondary or primary amines, producing degradation impurities that affect the quality of pharmaceuticals, food, and the environment. Furthermore, formaldehyde itself is toxic and will impact human health.

[0003] Literature review shows that there is currently no specific method for formaldehyde detection in the pharmaceutical field; methods for formaldehyde content detection in food are typically adopted. Commonly used analytical methods for formaldehyde content detection include spectrophotometry, liquid chromatography, gas chromatography, and fluorescent probe methods. Spectrophotometry is classified according to the colorimetric reagent, including the acetylacetone method, the phenol reagent method, and the chemical substance method, with the acetylacetone method being the most widely used. Its disadvantages include relatively low sensitivity, susceptibility to the turbidity of the reaction matrix, and certain requirements on the pigment content of the sample. Liquid chromatography and gas chromatography typically use 2,4-dinitrophenylhydrazine as a derivatizing agent. For example, a high-performance liquid chromatography method for formaldehyde analysis in ranitidine hydrochloride (application number 201911271909.X) reacts with formaldehyde in the sample to generate 2,4-dinitrophenylhydrazone, which has the characteristics of high precision and good separation effect. However, 2,4-dinitrophenylhydrazine is a substance listed in the national hazardous materials catalog, the procurement process is complicated, and it itself has high explosiveness, blood toxicity, and sensitizing properties, posing significant safety hazards. Fluorescent probe method utilizes the reaction between the amino group of the fluorescent probe molecule and formaldehyde. The fluorescence properties of the molecule change before and after the reaction, and the formaldehyde is quantitatively analyzed based on the change in fluorescence intensity. It has the characteristics of high sensitivity, high selectivity and simple operation, but its equipment is expensive and not widely used. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a novel liquid chromatography method and application for formaldehyde content detection. This method reduces the use of toxic reagents, is simple to operate, highly specific, highly sensitive, durable, and has good repeatability. It can be applied to the detection of formaldehyde in pharmaceutical raw materials, excipients, solvents, and formulations, as well as in food and the environment.

[0005] To achieve the primary objective, this invention provides a novel liquid chromatography method for the detection of formaldehyde content, comprising the following steps: Step 1: Prepare the reagent solution; Step 2: Add the reagent solution to the sample to be tested or place the reagent solution in the testing environment and dilute with solvent to obtain the test solution; Step 3: Accurately measure the reagent solution and dilute it with solvent to obtain the derivatized blank solution; Step 4: Take equal amounts of the test solution and the derivatized blank solution and inject them into a high-performance liquid chromatograph for quantitative detection. The high-performance liquid chromatography (HPLC) conditions are as follows: Column: ACE Excel 3 Super C18; Mobile phase A: 0.01 mol / L diammonium hydrogen phosphate solution - acetonitrile solution; Mobile phase B: Acetonitrile; Gradient elution is performed using mobile phase A and mobile phase B.

[0006] Preferably, the reagent solution in step one is prepared by accurately weighing an appropriate amount of derivatizing agent and using water to prepare a solution containing approximately 10 mg per 1 ml; the derivatizing agent is ambroxol hydrochloride.

[0007] As a preferred embodiment, the first method for preparing the test solution in step two is as follows: accurately weigh an appropriate amount of the sample to be tested, place it in a 10ml volumetric flask, accurately add 1ml of the reagent solution and an appropriate amount of solvent, seal, shake and sonicate to dissolve the sample, bathe in a 60℃ water bath for 10min, remove and cool, dilute with solvent to the mark, shake well and filter, and collect the filtrate.

[0008] As a preferred embodiment, the second method for preparing the test solution in step two is as follows: place the reagent solution in the detection environment, leave it open for 4 hours, accurately measure 1 ml of the reagent solution, place it in a 10 ml volumetric flask, dilute it to the mark with solvent, and shake well.

[0009] Preferably, the solvent is a mixture of mobile phase A and mobile phase B in a volume ratio of 1:1.

[0010] Preferably, the preparation method of mobile phase A in step four is as follows: accurately weigh diammonium hydrogen phosphate, add water and stir until dissolved, adjust the pH to 9.5 with phosphoric acid, and mix the above diammonium hydrogen phosphate solution with acetonitrile at a volume ratio of 8:2.

