Test methods for identification and content determination of two chemical drugs in nasal medical devices

Abstract

The present invention provides a method for identifying and determining the content of two chemical drugs in nasal medical devices, adopts high performance liquid chromatography (HPLC) for initial screening, high resolution liquid chromatography-mass spectrometry (HRMS / MS) or equivalent liquid chromatography-mass spectrometry for verification, and adopts high performance liquid chromatography (HPLC) for content determination of the verified positive samples, which can simply and efficiently obtain detection indicators with higher content, and ensure indicators such as specificity, detection limit, and durability.

Description

Technical Field

[0001] The invention belongs to the technical field of medical detection, and relates to a method for identifying and determining the contents of two chemical drugs in a nasal medical device. Background Art

[0002] The illegal addition of drugs to health products has a long history. In order to make their products achieve quick results, some companies illegally add drugs such as hormones, antibiotics or Chinese herbal extracts to their products. In recent years, it is not uncommon to detect chemical drug ingredients in medical device products.

[0003] Naphazoline, also known as naphazoline, nose-eye cleaner, and nose drops, is a vasoactive drug that acts on the circulatory system. It belongs to the class of adrenergic drugs and has pharmacological activity of vasoconstriction. Local application can relieve nasal mucosal swelling and congestion. Long-term use may cause rebound congestion, atrophic rhinitis, and symptoms such as local irritation, nausea, and headache. Excessive drops and excessive swallowing can cause poisoning. In addition, naphazoline is prohibited for use in patients with atrophic rhinitis and dry nasal cavity. Not only does it have no therapeutic effect, it is easy to cause drug-induced rhinitis and aggravate the patient's condition.

[0004] Chlorpheniramine, also known as chlorpheniramine, chlorpheniramine, chlorpheniramine, is a first-generation antihistamine drug that counteracts capillary dilation caused by allergic reactions, reduces capillary permeability, relieves nasal discharge, nasal congestion, sneezing, and relieves wheezing caused by bronchial smooth muscle contraction. The drug has a long-lasting antihistamine effect and also has a significant central inhibitory effect, which can increase the effects of anesthetics, analgesics, hypnotics, and local anesthetics. It is commonly used in the treatment of allergic diseases (allergic rhinitis, urticaria, etc.) and the common cold. It is inexpensive, and adverse reactions include dry mouth, dizziness, nausea, drowsiness, palpitations or skin ecchymosis, and bleeding tendency, but they are rare. The drug can induce epilepsy, so it is contraindicated for patients with a history of epilepsy.

[0005] Nasal medical devices include nasal liquid dressings, cold compress gels and cold compress gels, which are widely used in clinical practice. From the registration information of 21 batches of nasal medical device products collected by the inquiry center on the official website of the State Food and Drug Administration, none of the registration contents indicate that the products contain naphazoline and chlorpheniramine drug ingredients. In addition, most of them clearly state that the products do not contain pharmacological ingredients, and the treatment method is only physical therapy. From the perspective of national supervision, if medical devices contain chemical ingredients, Chinese medicinal materials or their extracts, and mainly rely on drugs to exert therapeutic effects, they are managed as drugs, and cannot be approved as medical devices. It is very urgent to establish inspection methods for naphazoline and chlorpheniramine in nasal medical devices. Summary of the invention

[0006] The present invention aims to provide a method for identifying and determining the content of two chemical drugs in nasal medical devices, which can simply and efficiently obtain detection indicators with higher content and ensure indicators such as specificity, detection limit, and durability.

[0007] The solution adopted by the present invention to achieve the technical purpose is:

[0008] Two chemical drug identification and content determination test methods in nasal medical devices, including:

[0009] Take the test solution and the reference solution of naphazoline hydrochloride and chlorpheniramine maleate, use high performance liquid chromatography (HPLC) to analyze the sample, and compare the results of the two. R ) and ultraviolet spectrum from 190nm to 400nm Figure 1 Consistency is used as the basis for initial screening;

[0010] Take the test solution and the reference solution mixed with naphazoline hydrochloride and chlorpheniramine maleate, use high resolution liquid chromatography-mass spectrometry (HRMS / MS) for sample analysis, compare the parent ion and fragment ion information of the test solution and the reference solution, and if the two are consistent, it is confirmed that the sample contains the corresponding drug component;

[0011] The reference substance stock solution was taken and prepared into a standard solution with a series of concentrations. The standard solution was analyzed by high performance liquid chromatography (HPLC) and a concentration-peak area standard curve was drawn. The amount of drug in the sample was calculated based on the peak area of ​​the positive sample.

