A method for detecting the content of lamotrigine and preservative in lamotrigine dry suspension

Abstract

The invention discloses a method for detecting the contents of lamotrigine and preservative in a lamotrigine dry suspension. The method adopts a high performance liquid chromatography method for separation and determination. A chromatographic column of the high performance liquid chromatography method uses octadecylsilane bonded silica gel as a filler, and gradient elution is performed using a mobile phase A and a mobile phase B. The mobile phase A is an acetate buffer solution, and the mobile phase B is an acetonitrile-methanol mixed solution with a volume ratio of 40-60:60-40. The total volume of the mobile phase is 100%, and the gradient elution program is as follows: 0-3 min, 80%→60% mobile phase A, 20%→40% mobile phase B; 3-12 min, 60%→40% mobile phase A, 40%→60% mobile phase B; 12-15 min, 40%→80% mobile phase A, 60%→20% mobile phase B; 15-20 min, 80% mobile phase A, 20% mobile phase B. The method for detecting the contents of lamotrigine and preservatives in the lamotrigine dry suspension of the present invention has strong specificity, the separation degrees between impurities and the raw material medicine and the preservative are all greater than 2, other auxiliary materials and the raw material medicine and the preservative do not interfere with each other, and the contents of lamotrigine and preservatives can be accurately determined.

Description

Technical Field

[0001] The invention relates to the technical field of drug quality detection, and in particular to a method for detecting the contents of lamotrigine and a preservative in a lamotrigine dry suspension. Background Art

[0002] Epilepsy is a chronic brain disease caused by abnormal discharges of brain neurons. During an attack, the patient experiences brief involuntary convulsions in a part of the body or the entire body, and may even be accompanied by loss of consciousness and urinary and fecal incontinence.

[0003] Lamotrigine is a new type of anti-epileptic drug that can treat secondary generalized tonic-clonic seizures and primary generalized tonic-clonic seizures. Its main function is to reduce the discharge frequency of neurons by blocking sodium channels, reducing the influx of sodium ions and the release of glutamate, thereby reducing epileptic seizures.

[0004] Lamotrigine is a triazine compound, its chemical name is 3,5-diamino-6-(2,3-dichlorophenyl)-triazine, and its molecular formula is C 9 H 7 N 5 Cl 2 , the structural formula is as follows

[0005]

[0006] Lamotrigine dry suspension is a new type of dosage form. As a liquid preparation, its advantage is that it can be taken by children and patients with dysphagia. Since it is a multi-dose liquid preparation, in order to ensure the quality and efficacy of the product during use after opening, preservatives sodium methylparaben and sodium propylparaben are added to the lamotrigine dry suspension. As one of the key items of product quality control, the content of preservatives needs to be tested. When using the detection method of lamotrigine tablets included in the United States Pharmacopoeia, whether it is the content detection method or the related substance detection method, the separation degree of lamotrigine impurities and raw materials is less than 2 and the baseline is not separated, which interferes with the accurate quantification of raw materials in lamotrigine dry suspension. At the same time, preservatives are also interfered with by impurities; when the detection method of the raw material manufacturer is used to detect lamotrigine dry suspension, the main peak shape is poor, and lamotrigine impurities interfere with the main peak. Therefore, neither the detection method of the United States Pharmacopoeia nor the method of the API manufacturer can simultaneously meet the requirements for the accurate quantification of the API and preservatives in the lamotrigine dry suspension. It is necessary to re-establish a content detection method that can simultaneously and accurately quantify the API and preservatives in the lamotrigine dry suspension. Summary of the invention

[0007] In view of the above-mentioned defects of the prior art, the present invention provides a method for detecting the content of lamotrigine and preservative in a lamotrigine dry suspension, by optimizing the composition and proportion of the mobile phase so that the separation degree between impurities and the raw material drug and the preservative is greater than 2, and the content of lamotrigine and preservative can be accurately determined.

[0008] In order to solve the above technical problems, the present invention provides a method for detecting the content of lamotrigine and preservatives in a lamotrigine dry suspension, wherein the separation and determination are performed by high performance liquid chromatography, wherein the chromatographic column of the high performance liquid chromatography uses octadecylsilane bonded silica gel as a filler, and gradient elution is performed by mobile phase A and mobile phase B, wherein mobile phase A is an acetate buffer solution, and mobile phase B is an acetonitrile-methanol mixed solution with a volume ratio of 40-60:60-40, and the total volume of the mobile phase is 100%, and the gradient elution program is:

[0009] 0-3min, 80%→60% mobile phase A, 20%→40% mobile phase B;

[0010] 3-12 min, 60% → 40% mobile phase A, 40% → 60% mobile phase B;

[0011] 12-15 min, 40% → 80% mobile phase A, 60% → 20% mobile phase B;

[0012] 15-20min, 80% mobile phase A, 20% mobile phase B.

[0013] As a preferred embodiment, the pH value of mobile phase A is 4.5-5.5.

[0014] As a more preferred embodiment, the pH value of mobile phase A is 5.2.

[0015] As a preferred embodiment, the preservatives are sodium methylparaben and sodium propylparaben.

[0016] As a preferred embodiment, the chromatographic column satisfies one or more of the following conditions:

[0017] 1) The chromatographic column is Waters XBridge C18, with specifications of 4.6×250mm, 5μm;

[0018] 2) The column temperature is 30-40°C.

[0019] As a more preferred embodiment, the temperature of the chromatographic column is 35°C.

[0020] As a preferred embodiment, mobile phase B is a mixed solution of acetonitrile and methanol in a volume ratio of 50:50.

[0021] As a preferred embodiment, the injection volume is 5 to 20 μl.

[0022] As a more preferred embodiment, the injection volume is 10 μl.

[0023] As a preferred embodiment, the flow rate of the mobile phase is 0.8-1.2 mL / min.

[0024] As a more preferred embodiment, the flow rate of the mobile phase is 1 mL / min.

[0025] As a preferred embodiment, the detection wavelength is 210-270 nm.

[0026] As a more preferred embodiment, the detection wavelength is 256 nm.