[0011] Preferably, in step four, the flow rate is 0.9 ml / min to 1.1 ml / min; the column temperature is 20℃ to 30℃; the injection volume is 20 μL; the injection plate temperature is 5℃; and the detection wavelength is 248 nm.

[0012] Preferably, the gradient elution procedure is as follows: Gradient elution for 0–30 minutes, with mobile phase A ranging from 100% to 79% and mobile phase B ranging from 0% to 21%; Gradient elution for 30–60 minutes, with mobile phase A comprising 79%–68% and mobile phase B comprising 21%–32%; Gradient elution for 60-62 minutes, with mobile phase A at 68% to 100% and mobile phase B at 32% to 0%; Elute at isocratic rate for 8 minutes, with mobile phase A at 100% and mobile phase B at 0%.

[0013] To achieve the second objective, this invention provides a novel liquid chromatography method for formaldehyde content detection, applicable to the detection of formaldehyde in pharmaceutical raw materials, excipients, solvents, and formulations, as well as in food and the environment.

[0014] The derivatizing agent of this invention is ambroxol hydrochloride. The sample to be tested is treated based on the fact that formaldehyde readily reacts with the derivatizing agent to generate trans-4-(6,8-dibromo-1,4-dihydroquinazolin-3(2H)-yl)cyclohexanol. The reaction temperature can be room temperature, or heating can be used to accelerate the reaction. The reaction is rapid, highly sensitive, the reagent is safe and non-toxic, and there is little harm to the human body.

[0015] The reaction mechanism is as follows: Using the ACE Excel 3 Super column, which offers superior separation performance and a wide applicable pH range, the mobile phase composition was adjusted, and the column temperature and gradient program were optimized to achieve better resolution. Particular attention was paid to screening the mobile phase pH to reduce interference from formaldehyde in the air during measurement. Additionally, the injection volume was determined based on the method's sensitivity requirements.

[0016] This invention, by adopting the above technical solutions, has significant technical effects: This invention is a novel analytical method for formaldehyde content detection in multiple fields, which can accurately detect the formaldehyde concentration or content in pharmaceutical raw materials, excipients, solvents, preparations, as well as in food and the environment.

[0017] The analytical method of this invention uses a reagent kit that is economical, safe, non-toxic, and readily available for sample preparation.

[0018] The detection method of the present invention reduces the use of toxic reagents, is simple to operate, highly specific, highly sensitive, durable and repeatable, and has good application prospects in controlling formaldehyde content. Attached Figure Description

[0019] Figure 1 This is the color spectrum of the formaldehyde content detection method used in the pharmaceutical field in Example 1.

[0020] Figure 2This is the color spectrum of the formaldehyde content detection method used in the food industry in Example 2.

[0021] Figure 3 This is the color spectrum of the formaldehyde content detection method used in the environmental field in Example 3.

[0022] Figure 4 The colorimetric spectrum is used to verify the specificity of the method in Experiment Example 1.

[0023] Figure 5 This is the mass spectrum of the reaction product of formaldehyde and the derivatizing reagent in Experiment Example 2.

[0024] Figure 6 This is the mass spectrum of the derivatives of the test sample solution in Experiment Example 3. Detailed Implementation

[0025] The present invention will be further illustrated below with reference to the accompanying drawings and embodiments. These embodiments are illustrative and exemplary of the invention and do not limit the scope of the invention in any way.

[0026] The present invention relates to the following sample information: L-tartaric acid from Hunan Erkang, pomegranate flavor from Shanghai Manshi, glycerin from Hedaxipu, sorbitol and propylene glycol from Gedian Renfu, and hydroxyethyl cellulose from Ashland.

[0027] Example 1 A novel method for formaldehyde content detection in pharmaceuticals employs high-performance liquid chromatography (HPLC) followed by derivatization with ambroxol hydrochloride. The HPLC conditions are as follows: Column: ACE Excel 3 Super C18, 4.6mm × 150mm; Mobile phase A: 0.01 mol / L diammonium hydrogen phosphate solution (pH adjusted to 9.5 with phosphoric acid) - acetonitrile (80:20); preparation method: accurately weigh about 1.32 g of diammonium hydrogen phosphate, add 1000 ml of water, stir to dissolve, adjust the pH to 9.5 with phosphoric acid, measure 800 ml of the solution and mix with 200 ml of acetonitrile. Mobile phase B: Acetonitrile; Flow rate: 0.9 ml / min ~ 1.1 ml / min; Column temperature: 20℃~30℃; Injection volume: 20 μl; Sample inlet temperature: 5℃; Detection wavelength: 248nm.