[0012] Preferably, the test solution is prepared according to the following method: add water to the test sample to a solution mass volume concentration of 0.04 g / mL, vortex for 3 minutes, ultrasonically treat for 20 minutes, cool, filter, and take the filtrate.

[0013] Preferably, the concentration of naphazoline hydrochloride and chlorpheniramine maleate in the mixed reference solution of naphazoline hydrochloride and chlorpheniramine maleate is 10 μg / mL.

[0014] Preferably, the conditions for high performance liquid chromatography include:

[0015] Column temperature: 30°C; Sample chamber temperature: 10°C;

[0016] Flow rate: 1.0 mL / min;

[0017] Detection wavelength: 280nm, scanning range: 190nm~400nm;

[0018] Injection volume: 10 μL;

[0019] Mobile phase: A (0.1% formic acid aqueous solution), B (acetonitrile), gradient conditions are as follows:

[0020]

[0021] More preferably, the high performance liquid chromatography method is carried out using a Phenomenex Gemini C 18 column.

[0022] Preferably, the chromatographic conditions of the high-resolution liquid chromatography-mass spectrometry method include:

[0023] Column temperature: 35°C, sample chamber temperature: 10°C;

[0024] Flow rate: 0.3 mL / min;

[0025] Injection volume: 1 μL;

[0026] Mobile phase: A (0.1% formic acid aqueous solution), B (acetonitrile), gradient conditions are as follows:

[0027]

[0028] Mass spectrometry conditions included:

[0029] Ion source: ESI positive ion mode

[0030] Dryer temperature: 325°C; Dryer flow rate: 8L / min;

[0031] Atomizer pressure: 35Psi;

[0032] Capillary voltage: 3500V;

[0033] Capillary outlet voltage: 140V;

[0034] The parameters for the two drugs are as follows:

[0035]

[0036] Note: * is the base peak.

[0037] More preferably, the high-resolution LC-MS method uses an Agilent Poroshell 120EC-C 18 Chromatographic column and Agilent 6530Q-TOF high-resolution liquid spectrometer.

[0038] Preferably, the reference substance stock solution is prepared according to the following method: respectively take naphazoline hydrochloride and chlorpheniramine maleate reference substances, accurately weigh them, place them in a brown volumetric flask, add water to dissolve them and then make up to volume, shake well, and prepare reference substance stock solutions with a concentration of 1 mg / mL each.

[0039] Preferably, the separation between the chromatographic peaks of the two reference substances and the adjacent chromatographic peaks is greater than 1.5; the signal-to-noise ratio (S / N) of the chromatographic peaks of the two reference substances is greater than 3:1.

[0040] The beneficial effects of the present invention are:

[0041] This method uses high performance liquid chromatography (HPLC) for initial screening, high resolution liquid chromatography-mass spectrometry (HRMS / MS) or equivalent liquid chromatography-mass spectrometry for verification, and the positive samples after verification are determined by high performance liquid chromatography (HPLC). The specificity, detection limit, durability and precision of the method currently developed all meet the requirements. This method was used in the experiment to detect naphazoline and chlorpheniramine, western medicine ingredients for the treatment of rhinitis, from multiple batches of nasal medical devices on the market. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Typical chromatograms of two drugs.

[0043] Figure 2 Chromatogram of positive sample solution (liquid dressing, Shandong Haibao Pharmaceutical Co., Ltd., batch number 20210601).

[0044] Figure 3 Chromatogram of negative sample solution (medical dressing nasal repair solution, Changchun Aolante Biochemical Pharmaceutical Equipment Research Base Co., Ltd., batch number 20220101).

[0045] Figure 4 Spectra of naphazoline hydrochloride and chlorpheniramine maleate reference substances.

[0046] Figure 5 Spectrum of positive sample solution (liquid dressing, Shandong Haibao Pharmaceutical Co., Ltd., batch number 20210601).

[0047] Figure 6 Chromatogram of detection limit of naphazoline hydrochloride reference substance.

[0048] Figure 7 Chromatogram and standard curve of naphazoline hydrochloride.

[0049] Figure 8 Chromatogram of detection limit of chlorpheniramine maleate reference substance.

[0050] Fig. 9 Chromatogram and standard curve of chlorpheniramine maleate.

[0051] Fig.10 Chromatographic column Superiorex ods (liquid dressing, Shandong Haibao Pharmaceutical Co., Ltd., batch number 20210601).