[0027] As a preferred embodiment, high performance liquid chromatography detection comprises the following steps:

[0028] S1. Prepare blank solution, reference solution, verification reference solution and test solution;

[0029] S2. Inject the blank solution, reference solution, recheck reference solution and test solution in step S1 into the chromatograph in sequence, and record the chromatograms of the blank solution, reference solution, recheck reference solution and test solution in sequence;

[0030] S3. Calculate the contents of lamotrigine and preservatives by the external standard method according to the peak areas of the chromatogram.

[0031] As a more preferred embodiment, the blank solution is an acetonitrile-water solution with a volume ratio of 50:50; the reference solution and the recheck reference solution are prepared by using the blank solution to prepare a solution containing 0.4 mg / mL lamotrigine, 0.08 mg / mL methylparaben, and 0.008 mg / mL propylparaben; and the test solution is prepared by dissolving the lamotrigine dry suspension powder to be tested with the blank solution.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] (1) The method for detecting the content of lamotrigine and preservative in the lamotrigine dry suspension of the present invention has strong specificity, the separation degree between impurities and the raw material drug and the preservative is greater than 2, other excipients do not interfere with the raw material drug and the preservative, and the content of lamotrigine and preservative can be accurately determined.

[0034] (2) The method for detecting the content of lamotrigine and preservative in the lamotrigine dry suspension of the present invention has excellent precision, accuracy, stability and durability.

[0035] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1-1 This is a typical chromatogram of the resolution solution determined using the United States Pharmacopoeia lamotrigine tablets assay method;

[0037] Figure 1-2 Typical chromatograms of solutions for the wavelength detection separation of impurities B, C, D, and G for adjusting the assay method for the content of lamotrigine tablets in the USP;

[0038] Figure 1-3 Typical chromatograms of the mobile phase ratio test diluent, sodium methylparaben positioning solution, and resolution solution for adjusting the test method for the content of lamotrigine tablets in the USP;

[0039] Figure 1-4 Typical chromatograms of sodium propylparaben fixative, sodium methylparaben fixative, and resolution solution were tested to adjust the mobile phase composition of the test method for the content of lamotrigine tablets in the United States Pharmacopoeia;

[0040] Figure 2 Typical chromatograms of the diluent, separation solution, and sodium propylparaben fixative solution determined using the United States Pharmacopoeia lamotrigine tablets related substance detection method;

[0041] Figure 3 Typical chromatograms of the diluent, resolution solution, and solution to be tested for determination using the API manufacturer's test method;

[0042] Figure 4 A chromatogram of lamotrigine, methylparaben sodium and propylparaben sodium in a test solution (lamotrigine dry suspension) detected by the method of the present invention;

[0043] Figure 5 A typical chromatogram obtained by detecting a blank solution using the method of the present invention;

[0044] Figure 6 The typical chromatogram obtained by detecting a blank auxiliary material solution using the method of the present invention;

[0045] Figure 7 A typical chromatogram obtained by detecting a 0.2% impurity B positioning solution using the method of the present invention;

[0046] Figure 8 A typical chromatogram obtained by detecting a 0.2% impurity C positioning solution using the method of the present invention;

[0047] Fig. 9A typical chromatogram obtained by detecting a 0.2% impurity D localization solution using the method of the present invention;

[0048] Fig.10 A typical chromatogram obtained by detecting a 0.2% impurity G localization solution using the method of the present invention;

[0049] Fig.11 A typical chromatogram obtained by detecting a 0.1% impurity spiked sample solution using the method of the present invention;

[0050] Fig.12 A typical chromatogram obtained by detecting a reference solution using the method of the present invention;

[0051] Fig.13 The linear relationship diagram obtained by detecting lamotrigine using the method of the present invention;

[0052] Fig.14 The linear relationship diagram obtained by detecting methylparaben using the method of the present invention;

[0053] Fig.15 The linear relationship diagram obtained by detecting propylparaben using the method of the present invention;

[0054] Fig.16 This is a typical chromatogram obtained by the method of Comparative Example 1;

[0055] Fig.17 This is a typical chromatogram obtained by the method of Comparative Example 2;

[0056] Fig.18 It is a typical chromatogram obtained by using the method of Comparative Example 3. DETAILED DESCRIPTION

[0057] In order to make the technical means, creative features, objectives and effects of the invention easier to understand, the invention is further described below with reference to specific illustrations. However, the invention is not limited to the following implementation cases.

[0058] It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings of this specification are only used to match the contents disclosed in the specification so as to facilitate understanding and reading by persons familiar with this technology. They are not used to limit the conditions under which the present invention can be implemented, and therefore have no substantive technical significance. Any structural modification, change in proportion or adjustment of size, without affecting the effects and purposes that can be achieved by the present invention, should still fall within the scope of the technical contents disclosed by the present invention.

[0059] Lamotrigine is a new type of anti-epileptic drug. Lamotrigine dry suspension, as a liquid preparation, is easy to take to children and patients with dysphagia. In order to ensure the quality and efficacy after opening, preservatives, sodium methylparaben and sodium propylparaben are added to the lamotrigine dry suspension. Lamotrigine dry suspension also contains lamotrigine impurities B, impurity C, impurity D and impurity G. If the content of lamotrigine and preservatives is to be accurately determined, the interference of impurities in the determination should be avoided. In addition, sodium methylparaben and sodium propylparaben themselves also contain impurity p-hydroxybenzoic acid, which will overlap with sodium methylparaben and sodium propylparaben during detection, thereby interfering with their accurate quantification. Therefore, for the accurate determination of lamotrigine and preservatives, it is necessary to solve the interference of lamotrigine impurities B, impurity C, impurity D and impurity G on lamotrigine and preservatives, and also solve the interference of p-hydroxybenzoic acid sodium methylparaben and sodium propylparaben.

[0060] Part I: Exploratory Experiment

[0061] Introduction to the raw materials and reagents involved in the exploratory experiment:

[0062] Phosphate buffer: Take 2.7 g of potassium dihydrogen phosphate and dissolve it in 1000 mL of water by ultrasonication, add 6.5 mL of triethylamine, and adjust the pH to 2.0±0.05 with phosphoric acid.

[0063] Acetate buffer: Take 0.8 g of ammonium acetate and dissolve it in 1000 mL of water by ultrasonication, and adjust the pH to 4.5±0.05 with acetic acid.

[0064] Diluent: Methanol and acetate buffer were mixed in a volume ratio (v / v) of 60:40.