[0028] The following gradient elution method was used: Time (mim) Mobile phase A (%) Mobile phase B (%) 0 100 0 30 79 21 60 68 32 62 100 0 70 100 0 Reagent solution: Accurately weigh an appropriate amount of ambroxol hydrochloride and prepare a solution containing approximately 10 mg per 1 ml with water.

[0029] Solvent: Mobile phase A: Mobile phase B = (50:50).

[0030] Derivatized blank solution: Accurately measure 1 ml of the reagent solution and place it in a 10 ml volumetric flask. Add an appropriate amount of solvent, seal, shake well, incubate in a 60°C water bath for 10 min, remove, cool, dilute to the mark with solvent, shake well, filter, and collect the filtrate.

[0031] Test solution 1: Accurately weigh an appropriate amount of L-tartaric acid and place it in a 10ml volumetric flask. Accurately add 1ml of the reagent solution and an appropriate amount of solvent. Seal the flask, shake and sonicate to dissolve the sample, incubate in a 60℃ water bath for 10min, remove and cool, dilute to the mark with solvent, shake well, filter, and collect the filtrate.

[0032] Test solution 2: Accurately weigh an appropriate amount of glycerol and place it in a 10ml volumetric flask. Accurately add 1ml of the reagent solution and an appropriate amount of solvent. Seal the flask, shake and sonicate to dissolve the sample, incubate in a 60℃ water bath for 10min, remove and cool, dilute to the mark with solvent, shake well, filter, and collect the filtrate.

[0033] Test solution 3: Accurately weigh an appropriate amount of hydroxyethyl cellulose and place it in a 10ml volumetric flask. Accurately add 1ml of the reagent solution and an appropriate amount of solvent. Seal the flask, shake and sonicate to completely disperse the sample, incubate in a 60℃ water bath for 10min, remove and cool, dilute to the mark with solvent, shake well, filter, and collect the filtrate.

[0034] See sample results Figure 1 The solvent does not interfere with the detection of formaldehyde in various pharmaceutical excipients.

[0035] Example 2 A novel method for detecting formaldehyde content in food products employs high-performance liquid chromatography (HPLC) followed by derivatization with ambroxol hydrochloride. The HPLC conditions are as follows: Column: ACE Excel 3 Super C18, 4.6mm × 150mm; Mobile phase A: 0.01 mol / L diammonium hydrogen phosphate solution (pH adjusted to 9.5 with phosphoric acid) - acetonitrile (80:20); preparation method: accurately weigh about 1.32 g of diammonium hydrogen phosphate, add 1000 ml of water, stir to dissolve, adjust the pH to 9.5 with phosphoric acid, measure 800 ml of the solution and mix with 200 ml of acetonitrile. Mobile phase B: Acetonitrile; Flow rate: 0.9 ml / min ~ 1.1 ml / min; Column temperature: 20℃~30℃; Injection volume: 20 μl; Sample inlet temperature: 5℃; Detection wavelength: 248nm.

[0036] The following gradient elution method was used: Time (mim) Mobile phase A (%) Mobile phase B (%) 0 100 0 30 79 21 60 68 32 62 100 0 70 100 0 Reagent solution: Accurately weigh an appropriate amount of ambroxol hydrochloride and prepare a solution containing approximately 10 mg per 1 ml with water.

[0037] Solvent: Mobile phase A: Mobile phase B = (50:50).

[0038] Derivatized blank solution: Accurately measure 1 ml of the reagent solution and place it in a 10 ml volumetric flask. Add an appropriate amount of solvent, seal, shake well, incubate in a 60°C water bath for 10 min, remove, cool, dilute to the mark with solvent, shake well, filter, and collect the filtrate.