[0052] Fig.11Chromatographic column Phenomenex Gemini C 18 (Liquid dressing, Shandong Haibao Pharmaceutical Co., Ltd., batch number 20210601).

[0053] Fig.12 Chromatography columnAgela technologies venusil XBP C 18 (Liquid dressing, Shandong Haibao Pharmaceutical Co., Ltd., batch number 20210601).

[0054] Fig.13 Column temperature 25°C (liquid dressing, Shandong Haibao Pharmaceutical Co., Ltd., batch number 20210601).

[0055] Fig.14 Column temperature 30°C (liquid dressing, Shandong Haibao Pharmaceutical Co., Ltd., batch number 20210601).

[0056] Fig.15 Column temperature 35°C (liquid dressing, Shandong Haibao Pharmaceutical Co., Ltd., batch number 20210601).

[0057] Fig.16 Flow rate: 0.9 mL min -1 (Liquid dressing, Shandong Haibao Pharmaceutical Co., Ltd., batch number 20210601).

[0058] Fig.17 Flow rate: 1.0 mL min -1 (Liquid dressing, Shandong Haibao Pharmaceutical Co., Ltd., batch number 20210601).

[0059] Fig.18 Flow rate: 1.1 mL min -1 (Liquid dressing, Shandong Haibao Pharmaceutical Co., Ltd., batch number 20210601).

[0060] Fig.19 Secondary mass spectrum of naphazoline hydrochloride reference substance.

[0061] Fig. 20 Secondary mass spectrum of positive sample - naphazoline hydrochloride component (liquid dressing (Bishutong), Shandong Haibao Pharmaceutical Co., Ltd., batch number 20210601).

[0062] Fig.21 Secondary mass spectrum of chlorpheniramine maleate reference substance.

[0063] Fig. 22 Secondary mass spectrum of positive sample - chlorpheniramine maleate ingredient (liquid dressing (Bishutong), Shandong Haibao Pharmaceutical Co., Ltd., batch number 20210601). DETAILED DESCRIPTION

[0064] In order to explain the present invention more clearly, the present invention is further described in detail below in conjunction with embodiments and with reference to the accompanying drawings. It should be understood by those skilled in the art that the content described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0065] The experimental design mainly considers two aspects: one is the sample processing method, which aims to obtain high-content detection indicators simply and efficiently; the other is the analysis method, which ensures indicators such as specificity, detection limit, and durability.

[0066] This method is applicable to nasal medical devices that form a protective layer on the wound surface, physically reduce fever, and provide a physical barrier. Examples of product names: liquid dressings, cold compress gels, cold compress gels, monomer silver rhinitis antibacterial sprays, etc.

[0067] The room temperature in this method, unless otherwise specified, should be 10℃~30℃.

[0068] The test water in this method should comply with the requirements of GB / T 6682.

[0069] Unless otherwise specified, the container used in this method should be a borosilicate glass container.

[0070] The measurements involved in this method should be taken using pipettes that meet the accuracy requirements specified in the relevant national standards.

[0071] Example

[0072] This method uses high performance liquid chromatography (HPLC) for initial screening, high resolution liquid chromatography-mass spectrometry (HRMS / MS) or equivalent liquid chromatography-mass spectrometry for verification, and high performance liquid chromatography (HPLC) for content determination of positive samples after verification. It mainly includes the following steps:

[0073] 1. Preparation of test solution

[0074] Take 1 g of the test sample, dilute to 25 mL with water, vortex for 3 minutes, ultrasonically treat for 20 minutes, cool, filter, and take the filtrate. If necessary, dilute it by appropriate multiples.

[0075] 2. High Performance Liquid Chromatography

[0076] 2.1 Instruments and reagents

[0077] 2.1.1 Instruments

[0078] High performance liquid chromatograph equipped with a diode array detector.

[0079] 2.1.2 Reagents

[0080] a) Acetonitrile: chromatographic grade, CAS#75-05-8;

[0081] b) Formic acid: chromatographic grade, CAS#64-18-6;

[0082] c) Information on the two reference substances is shown in Table 1.

[0083] Table 1. Information of two reference substances

[0084]

[0085] 2.2 Preparation of reference substance stock solution and mixed reference substance solution

[0086] Take about 10 mg of naphazoline hydrochloride and chlorpheniramine maleate reference substances respectively, weigh accurately, place in a 10 mL brown volumetric flask, add water to dissolve and make up to volume, shake well, and prepare a reference substance stock solution with a concentration of 1 mg / mL. Accurately measure 1 mL of each of the above two reference substance stock solutions, place in the same 100 mL brown volumetric flask, dilute to the scale with water, shake well, and obtain a mixed reference substance solution.