[0065] Separation solution: Use diluent to prepare a solution containing 0.8 μg / mL of lamotrigine impurities B, C, D, G and 0.4 mg / mL of lamotrigine.

[0066] Impurity B, C, D, G localization solutions: Use diluent to prepare 0.8 μg / mL lamotrigine impurity B, C, D, G solutions respectively.

[0067] Sodium methylparaben fixing solution (0.04mg / ml): accurately weigh 20mg of sodium methylparaben and place it in a 20ml volumetric flask, add diluent to dissolve it by ultrasonic, add diluent to make up the volume, and mix well; transfer 0.8ml of the above solution to a 20ml volumetric flask, add diluent to make up the volume, and mix well.

[0068] Sodium propylparaben fixing solution (0.04mg / ml): Accurately weigh 20mg of sodium propylparaben and place it in a 20ml volumetric flask, add diluent and ultrasonically dissolve it, add diluent to make up the volume, and mix well; transfer 0.8ml of the above solution to a 20ml volumetric flask, add diluent to make up the volume, and mix well.

[0069] Solution to be tested (lamotrigine dry suspension): prepared with diluent, containing approximately 0.4 mg / mL of lamotrigine, 0.08 mg / mL of methylparaben sodium, and 0.008 mg / mL of propylparaben sodium.

[0070] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 50" includes ±5% of 50, or from 47.5 to 52.5.

[0071] The ml and mL appearing in this application document are both volume units, referring to milliliters.

[0072] The United States Pharmacopoeia includes the detection method of the content of lamotrigine tablets and the detection method of related substances, among which the detection method of the content of lamotrigine tablets is to detect the content of the main drug lamotrigine; and the detection method of related substances of lamotrigine tablets is to detect the content of lamotrigine impurity B and impurity C. The inventors of the present application first tried to detect the content of lamotrigine and preservatives in lamotrigine dry suspension by using the detection method of the content of lamotrigine tablets or the detection method of related substances included in the United States Pharmacopoeia.

[0073] The test method for the content of lamotrigine tablets included in the United States Pharmacopoeia involves the following relevant parameters:

[0074] -HPLC column is octadecyl bonded porous silica gel (4.6*150mm, 5μm packing L1);

[0075] -The mobile phase is methanol: acetate buffer solution = 60:40 (V / V);

[0076] - Isocratic elution;

[0077] -Detection wavelength: 210nm; column temperature: room temperature; flow rate: 1mL / min; injection volume: 10μl; running time: 15 minutes;

[0078] In addition to using the above method to detect the content of lamotrigine and preservatives, the present application also adjusted the relevant parameters, as shown in Table 1.

[0079] Table 1

[0080]

[0081]

[0082] The experimental conditions in Table 1 were used to perform sample injection and detection, and chromatograms were obtained. The chromatogram corresponding to the United States Pharmacopoeia (the chromatogram corresponding to the resolution solution) is as follows: Figure 1-1 As shown, the chromatogram corresponding to the adjustment scheme 1 (corresponding to the separation solution, the comparison diagram of the impurity B, C, D, G positioning solution) is as follows Figure 1-2 As shown, the chromatogram corresponding to the adjustment scheme 2 (corresponding to the comparison diagram of the diluent, sodium hydroxybenzoate positioning solution, and separation solution) is as follows Figure 1-3 As shown, the chromatogram corresponding to the adjustment scheme three (corresponding to the comparison diagram of the sodium propylparaben positioning solution, the sodium methylparaben positioning solution, and the separation solution) is as follows Figure 1-4 shown.

[0083] from Figure 1-1 It can be seen that lamotrigine and lamotrigine impurity G and impurity B cannot be effectively separated; moreover, the lamotrigine peak is overloaded at the detection wavelength of 210nm and cannot be accurately quantified.

[0084] from Figure 1-2 It can be seen that although the problem of lamotrigine peak overload was overcome by adjusting the detection wavelength, the lamotrigine impurity G interfered with the determination of lamotrigine.

[0085] from Figure 1-3 , Figure 1-4 It can be seen that the lamotrigine impurity G interferes with the determination of lamotrigine, and the lamotrigine impurity C interferes with the determination of sodium methylparaben.

[0086] It can be seen that when using the detection method for the content of lamotrigine tablets included in the United States Pharmacopoeia to detect the content of lamotrigine and preservatives in lamotrigine dry suspension, there is a problem of impurity interference. Even if the detection wavelength, mobile phase composition or ratio is adjusted, the above problem cannot be solved.

[0087] To this end, we tried to use the detection method of related substances of lamotrigine tablets included in the United States Pharmacopoeia. The relevant parameters involved in this method are as follows:

[0088] -HPLC method, octadecyl bonded porous silica gel (4.6*250mm, 5μm packing L1);

[0089] -Mobile phase: acetonitrile:methanol:acetate buffer solution = 10:30:60 (V / V / V);

[0090] - Isocratic elution;

[0091] -Detection wavelength: 210nm; column temperature: room temperature; flow rate: 1mL / min; injection volume: 10μl;

[0092] Detection solution: diluent, separation solution, sodium propylparaben fixing solution.

[0093] After sampling and testing, the chromatogram obtained is as follows Figure 2 As shown, from Figure 2It can be seen that, on the one hand, the lamotrigine impurity D was not eluted, which may interfere with the analytes (lamotrigine, sodium methylparaben, sodium propylparaben); on the other hand, under this detection method, the peak time of propylparaben is 39 minutes, and the running time is too long, which is not conducive to detection in the experiment, increases the time cost, and reduces the detection efficiency.

[0094] In addition, an attempt was made to use the lamotrigine content detection method provided by the lamotrigine API manufacturer to determine the lamotrigine and preservative content in the lamotrigine dry suspension. The specific test conditions are as follows:

[0095] -HPLC method, the chromatographic column is octadecyl bonded porous silica gel (Cadenza CD-C18, 4.6*150mm, 3μm);

[0096] -Mobile phase A: phosphate buffer solution;

[0097] -Mobile phase B: acetonitrile

[0098] - Gradient elution, the elution program is shown in Table 2.

[0099] Table 2

[0100] Time (min) Mobile phase A (%) Mobile phase B (%) 0 80 20 2 80 20 17 55 45 25 55 45

[0101] -Detection wavelength: 230nm; column temperature: 40℃; flow rate: 1.0mL / min; injection volume: 20μl;

[0102] Detection solution: separation solution, solution to be detected (lamotrigine dry suspension).