[0039] Test solution 1: Accurately weigh an appropriate amount of pomegranate flavoring and place it in a 10ml volumetric flask. Accurately add 1ml of the reagent solution and an appropriate amount of solvent. Seal the flask, shake and sonicate to dissolve the sample, incubate in a 60℃ water bath for 10min, remove and cool, dilute to the mark with solvent, shake well, filter, and collect the filtrate.

[0040] Test solution 2: Accurately weigh an appropriate amount of sorbitol and place it in a 10ml volumetric flask. Accurately add 1ml of the reagent solution and an appropriate amount of solvent. Seal the flask, shake and sonicate to dissolve the sample, incubate in a 60℃ water bath for 10min, remove and cool, dilute to the mark with solvent, shake well, filter, and collect the filtrate.

[0041] Test solution 3: Accurately weigh an appropriate amount of propylene glycol and place it in a 10 ml volumetric flask. Accurately add 1 ml of the reagent solution and an appropriate amount of solvent. Seal the flask, shake and sonicate to dissolve or completely disperse the sample, bathe in a 60℃ water bath for 10 min, remove the flask, cool it, dilute it to the mark with solvent, shake well, filter, and collect the filtrate.

[0042] See sample results Figure 2 The solvent does not interfere with the analysis, and the analytical method of this invention can be used for the detection of formaldehyde in various food additives.

[0043] Example 3 A novel method for detecting formaldehyde content in the environment employs high-performance liquid chromatography (HPLC) followed by derivatization with ambroxol hydrochloride. The HPLC conditions are as follows: Column: ACE Excel 3 Super C18, 4.6mm × 150mm; Mobile phase A: 0.01 mol / L diammonium hydrogen phosphate solution (pH adjusted to 9.5 with phosphoric acid) - acetonitrile (80:20); preparation method: accurately weigh about 1.32 g of diammonium hydrogen phosphate, add 1000 ml of water, stir to dissolve, adjust the pH to 9.5 with phosphoric acid, measure 800 ml of the solution and mix with 200 ml of acetonitrile. Mobile phase B: Acetonitrile; Flow rate: 0.9 ml / min ~ 1.1 ml / min; Column temperature: 20℃~30℃; Injection volume: 20 μl; Sample inlet temperature: 5℃; Detection wavelength: 248nm.

[0044] The following gradient elution method was used: Time (mim) Mobile phase A (%) Mobile phase B (%) 0 100 0 30 79 21 60 68 32 62 100 0 70 100 0 Reagent solution: Accurately weigh an appropriate amount of ambroxol hydrochloride and prepare a solution containing approximately 10 mg per 1 ml with water.

[0045] Solvent: Mobile phase A: Mobile phase B = (50:50).

[0046] Derivatized blank solution: Accurately measure 1 ml of the reagent solution and place it in a 10 ml volumetric flask. Add an appropriate amount of solvent, seal, shake well, incubate in a 60°C water bath for 10 min, remove, cool, dilute to the mark with solvent, shake well, filter, and collect the filtrate.

[0047] When preparing the test solution, formaldehyde detection was performed in the same room at different times and in different rooms at the same time.

[0048] Test solution 1: Place the reagent solution in the test room 1 with the opening open for 4 hours, accurately measure 1 ml, place it in a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0049] Test solution 2: Place the reagent solution in the test room 1 with the air vent open for 4 hours (the next day), accurately measure 1 ml, place it in a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0050] Test solution 3: Place the reagent solution in the test chamber 2 with the opening open for 4 hours, accurately measure 1 ml, place it in a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0051] The test results are shown in Table 1: Table 1 Serial Number sample Detection amount 1 Test solution 1 (room 1) 0.053% 2 Test solution 2 (room 1, the next day) 0.011% 3 Test solution 3 (room 2) 0.003% See sample results Figure 3Formaldehyde levels in the same room at different times and in different rooms at the same time vary in laboratory environments, allowing for the monitoring of formaldehyde changes in the environment. The analytical method of this invention can be used to detect formaldehyde levels in the air.

[0052] I. Taking glycerol as an example, the analytical method of the present invention was verified, as detailed below.

[0053] (1) Exclusivity ①Preparation of test solution Reagent solution: Accurately weigh an appropriate amount of ambroxol hydrochloride and prepare a solution containing approximately 10 mg per 1 ml with water.

[0054] Solvent: Mobile phase A: Mobile phase B = (50:50).