[0087] 2.3 HPLC conditions

[0088] Column: Phenomenex Gemini C 18 Column (250×4.6 mm, 5 μm) or other equivalent chromatographic columns.

[0089] Column temperature: 30°C, sample chamber temperature: 10°C;

[0090] Flow rate: 1.0 mL / min;

[0091] Detection wavelength: 280nm, scanning range: 190nm~400nm;

[0092] Injection volume: 10 μL.

[0093] Mobile phase: A (0.1% formic acid aqueous solution), B (acetonitrile), gradient conditions see Table 2.

[0094] Table 2. HPLC mobile phase gradient conditions

[0095]

[0096] 2.4 System suitability test

[0097] 2.4.1 Separation

[0098] Take 1 mL of naphazoline hydrochloride stock solution and 2.5 mL of chlorpheniramine maleate reference substance stock solution and place them in a 50 mL brown volumetric flask, add water to dilute to the scale, shake well to make a mixed reference solution containing 20 mg / L naphazoline hydrochloride and 50 mg / L chlorpheniramine maleate, take 10 μL and inject it into the liquid chromatograph, record the chromatogram, the separation between the chromatographic peaks of the two reference substances and the adjacent chromatographic peaks should be greater than 1.5.

[0099] 2.4.2 Sensitivity

[0100] Take appropriate amount of the two reference stock solutions in 2.2, dilute with water to the concentration of "limit of detection (LOD)" in Table 3, take 10 μL and inject into liquid chromatograph, record the chromatogram, the signal-to-noise ratio (S / N) of the chromatographic peaks of the two reference substances should be greater than 3:1.

[0101] Table 3. Sensitivity requirements for two drugs

[0102]

[0103] 3. High-resolution LC-MS

[0104] 3.1 Instruments and reagents

[0105] 3.1.1 Instruments

[0106] High Resolution Liquid Spectrometry

[0107] 3.1.2 Reagents

[0108] a) Acetonitrile: chromatographic grade, CAS#75-05-8;

[0109] b) Formic acid: chromatographic grade, CAS#64-18-6;

[0110] c) The two reference substances are the same as 2.1.2.

[0111] 3.2 Preparation of reference solution

[0112] Accurately measure 1 mL of each reference substance stock solution under 2.2, place it in the same 100 mL volumetric flask, dilute it to the mark with water, and shake well. Accurately measure 1 mL, place it in a 100 mL volumetric flask, dilute it to the mark with water, and shake well to prepare a 100 ng / mL reference substance solution.

[0113] 3.3 Chromatographic conditions

[0114] Column: Agilent Poroshell 120EC-C 18 50×2.1mm 2.7μm

[0115] Column temperature: 35°C, sample chamber temperature: 10°C;

[0116] Flow rate: 0.3 mL / min;

[0117] Injection volume: 1 μL;

[0118] Mobile phase: A (0.1% formic acid aqueous solution), B (acetonitrile), gradient conditions see Table 4.

[0119] Table 4. Mobile phase gradient conditions for HPLC-HRMS / MS method

[0120]

[0121] 3.4 Mass spectrometry conditions

[0122] Instrument model: Agilent 6530Q-TOF high-resolution liquid spectrometer.

[0123] Ion source: ESI positive ion mode

[0124] Dryer temperature: 325°C;

[0125] Dryer flow rate: 8L / min;

[0126] Atomizer pressure: 35Psi;

[0127] Capillary voltage: 3500V;

[0128] Capillary outlet voltage: 140V;

[0129] The test parameters of the two drugs are shown in Table 5.

[0130] Table 5. Two drug detection modes, secondary energies and fragment ions

[0131]

[0132] Note: * is base peak

[0133] IV. Drug identification and content determination

[0134] Take the test solution prepared in step 1 and the mixed reference solution prepared in step 2.2, and analyze them according to the method in step 2.3. Compare the results of the two and use the retention time (t R ) and ultraviolet spectrum from 190nm to 400nm Figure 1 Consistency is used as the basis for initial screening.

[0135] Take the test solution that may contain drugs and the mixed reference solution prepared in 3.2, and analyze them according to the methods in 3.3 and 3.4. Compare the parent ion and fragment ion information of the test solution and the reference solution. If the two are consistent, it is confirmed that the sample contains the corresponding drug component.