[0103] After injection testing, the chromatogram obtained is as follows Figure 3 As shown. Figure 3 It can be seen that the peak shape of lamotrigine is poor, the tailing is serious, and the impurity G and lamotrigine cannot be effectively separated.

[0104] It can be seen that neither the United States Pharmacopoeia test method nor the test method of the API manufacturer can achieve quantitative detection of the API and preservatives in lamotrigine dry suspension. Even if the pharmacopoeia method is optimized, it cannot meet the simultaneous quantitative detection of lamotrigine and preservatives. In addition, it was found in the experiment that p-hydroxybenzoic acid would interfere with the quantification of sodium propylparaben and sodium methylparaben, and p-hydroxybenzoic acid is an impurity of sodium methylparaben and sodium propylparaben. Therefore, when establishing the test method, it is necessary to meet the simultaneous and accurate quantification of lamotrigine and preservatives, and also to ensure that the excipients, impurities, p-hydroxybenzoic acid, lamotrigine and preservatives do not interfere with each other.

[0105] Part II Detection Method of the Application

[0106] The raw materials and reagents involved are introduced as follows:

[0107] Acetate buffer: Take 0.8 g of ammonium acetate and dissolve it in 1000 mL of water by ultrasonication, and adjust the pH to 5.2±0.05 with acetic acid.

[0108] Diluent: Methanol and acetate buffer were mixed in a volume ratio (v / v) of 60:40.

[0109] Blank solution: Mix acetonitrile and water in a volume ratio of 50 / 50 and shake well.

[0110] Methylparaben reference stock solution (0.8mg / mL): Accurately weigh 8mg of methylparaben reference into a 10mL volumetric flask, add an appropriate amount of blank solution and sonicate to completely dissolve. Add blank solution to volume and mix well. (Prepare in duplicate)

[0111] Propylparaben reference stock solution (0.08mg / mL): Accurately weigh 8mg of propylparaben reference into a 100mL volumetric flask, add an appropriate amount of blank solution and sonicate to completely dissolve. Add blank solution to volume and mix well. (Prepare in duplicate)

[0112] Reference substance solution (lamotrigine: 0.4 mg / mL, methylparaben: 0.08 mg / mL, propylparaben: 0.008 mg / mL): Accurately weigh 4 mg of lamotrigine reference substance and transfer 1.0 mL of methylparaben reference substance stock solution and 1.0 mL of propylparaben reference substance stock solution into the same 10 mL volumetric flask, add appropriate amount of blank solution and sonicate to completely dissolve, add blank solution to make up to volume, and mix well.

[0113] Verify the reference solution (lamotrigine: 0.4 mg / mL, methylparaben: 0.08 mg / mL, propylparaben: 0.008 mg / mL): Accurately weigh 4 mg of lamotrigine reference substance and transfer 1.0 mL of methylparaben reference substance stock solution and 1.0 mL of propylparaben reference substance stock solution into the same 10 mL volumetric flask, add an appropriate amount of blank solution and sonicate to completely dissolve, add blank solution to make up to volume, and mix well.

[0114] Test solution: Accurately weigh 1.65g of test powder and place it in a 100mL volumetric flask (equivalent to 200mg of lamotrigine, 40mg of sodium methylparaben, and 4mg of sodium propylparaben), add 50mL of acetonitrile to disperse the powder, ultrasonicate for 40 minutes, cool to room temperature, add water to volume, and mix. Accurately pipette 2.0mL of the above test solution into a 10mL volumetric flask, add blank solution to volume, and mix. Filter with a 0.45μm PTFE filter membrane, discard the first mL, and take the filtrate. (Prepare in duplicate)

[0115] Blank excipient solution: Accurately weigh 1.4g of excipient (without lamotrigine, sodium methylparaben and sodium propylparaben, and composed of xanthan gum, anhydrous disodium hydrogen phosphate, anhydrous citric acid, sucralose, lactose monohydrate, polysorbate 80, simethicone emulsion, colloidal silicon dioxide, orange powder flavor and microcrystalline cellulose mixed in the proportion of self-developed prescription) and place it in a 100mL volumetric flask, add acetonitrile dispersion powder of 50% of the volume of the volumetric flask, ultrasonicate for 40 minutes, add water to the volume, and shake well. Accurately transfer 2.0mL of the above solution to a 10mL volumetric flask, add blank solution to the volume, and shake well. Filter with a 0.45μm PTFE filter membrane, discard 1mL, and take the filtrate.

[0116] Impurity B / C / D / G reference substance stock solution (0.08 mg / mL): Accurately weigh 2 mg of impurity B / C / D / G reference substance and place it in different 25 mL volumetric flasks, add appropriate amount of blank solution and ultrasonically dissolve it, add blank solution to make up to volume, and shake well.

[0117] 0.2% Impurity B / C / D / G positioning solution (0.8 μg / mL): Pipette 1.0 mL of Impurity B / C / D / G reference substance stock solution into different 100 mL volumetric flasks, add blank solution to make up to volume, and shake well.

[0118] 0.1% impurity spiked sample solution: Accurately weigh 1.65g of the test sample powder and place it in a 100mL volumetric flask. Add 50% of the volume of the flask's acetonitrile dispersion powder, and pipette 5.0mL of the impurity B / C / D / G reference substance stock solution respectively. Ultrasonicate for 40 minutes, add water to the volume, and shake well. Accurately pipette 2.0mL of the above solution and place it in a 10mL volumetric flask, add blank solution to the volume, and shake well. Filter with a 0.45μm PTFE filter membrane, discard 1mL, and take the filtrate.

[0119] Propylparaben reference substance stock solution (1 mg / mL): Accurately weigh 20 mg of propylparaben reference substance and place it in a 20 mL volumetric flask, add blank solution and ultrasonically dissolve it completely, add blank solution to make up to volume, and shake well.

[0120] Linear stock solution (lamotrigine 0.8 mg / ml, methylparaben 0.16 mg / ml, propylparaben 0.016 mg / ml): Accurately weigh 40 mg of lamotrigine reference substance and 8 mg of methylparaben reference substance and place them in a 50 ml volumetric flask, transfer 10 ml of propylparaben reference substance stock solution (1 mg / mL) into the above volumetric flask, add blank solution and ultrasonically dissolve, make up to volume of blank solution, and mix well.