[0055] trans-4-(6,8-dibromo-1,4-dihydroquinazoline-3(2H)-yl)cyclohexanol positioning solution: Accurately weigh 2.078 mg of trans-4-(6,8-dibromo-1,4-dihydroquinazoline-3(2H)-yl)cyclohexanol, place it in a 20 ml volumetric flask, dissolve and dilute to the mark with methanol, shake well, accurately measure 1 ml, place it in a 20 ml volumetric flask, dilute to the mark with solvent, shake well, measure 1.5 ml, place it in a 10 ml volumetric flask, dilute to the mark with solvent, shake well.

[0056] Derivatized blank solution: Accurately measure 1 ml of the reagent solution and place it in a 10 ml volumetric flask. Add an appropriate amount of solvent, seal, shake well, incubate in a 60°C water bath for 10 min, remove, cool, dilute to the mark with solvent, shake well, filter, and collect the filtrate.

[0057] Test solution: Accurately weigh 2.4788 g of glycerol and place it in a 10 ml volumetric flask. Accurately add 1 ml of the reagent solution and an appropriate amount of solvent. Seal the flask, shake and sonicate to dissolve the sample, incubate in a 60 °C water bath for 10 min, remove and cool, dilute to the mark with solvent, shake well, filter, and collect the filtrate.

[0058] ②The test results are as follows Figure 4 As shown, the results indicate that the solvent and other components in the sample do not interfere with the detection of the target component, and the method has good specificity.

[0059] (2) Sensitivity ①Preparation of test solution Solvent: Mobile phase A: Mobile phase B = (50:50).

[0060] Limit of Quantitation Solution: Accurately weigh 5.003 mg of trans-4-(6,8-dibromo-1,4-dihydroquinazolin-3(2H)-yl)cyclohexanol, place it in a 20 ml volumetric flask, dissolve and dilute to the mark with methanol, and shake well. Accurately measure 1.5 ml of the solution and place it in a 50 ml volumetric flask, dilute to the mark with solvent, and shake well. Measure 1 ml of the solution and place it in a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0061] ② The experimental results show that the signal-to-noise ratio of the trans-4-(6,8-dibromo-1,4-dihydroquinazolin-3(2H)-yl)cyclohexanol peak in the limit of quantitation solution chromatogram is 15.8, indicating that this concentration (0.0751 μg / ml) can be accurately quantified, which is equivalent to a formaldehyde concentration of 5.78 ng / ml; the method has good sensitivity.

[0062] (3) Accuracy ①Preparation of test solution Reagent solution: Accurately weigh an appropriate amount of ambroxol hydrochloride and prepare a solution containing approximately 10 mg per 1 ml with water.

[0063] Solvent: Mobile phase A: Mobile phase B = (50:50).

[0064] Reference solution: Accurately weigh 2.078 mg of trans-4-(6,8-dibromo-1,4-dihydroquinazolin-3(2H)-yl)cyclohexanol, place it in a 20 ml volumetric flask, dissolve and dilute to the mark with methanol, shake well, accurately measure an appropriate amount, and dilute with solvent to prepare a solution containing 0.3990 μg per ml.

[0065] Formaldehyde stock solution: Accurately weigh an appropriate amount of formaldehyde solution and dilute it with solvent to prepare a solution containing 0.3068 μg per 1 ml.

[0066] Stock solution for test sample: Accurately weigh 15.4615 g of glycerol, place it in a 25 ml volumetric flask, add solvent to dissolve and dilute to the mark, and shake well.

[0067] Test solution: Accurately measure 5 ml of the test solution stock solution and place it in a 10 ml volumetric flask. Accurately add 1 ml of the reagent kit and an appropriate amount of solvent. Seal the flask, shake and sonicate to dissolve the sample, incubate in a 60°C water bath for 10 min, remove and cool, dilute to the mark with solvent, shake well, filter, and collect the filtrate.

[0068] 50% spiked test solution: Accurately measure 5 ml of the test solution stock solution and place it in a 10 ml volumetric flask. Accurately add 1 ml of the reagent kit, 0.5 ml of formaldehyde stock solution, and an appropriate amount of solvent. Seal the flask, shake and sonicate to dissolve the sample, incubate in a 60°C water bath for 10 min, remove the flask, cool it, dilute it to the mark with solvent, shake well, filter, and collect the filtrate.