[0136] Take the reference substance stock solution in 2.2 and prepare a series of standard solutions, and perform sample analysis according to the method in 2.3. Draw a concentration-peak area standard curve, and calculate the amount of drug in the sample based on the peak area of ​​the positive sample. Figure 1 Typical chromatograms of two drugs are given.

[0137] This method provides the identification and content determination methods of two drugs, naphazoline and chlorpheniramine. If other drugs that may be added are of concern, methodologically validated methods can also be used for analysis.

[0138] Application Examples

[0139] 1. HPLC-DAD Methodology Validation

[0140] 1. Instruments and test drugs

[0141] Waters e2695 high performance liquid chromatograph, diode array detector (Waters Corporation, USA); Sartorius BP211D electronic analytical balance (Sartorius, Germany); KH-700DE digital control ultrasonic instrument (Kunshan Hechuang Ultrasonic Instrument Co., Ltd.).

[0142] Naphazoline hydrochloride reference substance (source: China Food and Drug Inspection Institute, batch number: 100111-202105, content: 99.9%); Chlorpheniramine maleate reference substance (source: China Food and Drug Inspection Institute, batch number: 100047-202008, content: 99.7%); Acetonitrile (chromatographic grade); Formic acid (chromatographic grade); Water is ultrapure water. Specific information of nasal medical device samples is shown in the table below:

[0143] Table 6. Nasal medical device sample information

[0144]

[0145] 2. Chromatographic conditions

[0146] Column: Phenomenex Gemini C 18 Column (250×4.6 mm, 5 μm), 0.1% formic acid aqueous solution as mobile phase A, acetonitrile as mobile phase B, gradient elution as specified in the table below; detection wavelength 280 nm; column temperature 30°C; injection volume 10 μL. The theoretical plate number calculated based on the naphazoline peak is not less than 2000.

[0147] Table 7. Gradient elution program

[0148]

[0149]

[0150] 3. Solution preparation

[0151] 3.1 Preparation of reference solution: Take about 10 mg of naphazoline hydrochloride and chlorpheniramine maleate reference substances, weigh accurately, place in a 10 mL brown volumetric bottle, dissolve with water and dilute to scale, shake well, and make a solution containing about 1 mg of each substance per 1 mL as two reference substance stock solutions. Accurately measure 1 mL of each of the above two reference substance stock solutions, place in the same 100 mL brown volumetric bottle, dilute to scale with water, shake well, and obtain a mixed reference substance solution.

[0152] 3.2 Preparation of test solution: Take 1 g of the test sample, dilute to 25 mL with water, vortex for 3 minutes, ultrasonically treat for 20 minutes, cool, filter, and take the filtrate.

[0153] 4. Specificity test

[0154] Take 10 μL of the mixed reference solution and the test solution, and measure them according to the chromatographic conditions under item 2. The results show that the positive sample of nasal medical device presents a chromatographic peak with the same retention time as the naphazoline hydrochloride and chlorpheniramine maleate reference substances, and the UV-visible absorption spectrum of the corresponding chromatographic peak is the same as that of the naphazoline hydrochloride and chlorpheniramine maleate reference substances, while the negative sample has no corresponding chromatographic peak. Figure 1 to Figure 5 .

[0155] 5. Limit of detection and limit of quantification

[0156] Refer to the signal-to-noise ratio method in the 9101 Analytical Method Validation Guidelines of the 2020 edition of the Chinese Pharmacopoeia. The naphazoline hydrochloride reference solution and chlorpheniramine maleate solution were gradually diluted, and the minimum detection concentration of naphazoline hydrochloride (LOD) was 0.05 μg / mL, and the minimum detection concentration of chlorpheniramine maleate (LOD) was 0.2 μg / mL at the maximum absorption wavelength of 280 nm and 262 nm, respectively, with a signal-to-noise ratio of about 3. The instrument detection limit (IDL) was 0.5 ng, and the corresponding method detection limit (MDL) was 1.25 μg / g, see Figure 6 , Figure 8 .

[0157] 6. Linear Relationship

[0158] Accurately weigh an appropriate amount of naphazoline hydrochloride reference substance, and use water to prepare a series of linear reference substance solutions with naphazoline hydrochloride mass concentrations of 1.03, 5.13, 10.27, 20.53, 51.34, and 102.67 μg / mL, respectively. Inject and analyze in sequence, record the peak area, and perform linear regression with the reference substance mass concentration (μg / mL) as the abscissa and the peak area as the ordinate. The regression equation of naphazoline hydrochloride is: y=20965x–11426, r=0.9999. The results show that naphazoline hydrochloride has a linear linear relationship with the peak area.