[0121] Sodium methylparaben solution (0.08 mg / mL): Accurately weigh 20 mg of sodium methylparaben and place it in a 20 mL volumetric flask, add diluent and ultrasonically dissolve it, add diluent to the volume, and mix well; transfer 0.8 mL of the above solution to a 10 mL volumetric flask, add diluent to the volume, and mix well.

[0122] Sodium propylparaben solution (0.008 mg / mL): Accurately weigh 20 mg of sodium propylparaben and place it in a 20 mL volumetric flask, add diluent and ultrasonically dissolve it, add diluent to make up the volume, and mix well; transfer 0.8 mL of the above solution to a 100 mL volumetric flask, add diluent to make up the volume, and mix well.

[0123] Mixed solution (containing lamotrigine, impurities and preservatives): Use a diluent to prepare a solution containing 0.8 μg / mL of lamotrigine impurities B, C, D, G and 0.4 mg / mL of lamotrigine, 0.08 mg / mL of methylparaben sodium, and 0.008 mg / mL of propylparaben sodium.

[0124] The present invention provides a method for detecting the contents of lamotrigine and preservatives in a lamotrigine dry suspension, wherein the method adopts a high performance liquid chromatography method for separation and determination, wherein a chromatographic column of the high performance liquid chromatography method uses octadecylsilane bonded silica gel as a filler, and a mobile phase A and a mobile phase B are used for gradient elution, wherein the mobile phase A is an acetate buffer solution, and the mobile phase B is an acetonitrile-methanol mixed solution with a volume ratio of 40-60:60-40, and the total volume of the mobile phase is 100%, and the gradient elution procedure is:

[0125] 0-3min, 80%→60% mobile phase A, 20%→40% mobile phase B;

[0126] 3-12 min, 60% → 40% mobile phase A, 40% → 60% mobile phase B;

[0127] 12-15 min, 40% → 80% mobile phase A, 60% → 20% mobile phase B;

[0128] 15-20min, 80% mobile phase A, 20% mobile phase B.

[0129] Wherein, the pH value of mobile phase A is 4.5 to 5.5. Preferably, the pH value of mobile phase A is 5.2.

[0130] Among them, the preservatives are sodium methylparaben and sodium propylparaben.

[0131] The chromatographic column is Waters XBridge C18, with a specification of 4.6×250 mm, 5 μm; the temperature of the chromatographic column is 30-40° C., preferably, the temperature of the chromatographic column is 35° C.

[0132] The mobile phase B was a mixed solution of acetonitrile and methanol in a volume ratio of 50:50.

[0133] The injection volume is 5 to 20 μl, preferably, the injection volume is 10 μl.

[0134] The flow rate of the mobile phase is 0.8-1.2 mL / min, preferably, the flow rate of the mobile phase is 1 mL / min.

[0135] The detection wavelength is 210-270 nm, preferably, the detection wavelength is 256 nm.

[0136] Example 1

[0137] The test methods, conditions and reagents are as follows:

[0138] -HPLC method, octadecylsilane bonded silica gel column (Waters XBridge C18 4.6×250mm, 5μm);

[0139] - Mobile phase A: pH 5.2 acetate buffer solution;

[0140] - Mobile phase B: a mixed solution of acetonitrile-methanol = 50 / 50 (V / V);

[0141] - Flow rate: 1.0 mL / min;

[0142] - Column temperature: 35℃

[0143] -Detection wavelength: 256nm

[0144] -Injection volume: 10μl

[0145] - Elution procedure:

[0146] 0-3min, 80%→60% is mobile phase A, 20%→40% is mobile phase B;

[0147] 3-12min, 60%→40% mobile phase A, 40%→60% mobile phase B;

[0148] 12-15min, 40%→80% mobile phase A, 60%→20% mobile phase B;

[0149] 15-20min, 80% mobile phase A, 20% mobile phase B;

[0150] - The samples to be tested, lamotrigine dry suspension samples, three batches in total (batch numbers: 2403011, 2403012, 2403013), were prepared using the test solution method.

[0151] The specific detection steps are as follows:

[0152] S1. Prepare blank solution, reference solution, verification reference solution and test solution (refer to the introduction of raw materials and reagents for specific preparation methods).

[0153] S2. Continuously and sequentially inject the blank solution, reference solution, recheck reference solution and test solution in step S1 into the chromatograph, and record the chromatograms of the blank solution, reference solution, recheck reference solution and test solution in turn.

[0154] The specific injection process is as follows: (1) Blank solution (injection number ≥ 1) (2) Reference solution (injection number 6 injections) (3) Verification reference solution (injection number 2 injections) (4) Test solution (injection number 1 injection, maximum 12 injections) (5) Reference solution (injection number 1 injection, for every 12 injections of test solution, 1 injection of reference solution is required).

[0155] S3. Calculate the contents of lamotrigine and preservatives in the test solution by the external standard method based on the peak areas of the chromatogram.

[0156] Lamotrigine calculation formula:

[0157]

[0158] Methylparaben calculation formula:

[0159]

[0160] Propylparaben calculation formula:

[0161]

[0162] Where:

[0163] A SPL : The peak area of ​​lamotrigine or methylparaben or propylparaben in the test solution;

[0164] A STD : The peak area of ​​lamotrigine or methylparaben or propylparaben in all reference solutions;

[0165] W STD : Weighing of lamotrigine or methylparaben or propylparaben reference substances;

[0166] W SPL : Weighing of the test sample;

[0167] V STD : dilution volume of reference solution;

[0168] V SPL : dilution volume of the test solution;

[0169] P: Content of lamotrigine or methylparaben or propylparaben reference substance

[0170] LC: labeled amount

[0171] Figure 4 The chromatogram obtained by sampling and testing the test solution using the method of the present invention is shown (the chromatogram corresponding to batch number 2403011, and the chromatograms corresponding to other batch numbers are similar). Using the above formula, the percentage results of the actual measured values ​​of lamotrigine, methylparaben sodium and propylparaben sodium in three batches of test solution to the theoretical values ​​are obtained, as shown in Table 3.