[0069] 100% spiked test solution: Accurately weigh 3.0923 g of glycerol and place it in a 10 ml volumetric flask. Accurately add 1 ml of the reagent kit, 1 ml of formaldehyde stock solution, and an appropriate amount of solvent. Seal the flask, shake and sonicate to dissolve the sample, incubate in a 60 °C water bath for 10 min, remove the flask, cool it, dilute it to the mark with solvent, shake well, filter, and collect the filtrate.

[0070] 200% spiked test solution: Accurately weigh 3.0923 g of glycerol and place it in a 10 ml volumetric flask. Accurately add 1 ml of the reagent kit, 2 ml of formaldehyde stock solution, and an appropriate amount of solvent. Seal the flask, shake and sonicate to dissolve the sample, incubate in a 60°C water bath for 10 min, remove the flask, cool it, dilute it to the mark with solvent, shake well, filter, and collect the filtrate.

[0071] ②The experimental results are shown in Table 2: Table 2 The results show that the method has good accuracy.

[0072] (4) Repeatability ①Preparation of test solution Reagent solution: Accurately weigh an appropriate amount of ambroxol hydrochloride and prepare a solution containing approximately 10 mg per 1 ml with water.

[0073] Solvent: Mobile phase A: Mobile phase B = (50:50).

[0074] Test solution: Accurately weigh an appropriate amount of glycerol and place it in a 10 ml volumetric flask. Accurately add 1 ml of the reagent kit and an appropriate amount of solvent. Seal the flask, shake and sonicate to dissolve the sample, incubate in a 60°C water bath for 10 min, remove, cool, dilute to the mark with solvent, shake well, filter, and collect the filtrate. Prepare 6 parallel solutions.

[0075] ②The experimental results are shown in Table 3: Table 3 The results show that the method has good reproducibility.

[0076] II. Qualitative analysis of derivatives (1) High performance liquid chromatography (HPLC) conditions: The chromatographic column was an ACE Excel 3 super C18, 150 mm × 4.6 mm, 3 μm; mobile phase A was 0.02 mol / L ammonium acetate (pH adjusted to 10.6 with ammonia) - acetonitrile (volume ratio 80:20); mobile phase B was acetonitrile; the flow rate was 1.0 mL / min; the column temperature was 25 ℃; the detection wavelength was 248 nm; and the injection volume was 20 µL. Gradient elution was performed using the gradient elution program shown in Table 4 below.

[0077] Table 4 Mass spectrometry conditions: Ion source: ESI, Q1 positive ion mode; curtain gas (CUR) 35 psi; ionization voltage (IS) 5500 V; collider (CAD) Medium; ion source temperature (TEM) 550 °C; spray gas (GS1) 60 psi; declustering voltage (DP) 60 V; scan range 50–800 Da.

[0078] (2) Preparation of test solution: Reagent solution: Accurately weigh an appropriate amount of ambroxol hydrochloride and prepare a solution containing approximately 10 mg per 1 ml with water.

[0079] Solvent: Mobile phase A: Mobile phase B = (50:50).

[0080] trans-4-(6,8-dibromo-1,4-dihydroquinazoline-3(2H)-yl)cyclohexanol reference solution: Accurately weigh 4.532 mg of trans-4-(6,8-dibromo-1,4-dihydroquinazoline-3(2H)-yl)cyclohexanol, place it in a 20 ml volumetric flask, dissolve and dilute to the mark with methanol, accurately measure 1 ml, place it in a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0081] Formaldehyde solution: Measure 20 μl of formaldehyde (37%), dilute with water to 100 ml, and shake well.

[0082] Test solution: Accurately weigh 10.27 mg of derivatizing agent, place it in a 10 ml volumetric flask, add 1 ml of solvent and 1 ml of formaldehyde solution, seal, incubate in a 60 °C water bath for 10 min, remove, cool, dilute to the mark with solvent, and shake well.

[0083] (3) Take the above reference solution and test solution, and analyze them using high performance liquid chromatography-mass spectrometry. The results are as follows: Figure 5 , 6 As shown. From Figure 5 and Figure 6 It can be seen that the main M / Z ratios of the mass spectrum are basically consistent, which is the same as the actual molecular weight of 390.