[0159] The linear relationship is good in the range of 1.03 to 102.67 μg / mL. Figure 7 .

[0160] Accurately weigh an appropriate amount of chlorpheniramine maleate reference substance, and prepare a series of linear reference substance solutions with chlorpheniramine maleate mass concentrations of 5.09, 10.18, 20.36, 50.90, 101.80, and 203.60 μg / mL with water. Inject and analyze in sequence, record the peak area, and perform linear regression with the reference substance mass concentration (μg / mL) as the abscissa and the peak area as the ordinate. The regression equation of naphazoline hydrochloride is: y=3313X+1041, r=0.9999. The results show that chlorpheniramine maleate has a good linear relationship in the range of 5.09-203.60 μg / mL, as shown in the results. Fig. 9 .

[0161] 7. Repeatability test

[0162] Take 10 μL of the positive sample solution (liquid dressing, Shandong Haibao Pharmaceutical Co., Ltd., batch number 20210601) and inject it under the above chromatographic conditions. Prepare 6 copies in parallel according to the method under "Preparation of test solution". Determine naphazoline according to the proposed determination method. The method has good repeatability. The results are shown in Table 8.

[0163] Table 8. Repeatability test

[0164]

[0165] 8. Recovery rate test

[0166] Accurately measure 4 mL of positive sample with known content (liquid dressing, Shandong Haibao Pharmaceutical Co., Ltd., batch number 20210601 containing 0.3536 mg / mL naphazoline hydrochloride and 1.5648 mg / mL chlorpheniramine maleate), a total of 6 portions, respectively placed in 100 mL volumetric bottles, respectively add 1.8 mL of naphazoline hydrochloride reference substance stock solution and 5 mL of chlorpheniramine maleate reference substance stock solution, prepare according to the method under "Test solution preparation", determine and calculate according to the proposed determination method, and the results are shown in Table 9. The results show that the average recovery rate of naphazoline is 94.5%, RSD is 0.8%, and the average recovery rate of chlorpheniramine is 99.8%, RSD is 0.3%. The sample recovery rate of this method is good.

[0167] Table 9. Recovery test

[0168]

[0169] 9. Durability inspection

[0170] Three different types of chromatographic columns were selected: Superiorex ods (250×4.6 mm, 5 μm); Phenomenex Gemini C 18 (250×4.6mm, 5μm); Agela technologies venusil XBP C 18 (250×4.6mm, 5μm), inject according to the two chromatographic conditions, and record the chromatograms, see Table 10 and Figure 10 to Figure 12 The results showed that naphazoline could be effectively separated from other components in the positive sample solution of nasal medical devices on all three chromatographic columns, and the retention behavior was stable and reliable, with high durability of the chromatographic columns. In addition, the chromatographic retention behavior at different column temperatures and flow rates was investigated, as shown in Tables 11 to 12 and Figure 13 to Figure 18 The column efficiency can meet the requirements and the durability is good.

[0171] Table 10. Chromatographic column durability (liquid dressing, Shandong Haibao Pharmaceutical Co., Ltd., batch number 20210601)

[0172]

[0173] Table 11. Column temperature durability (liquid dressing, Shandong Haibao Pharmaceutical Co., Ltd., batch number 20210601)

[0174]

[0175] Table 12. Flow rate durability (liquid dressing, Shandong Haibao Pharmaceutical Co., Ltd., batch number 20210601)

[0176]

[0177] In summary, the specificity, detection limit, durability and precision of the currently developed method all meet the requirements.

[0178] 2. HPLC-MS / MS Method Confirmation

[0179] 1. Instruments and test drugs

[0180] Agilent 6530B Accurate-Mass Q-TOF, Sartorius BP211D electronic analytical balance (Sartorius, Germany); KH-700DE digital control ultrasonic instrument (Kunshan Hechuang Ultrasonic Instrument Co., Ltd.).

[0181] Naphazoline hydrochloride reference substance (source: China Food and Drug Inspection Institute, batch number: 100111-202105, content: 99.9%); chlorpheniramine maleate reference substance (source: China Food and Drug Inspection Institute, batch number: 100047-202008, content: 99.7%); acetonitrile (chromatographic grade); formic acid (chromatographic grade); ethanol (chromatographic grade); water is ultrapure water. Specific information of nasal medical device samples is the same as Table 6.