[0172] Table 3

[0173]

[0174] from Figure 4 As can be seen from Table 3, the present invention can quantitatively detect the contents of lamotrigine, sodium methylparaben and sodium propylparaben in the lamotrigine dry suspension, and p-hydroxybenzoic acid does not interfere with the detection of methylparaben and propylparaben.

[0175] By conversion, the content ratios of lamotrigine, methylparaben sodium, and propylparaben sodium in the test sample were obtained, as shown in Table 4.

[0176] Table 4

[0177]

[0178] It can be seen that the method of the present invention can accurately quantify the contents of lamotrigine, methylparaben sodium and propylparaben sodium in the lamotrigine dry suspension.

[0179] Example 2

[0180] 1. System suitability test

[0181] The purpose of this test is to determine the system applicability of the high performance liquid chromatography method used in the present invention for the quantitative detection of lamotrigine, sodium methylparaben and sodium propylparaben in lamotrigine dry suspension.

[0182] According to the ultraviolet spectra of lamotrigine, methylparaben and propylparaben, the absorption wavelength is selected as 256nm.

[0183] The test methods, conditions and reagents are as follows:

[0184] -Chromatographic system: Waters e2695 or Agilent 1260 or other similar instruments;

[0185] -Chromatographic column: octadecylsilane bonded silica column Waters Xbridge C18, 4.6*250mm, 5μm;

[0186] -Mobile phase A: 0.8 g / L acetate buffer, pH adjusted to 5.2;

[0187] - Mobile phase B: acetonitrile: methanol = 50:50 (V / V);

[0188] - Gradient elution

[0189] Table 5

[0190] Time (min) Mobile phase A (%) Mobile phase B (%) 0 80 20 3 60 40 12 40 60 15 80 20 20 80 20

[0191] - Flow rate: 1 mL / min;

[0192] - Column temperature: 35°C;

[0193] - Detection wavelength: 256nm;

[0194] -Injection volume: 10 μl.

[0195] The above-mentioned gradient elution is achieved by setting the starting value and end value of the mobile phase concentration change in the high performance liquid chromatography. For example, within 0-3 minutes, the starting value is set to 80% of mobile phase A and 20% of mobile phase B, and the end value is set to 60% of mobile phase A and 40% of mobile phase B.

[0196] Take blank solution, reference solution and verification reference solution for analysis respectively, and record the chromatogram.

[0197] System suitability requirements: (1) The blank solution should be free of interference except for the solvent peak; (2) The RSD of the lamotrigine peak area in all reference solutions should be no more than 2.0%, and the RSD of the peak area of ​​methylparaben and propylparaben should be no more than 5.0%; (3) The recovery rate of lamotrigine in the reference solution should be between 98.0-102.0%, and the recovery rate of methylparaben and propylparaben should be between 95.0%-105.0%.

[0198] The test results are shown in Table 6.

[0199] Table 6

[0200]

[0201] The results showed that the system applicability met the acceptable standards, indicating that the system applicability of the method of the present invention was good.

[0202] 2. Specificity test

[0203] The purpose of this experiment is to examine the ability of the HPLC method to correctly determine the analyte in the possible presence of other components (such as blank solution, excipients, impurities, etc.).

[0204] The specific chromatographic conditions of this experiment were the same as those of the system suitability test.

[0205] Take blank solution, blank auxiliary material solution, 0.2% impurity B / C / D / G positioning solution, 0.1% impurity spiked sample solution, and reference solution (prepare according to the method in the raw materials and reagents introduction section), inject and analyze according to the above chromatographic conditions, and record the chromatogram. Figure 5 A typical chromatogram obtained by detecting a blank solution using the method of the present invention is shown. Figure 6 The typical chromatogram obtained by detecting a blank auxiliary material solution using the method of the present invention is shown. Figure 7-10 The typical chromatogram obtained by detecting 0.2% impurity B, C, D, G positioning solution using the method of the present invention is shown. Fig.11 The typical chromatogram obtained by detecting a 0.1% impurity spiked sample solution using the method of the present invention is shown. Fig.12 Figure 2 shows a typical chromatogram obtained by detecting a reference solution using the method of the present invention. Figure 5-12 As shown in the figure, the blank solution, blank excipient solution and impurities did not interfere with the determination of lamotrigine, methylparaben and propylparaben. Even though there were lamotrigine impurity G and lamotrigine impurity C between lamotrigine and methylparaben, the separation between impurity G and impurity C and lamotrigine and methylparaben was good and did not interfere with the accurate quantification of lamotrigine and methylparaben. The separation between lamotrigine, methylparaben and propylparaben was excellent, with the separation being greater than 2. The above results showed that the method had excellent specificity.

[0206] 3. Linearity and range tests

[0207] The purpose of this experiment is to investigate the ability of HPLC to show a proportional relationship between the peak area and concentration of the analyte within the designed range, as well as the accuracy of content calculation by external standard method.

[0208] The specific chromatographic conditions of this experiment were the same as those of the system suitability test.

[0209] The linear stock solution was prepared according to the method in the raw materials and reagents introduction section.

[0210] According to Table 7, a certain amount of linear stock solution was respectively pipetted to prepare standard solutions at 5 concentration levels, which were 40%, 80%, 100%, 120% and 160% of the target sample concentration, respectively.

[0211] Table 7

[0212]

[0213] Take the above solutions and inject them respectively, record the chromatogram, use the concentration as the horizontal axis and the peak area as the vertical axis to make a linear recovery equation, requiring the correlation coefficient r to be no less than 0.999, the residual to be no more than 2%, and the intercept of the Y axis to be within 2% of the 100% response value. The results are shown in Table 8:

[0214] Table 8

[0215]

[0216]

[0217] The results showed that lamotrigine had a good linear relationship in the concentration range of 0.16mg / mL-0.64mg / mL; methylparaben had a good linear relationship in the concentration range of 0.03mg / mL-0.13mg / mL; and propylparaben had a good linear relationship in the concentration range of 0.003mg / mL-0.013mg / mL.

[0218] Fig.13 The linear relationship diagram obtained by detecting lamotrigine using the method of the present invention is shown. Fig.14 The linear relationship diagram obtained by detecting methylparaben using the method of the present invention is shown. Fig.15 The linear relationship diagram obtained by detecting propylparaben using the method of the present invention is shown.