[0084] The above description is merely one embodiment of the present invention. It should be understood that the present invention is not limited to the forms disclosed herein and should not be construed as excluding other embodiments. It can be used in various other combinations, modifications, and environments, and can be altered within the scope of the concept described herein through the above teachings or related technologies or knowledge. Modifications and variations made by those skilled in the art that do not depart from the spirit and scope of the present invention should be within the protection scope of the appended claims.

Claims

1. A completely new method for detecting the formaldehyde content by liquid chromatography, characterized in that, It comprises the following steps: Step one, preparation of kit solution; Step two, adding the kit solution to the sample to be tested or placing the kit solution in the detection environment, diluting with solvent to obtain a test solution; Step three, precisely measuring the kit solution, diluting with solvent to obtain a derivatization blank solution; Step four, respectively taking the same amount of test solution and derivatization blank solution, injecting into high performance liquid chromatography for quantitative detection; The high performance liquid chromatography conditions are as follows: Chromatographic column: ACE Excel 3 Super C18; Mobile phase A: 0.01 mol / L diammonium hydrogen phosphate solution-acetonitrile solution; Mobile phase B: acetonitrile; Gradient elution is performed on mobile phase A and mobile phase B.

2. The formaldehyde content liquid chromatography detection method according to claim 1, characterized in that, The preparation method of the kit solution in step one is as follows: precisely weighing a certain amount of derivatizing agent, and preparing a solution containing about 10 mg per 1 ml with water; the derivatizing agent is amlexanox hydrochloride.

3. The formaldehyde content liquid chromatography detection method of claim 1, wherein, The first preparation method of the test solution in step two is as follows: precisely weighing a certain amount of sample to be tested, placing it in a 10 ml volumetric flask, precisely adding 1 ml of kit solution and a certain amount of solvent, sealing, shaking and ultrasonicating to dissolve the sample, placing in a 60°C water bath for 10 minutes, taking out and cooling, diluting to the mark with solvent, shaking well and filtering, and taking the filtrate.

4. The formaldehyde content liquid chromatography detection method of claim 1, wherein, The second preparation method of the test solution in step two is as follows: placing the kit solution in the detection environment, placing it open for 4 hours, precisely measuring 1 ml of kit solution, placing it in a 10 ml volumetric flask, and diluting to the mark with solvent, and shaking well.

5. The formaldehyde content liquid chromatography detection method according to claim 3 or 4, characterized in that, The solvent is a mixture of mobile phase A and mobile phase B in a volume ratio of 1:

1.

6. The formaldehyde content liquid chromatography detection method according to claim 5, characterized in that, The preparation method of mobile phase A in step four is as follows: precisely weighing diammonium hydrogen phosphate, adding water to stir to dissolve, adjusting the pH to 9.5 with phosphoric acid, and mixing the above diammonium hydrogen phosphate solution with acetonitrile in a volume ratio of 8:

2.

7. The formaldehyde content liquid chromatography detection method according to claim 6, characterized in that, The flow rate in step four is 0.9 ml / min to 1.1 ml / min; the column temperature is 20°C to 30°C; the injection volume is 20 μL; the injection tray temperature is 5°C; and the detection wavelength is 248 nm.

8. The formaldehyde content liquid chromatography detection method of claim 6, wherein, The gradient elution program is as follows: Gradient elution for 0 to 30 minutes, with the proportion of mobile phase A being 100% to 79% and the proportion of mobile phase B being 0% to 21%; Gradient elution for 30 to 60 minutes, with the proportion of mobile phase A being 79% to 68% and the proportion of mobile phase B being 21% to 32%; Gradient elution for 60 to 62 minutes, with the proportion of mobile phase A being 68% to 100% and the proportion of mobile phase B being 32% to 0%; Isocratic elution for 8 minutes, with the proportion of mobile phase A being 100% and the proportion of mobile phase B being 0%.

9. Use of a formaldehyde content liquid chromatography detection method according to any one of claims 1 to 8, characterized in that, It is used for detecting formaldehyde in raw materials for drugs, excipients, solvents, preparations, and food and environment.

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