[0182] 2. Chromatographic mass spectrometry conditions

[0183] Column: Agilent Poroshell 120EC-C 18 (3.0×50mm, 2.7μm), mobile phase: 0.1% formic acid-acetonitrile (70:30); injection volume: 1μL; flow rate: 0.3mL / min. ESI + Scanning: capillary voltage 3500 V, capillary outlet voltage 140 V, drying gas temperature: 325°C, drying gas flow rate: 8 L / min, nebulizer pressure: 35 psi, collision energy 10 V; scanning range 50-2000 m / z.

[0184] 3. Solution preparation

[0185] The solution preparation was the same as that in HPLC-DAD Methodology Validation 3.

[0186] 4. Confirmation experiment

[0187] The mixed reference solution and the test solution were injected according to the HPLC-MS / MS method to confirm the chromatographic mass spectrometric conditions under item 2. Results The positive samples in the nasal medical device showed ion peaks with the same retention time as the two reference substances, and the secondary mass spectra were consistent with those of naphazoline hydrochloride and chlorpheniramine maleate reference substances. The negative samples did not have the same mass spectrum peaks, see Figure 19 to Figure 22 and Tables 13 to 14.

[0188] Table 13. Data table of naphazoline hydrochloride reference and nasal medical device positive samples

[0189]

[0190] Table 14. Data table of chlorpheniramine maleate reference substance and nasal medical device positive samples

[0191]

[0192]

[0193] 3. Sample test results

[0194] Principles for judging positive results: In the chromatogram of the test product, there should be no chromatographic peak with the same retention time as the naphazoline hydrochloride reference product. If there is a chromatographic peak with the same retention time, use a diode array detector to compare the UV-visible absorption spectra of the corresponding chromatographic peaks. The absorption spectra should be different in the wavelength range of 210-400nm. If the absorption spectra are the same, it is considered a positive detection.

[0195] Determination results: According to the inspection method of this application, 21 batches of nasal medical devices collected by the center were inspected. As a result, naphazoline was detected in 12 batches of samples, and naphazoline and chlorpheniramine were simultaneously detected in 3 batches of samples; the positive samples detected were confirmed by mass spectrometry. As a result, the parent ion peaks and the main fragment ion peaks of the secondary mass spectra of 15 batches of positive samples were consistent with those of the naphazoline hydrochloride reference substance, and the parent ion peaks and the main fragment ion peaks of the secondary mass spectra of the 3 batches of positive samples were consistent with those of the chlorpheniramine reference substance, as shown in Tables 15 to 18.

[0196] Table 15. Determination results of naphazoline in nasal medical devices

[0197]

[0198]

[0199] Table 16. Mass spectrometry confirmation results of naphazoline in nasal medical devices

[0200]

[0201] Table 17. Determination results of chlorpheniramine in nasal medical devices

[0202]

[0203]

[0204] Table 18. Mass spectrometry confirmation results of chlorpheniramine in nasal medical devices

[0205]

[0206] The commercially available nasal preparation of naphazoline with a national medicine standard is naphazoline hydrochloride nasal drops, which is included in the 2020 edition of the Chinese Pharmacopoeia. The mobile phase system used is methanol-water-triethylamine-phosphoric acid (50:50:0.25:0.75). The mobile phase system of naphazoline hydrochloride nasal drops can also be used for the detection of naphazoline in nasal medical devices. The disadvantage is that it is difficult to meet the throughput detection because the prescriptions and production processes of nasal medical devices (dressings, gels, gels, etc.) are different, and the matrix and chemical components contained are more complex than the drug naphazoline hydrochloride nasal drops. Once continuously sampled, the difficult-to-elute components in the sample will clog the chromatographic column and cross-contaminate with the residual components in the chromatographic column. This center uses acetonitrile-0.1% formic acid aqueous solution gradient elution, which has the advantage that the mobile phase is only two phases, which is simple and easy to prepare and has good stability. Naphazoline and chlorpheniramine can be effectively separated. After naphazoline and chlorpheniramine peak, other chemical components can be eluted with a high proportion of acetonitrile, which is suitable for large-throughput detection. Experiments have shown that 21 batches of samples produced by more than ten companies were continuously injected, and the data were stable, no cross-contamination of chromatographic peaks occurred, and the durability and stability were good.