[0219] 4. Accuracy test

[0220] In this test, HPLC was used to add lamotrigine, methylparaben and propylparaben of known different concentrations to the sample solution containing excipients to examine whether the added analytes can be completely extracted and the degree of proximity between the measured results and the true values, which is generally expressed as recovery rate (%).

[0221] The specific chromatographic conditions of this experiment were the same as those of the system suitability test.

[0222] The stock solution of propylparaben reference substance was prepared according to the method described in the section on raw materials and reagents.

[0223] According to Table 9 below, weigh blank excipients (no lamotrigine, sodium methylparaben, sodium propylparaben, xanthan gum, anhydrous disodium hydrogen phosphate, anhydrous citric acid, sucralose, lactose monohydrate, polysorbate 80, simethicone emulsion, colloidal silicon dioxide, orange powder flavor and microcrystalline cellulose mixed according to the ratio of self-developed prescription), lamotrigine reference substance and methylparaben reference substance, placed in a 25mL volumetric flask, add acetonitrile of 50% of the volume of the volumetric flask, disperse the powder, then pipette propylparaben reference substance stock solution (1mg / ml) and place it in the above-mentioned volumetric flask, ultrasonicate for 40 minutes, add water to volume, and shake up. Accurately pipette 2.0mL of the above-mentioned solution and place it in a 10mL volumetric flask, add blank solution to volume, and shake up. Filter with a 0.45μm PTFE filter membrane, discard 1mL, take the subsequent filtrate, and obtain solutions of three concentrations. Prepare three copies of each concentration in parallel, and inject one needle per solution.

[0224] Table 9

[0225]

[0226]

[0227] It is required that the recovery rate and average recovery rate of lamotrigine at each concentration should be between 98.0% and 102.0%, the recovery rate and average recovery rate of methylparaben and propylparaben at each concentration should be between 95.0% and 105.0%, the RSD of the recovery rate of lamotrigine at each concentration should not be greater than 2.0%, and the RSD of methylparaben and propylparaben at each concentration should not be greater than 5.0%.

[0228] Each of the above solutions was sampled and analyzed, and the chromatogram was recorded. The recovery rate of each impurity was calculated by the external standard method, and the results are shown in Tables 10 and 11. Table 10 shows the accuracy results of lamotrigine and methylparaben, and Table 11 shows the accuracy results of propylparaben.

[0229] Table 10

[0230]

[0231] Table 11

[0232]

[0233]

[0234] The results showed that the recoveries, average recoveries and RSDs of lamotrigine, methylparaben and propylparaben at each concentration level all met the requirements; the above results indicated that the method of the present invention had high accuracy.

[0235] 5. Precision test

[0236] The purpose of this experiment is to test the closeness of the results obtained by multiple sampling measurements under certain conditions using the high performance liquid chromatography method.

[0237] The specific chromatographic conditions of this experiment were the same as those of the system suitability test.

[0238] The test solution was prepared according to the method described in the section on raw materials and reagents.

[0239] The above solution was sampled and analyzed, the chromatogram was recorded, and the recovery was calculated by the external standard method. The results are shown in Table 12.

[0240] Table 12

[0241]

[0242] The results showed that the RSDs of lamotrigine content in 6 repeated sample solutions were all less than 2.0%, and the RSDs of sodium methylparaben and sodium propylparaben content were all less than 5.0%, indicating good repeatability.

[0243] In another laboratory, another experimenter prepared 6 test sample solutions (lamotrigine dry suspension) according to the same preparation method, injected the samples for analysis, recorded the chromatograms, calculated the recovery rate according to the external standard method, and compared the test results of the two laboratories, as shown in Table 13.

[0244] Table 13

[0245]

[0246]

[0247] The results showed that the RSD of lamotrigine content in 6 sample solutions was less than 2.0%, the relative deviation of the average value of lamotrigine content between the two laboratories was no more than 3%, the RSD of sodium methylparaben and sodium propylparaben content was less than 5.0%, the relative deviation of the average value of sodium methylparaben and sodium propylparaben content between the two laboratories was no more than 5%, indicating good precision.

[0248] 6. Stability test

[0249] The purpose of this experiment is to examine the stability of the sample solution and the reference solution at room temperature and 2-8°C after preparation, so as to support the accuracy and reliability of the data during the test process.

[0250] The specific chromatographic conditions of this experiment were the same as those of the system suitability test.

[0251] The reference solution and test solution were prepared according to the methods in the introduction of raw materials and reagents.

[0252] The reference solution and the test solution were placed at room temperature and 2-8°C for 3 days for testing to examine the stability of the solutions. The stability results of the reference solution are shown in Table 14, and the stability results of the test solution are shown in Table 15.

[0253] Table 14

[0254]

[0255] Table 15

[0256]

[0257]

[0258] The results showed that the lamotrigine reference solution was placed at 2-8℃ for 3 days, and the content changed within ±2.0% compared with the zero point, indicating that it was stable within 3 days at 2-8℃; it was placed at room temperature for 2 days, and the content changed within ±2.0% compared with the zero point, indicating that it was stable within 2 days at room temperature. The methylparaben and propylparaben reference solution was placed at 2-8℃ for 3 days, and the content changed within ±5%, indicating that it was stable within 3 days at 2-8℃; it was placed at room temperature for 2 days, and the content changed within ±5%, indicating that it was stable within 2 days at room temperature. The test solution was placed at 2-8℃ for 3 days, and at room temperature for 1 day, and the content of lamotrigine changed within ±2.0%, and the content of methylparaben sodium and propylparaben sodium changed within ±5%, indicating that it was stable within 3 days at 2-8℃ and within 1 day at room temperature.

[0259] 7. Durability test

[0260] The purpose of this experiment is to find the optimal chromatographic parameters and determine the tolerance level of the results without being affected by small changes in target parameters by examining the changes in chromatographic conditions such as flow rate, column temperature, mobile phase pH, column batch number, and detection wavelength. The specific inspection items are shown in Table 16, and the inspection results are shown in Table 17.

[0261] The specific chromatographic conditions of this experiment were the same as those of the system suitability test.

[0262] Table 16

[0263]

[0264] Table 17

[0265]

[0266]

[0267] The results show that when different batches of chromatographic columns are changed and the pH of the mobile phase changes slightly, the difference between the content of lamotrigine and the initial value is no more than 2.0%, and the difference between the content of sodium methylparaben and sodium propylparaben and the initial value is no more than 5.0%, indicating that the method of the present invention has good durability.