[0207] The concentration of naphazoline hydrochloride nasal drops for nasal preparations is 0.1% or 0.05%. In practice, it is found that the concentration of naphazoline illegally added to nasal medical devices is about 0.1% to 0.4% in terms of naphazoline hydrochloride. The added amount generally reaches or exceeds the therapeutic dose, and all are strong positive samples. Considering the increasing popularity of liquid phase in grassroots laboratories, the developed test method is mainly based on liquid chromatography (HPLC-DAD), and liquid-to-mass confirmation is required. Naphazoline has a 1-naphthylmethyl structure and strong ultraviolet absorption, and the liquid phase method has high sensitivity. The instrument detection limit (IDL) of the HPLC-DAD method is 0.5ng. The method developed by the center allows the sample to be diluted 25 times and injected with 10μL, with high column efficiency and good peak shape. This method saves reagents and can also ensure high sensitivity. The method is highly durable and applicable not only to normal pressure liquid phase but also to ultra-high performance liquid phase. In addition, the existing liquid phase method can be directly connected in series with mainstream single-rod mass spectrometers such as Waters QDA. The mobile phase system of this method is also applicable to triple quadrupole mass spectrometers, high-resolution mass spectrometers, etc., and the method has high applicability.

[0208] Obviously, the above embodiments of the present invention are merely examples to more clearly illustrate the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. Two chemical drug identification and content determination test methods in nasal medical devices, including: Take the test solution and the reference solution of naphazoline hydrochloride and chlorpheniramine maleate, use high performance liquid chromatography to analyze the sample, compare the results of the two, and use the consistency of the retention time and the ultraviolet spectrum of 190 nm~400 nm as the basis for preliminary screening. The test solution is prepared according to the following method: take the test sample and add water to the solution mass volume concentration of 0.04g / mL, vortex for 3 minutes, ultrasonically treat for 20 minutes, cool, filter, and take the filtrate. The concentrations of naphazoline hydrochloride and chlorpheniramine maleate in the reference solution of naphazoline hydrochloride and chlorpheniramine maleate are both 10μg / mL. The conditions of high performance liquid chromatography include: Column: Phenomenex Gemini C 18 column; Column temperature: 30°C; Sample chamber temperature: 10°C; Flow rate: 1.0 mL / min; Detection wavelength: 280 nm, scanning range: 190 nm~400 nm; Injection volume: 10 μL; Mobile phase: A (0.1% formic acid in water), B (acetonitrile), gradient conditions are as follows: ； Take the test solution and the reference solution of naphazoline hydrochloride and chlorpheniramine maleate, use high-resolution liquid chromatography-mass spectrometry to analyze, compare the parent ion and fragment ion information of the test solution and the reference solution, and if the two are consistent, it is confirmed that the sample contains the corresponding drug component. The chromatographic conditions of high-resolution liquid chromatography-mass spectrometry include: Column: Agilent Poroshell 120 EC-C 18 ; Column temperature: 35 °C, sample chamber temperature: 10 °C; Flow rate: 0.3 mL / min; Injection volume: 1 μL; Mobile phase: A (0.1% formic acid in water), B (acetonitrile), gradient conditions are as follows: ； Mass spectrometry conditions included: Ion source: ESI positive ion mode Dryer temperature: 325°C; Dryer flow rate: 8L / min; Atomizer pressure: 35Psi; Capillary voltage: 3500V; Capillary outlet voltage: 140V; Collision voltage: 20V; The parameters for the two drugs are as follows: Note: * is the base peak; Take the reference substance stock solution and prepare a series of standard solutions. Use high performance liquid chromatography for sample analysis, draw a concentration-peak area standard curve, and calculate the amount of drug in the sample based on the peak area of ​​the positive sample. Sample information of nasal medical devices is as follows: 。 2. The method according to claim 1, characterized in that Agilent 6530Q-TOF high-resolution liquid spectrometer was used for high-resolution liquid spectrometry.

3. The method according to claim 1, characterized in that The reference substance stock solution was prepared as follows: naphazoline hydrochloride and chlorpheniramine maleate reference substances were taken separately, accurately weighed, placed in a brown volumetric flask, dissolved with water and then fixed to volume, shaken well, and prepared into reference substance stock solutions with a concentration of 1 mg / mL each.

4. The method according to claim 1, characterized in that: The separation between the chromatographic peaks of the two reference substances and the adjacent chromatographic peaks is greater than 1.5; the signal-to-noise ratio of the chromatographic peaks of the two reference substances is greater than 3:1.

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