[0268] Comparative Example 1

[0269] The chromatographic columns of the same specifications from other brands were used for liquid phase analysis of lamotrigine dry suspension.

[0270] -HPLC method, octadecylsilane bonded silica gel column (ODS-3Intersil 4.6×250mm, 5μm);

[0271] -Mobile phase A: pH 4.5 acetate buffer solution

[0272] -Mobile phase B: methanol: acetonitrile = 1:1

[0273] - Flow rate: 1.0 mL / min;

[0274] - Column temperature: 35℃

[0275] -Detection wavelength: 256nm

[0276] -Injection volume: 10μl

[0277] - Elution method: same as Example 1

[0278] Test solution: mixed solution (containing lamotrigine, impurities and preservatives) and sodium propylparaben solution. Fig.16 A typical chromatogram obtained in Comparative Example 1 is shown.

[0279] Comparative Example 2

[0280] Other mobile phase systems were used to perform liquid phase analysis of lamotrigine dry suspension.

[0281] -HPLC method, octadecylsilane bonded silica gel column (Waters XBridge C18 4.6×250mm, 5μm);

[0282] - Mobile phase A: 0.1% phosphoric acid solution: acetonitrile = 70:30

[0283] - Mobile phase B: 0.1% phosphoric acid solution: acetonitrile = 20:80

[0284] - Flow rate: 1.0 mL / min;

[0285] - Column temperature: 25℃

[0286] - Detection wavelength: 230nm

[0287] -Injection volume: 10μl

[0288] - Elution method: Mobile phase A: Mobile phase B = 70:30

[0289] Test solutions: sodium methylparaben solution, sodium propylparaben solution and mixed solution (containing lamotrigine, impurities and preservatives).

[0290] Fig.17 A typical chromatogram obtained in Comparative Example 2 is shown.

[0291] Comparative Example 3

[0292] Liquid phase analysis of lamotrigine dry suspension was performed using other pH buffer salts.

[0293] Mobile phase A: pH 4.0 acetate buffer solution: Take 0.8 g of ammonium acetate and dissolve it in 1000 mL of water by ultrasonication, and adjust the pH to 4.0±0.05 with acetic acid.

[0294] -Mobile phase B: methanol: acetonitrile = 1:1 (V:V)

[0295] - Flow rate: 1.0 mL / min;

[0296] - Column temperature: 35℃

[0297] -Detection wavelength: 256nm

[0298] -Injection volume: 10μl

[0299] - Elution method: same as Example 2

[0300] Detection solutions: blank solution, separation solution, mixed solution (containing lamotrigine, impurities and preservatives).

[0301] Fig.18 A typical chromatogram obtained in Comparative Example 3 is shown.

[0302] from Fig.16 It can be seen that the peak position of sodium propylparaben interferes with sodium methylparaben; Fig.17 It can be seen that lamotrigine elutes too quickly and overlaps with the diluent peak; Fig.18 It can be seen that there is an unknown peak in the blank solution that overlaps with sodium propylparaben.

[0303] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A method for detecting the content of lamotrigine and preservative in lamotrigine dry suspension, characterized in that: The separation and determination were carried out by high performance liquid chromatography. The chromatographic column of the high performance liquid chromatography was filled with octadecylsilane bonded silica gel. The mobile phase A and mobile phase B were used for gradient elution. The mobile phase A was an acetate buffer solution, and the mobile phase B was an acetonitrile-methanol mixed solution with a volume ratio of 40-60:60-40. The total volume of the mobile phase was 100%. The gradient elution program was: 0-3min, 80%→60% mobile phase A, 20%→40% mobile phase B; 3-12 min, 60% → 40% mobile phase A, 40% → 60% mobile phase B; 12-15 min, 40% → 80% mobile phase A, 60% → 20% mobile phase B; 15-20min, 80% mobile phase A, 20% mobile phase B.

2. The method for detecting the content of lamotrigine and preservative in the lamotrigine dry suspension according to claim 1, characterized in that: The pH value of mobile phase A is 4.5-5.

5.

3. The method for detecting the content of lamotrigine and preservative in the lamotrigine dry suspension according to claim 1, characterized in that: The preservatives are sodium methylparaben and sodium propylparaben.

4. The method for detecting the content of lamotrigine and preservative in the lamotrigine dry suspension according to claim 1, characterized in that: The chromatographic column meets one or more of the following conditions: 1) The chromatographic column is Waters XBridge C18, with specifications of 4.6×250mm, 5μm; 2) The column temperature is 30-40°C.

5. The method for detecting the content of lamotrigine and preservative in the lamotrigine dry suspension according to claim 1, characterized in that: Mobile phase B was a mixed solution of acetonitrile and methanol in a volume ratio of 50:

50.

6. The method for detecting the content of lamotrigine and preservative in the lamotrigine dry suspension according to claim 1, characterized in that: The injection volume is 5 to 20 μl.

7. The method for detecting the content of lamotrigine and preservative in the lamotrigine dry suspension according to claim 1, characterized in that: The flow rate of the mobile phase is 0.8-1.2 mL / min.

8. The method for detecting the content of lamotrigine and preservative in lamotrigine dry suspension according to claim 1, characterized in that: The detection wavelength is 210~270nm.

9. The method for detecting the content of lamotrigine and preservative in the lamotrigine dry suspension according to claim 1, characterized in that: The HPLC method includes the following steps: S1. Prepare blank solution, reference solution, verification reference solution and test solution; S2. Inject the blank solution, reference solution, recheck reference solution and test solution in step S1 into the chromatograph in sequence, and record the chromatograms of the blank solution, reference solution, recheck reference solution and test solution in sequence; S3. Calculate the contents of lamotrigine and preservatives by the external standard method according to the peak areas of the chromatogram.

10. The method for detecting the content of lamotrigine and preservative in lamotrigine dry suspension according to claim 9, characterized in that: The blank solution is an acetonitrile-water solution with a volume ratio of 50:50; the reference solution and the recheck reference solution are prepared by using the blank solution to prepare a solution containing 0.4 mg / mL lamotrigine, 0.08 mg / mL methylparaben, and 0.008 mg / mL propylparaben; the test solution is prepared by dissolving the lamotrigine dry suspension powder to be tested with the blank solution.

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