Method for detecting formamide residue in allopurinol

Abstract

The invention discloses a method for detecting formamide residues in allopurinol. The method comprises the following steps: S1, preparing a reference substance stock solution; s2, preparing a reference substance solution; s3, preparing a test solution; and S4, detecting. According to the detection method, the dissolvability difference of allopurinol and formamide in different solvents is utilized, a solvent which is easy to dissolve in the formamide and a solvent which is difficult to dissolve in allopurinol are selected, an allopurinol sample is dissolved in a dimethyl sulfoxide solvent at a high concentration, then a small amount of dichloromethane solvent is added, the temperature is reduced, allopurinol is separated out, and the content of allopurinol is detected. And the precipitated allopurinol is removed through centrifugation, and the finally obtained test solution only contains residual formamide to be detected (if any). According to the method, the amount of a target object entering a chromatographic system can be increased, and the interference of too high allopurinol sample concentration on the chromatographic system is reduced, so that the problem of too low sensitivity of a method for detecting the formamide residue in allopurinol is solved. Moreover, helium is selected as carrier gas, so that the detection sensitivity can be further improved.

Description

Technical Field

[0001] The invention relates to the technical field of impurity detection, and in particular to a method for detecting formamide residues in allopurinol. Background Art

[0002] Allopurinol is a white or off-white crystalline powder. Its chemical structure is similar to that of hypoxanthine. It can inhibit xanthine oxidase, reduce uric acid synthesis, and thus reduce the concentration of uric acid in the blood and the deposition of urate in bones, joints and kidneys. It can be used clinically to treat gout, as well as hyperuricemia caused by chemotherapy or radiotherapy for leukemia, polycythemia vera, multiple myeloma and other malignant tumors.

[0003] Formamide is a solvent used in the synthesis of allopurinol API. According to the Q3C(R8)-Impurities: Residual Solvents Guidelines issued by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH), the residual formamide in allopurinol API should not exceed 0.022%, which is a relatively low residual amount.

[0004] Gas chromatography is a common method for detecting residual solvents. A suitable gas chromatography detection method usually requires a certain degree of sensitivity and precision. Especially for the detection of solvents with low residual amounts, whether the sensitivity requirements can be met is an important requirement for determining whether a method is feasible. At present, improving the sensitivity of the detection method can usually be started from two directions: one is to increase the concentration of the test sample, and the other is to reduce the interference of other substances on the detection instrument. For gas chromatography, it is usually necessary to first dissolve the test sample in a suitable medium to make a test solution, and then put the test solution into the instrument for detection.

[0005] Since allopurinol has poor solubility in conventional solvents, the concentration of the prepared test solution is usually difficult to meet the detection requirement that the residual formamide should not exceed 0.022%, and there is a limit to dissolution, so the concentration of the test solution cannot be increased infinitely. For example, the paper "Gas Chromatography Determination of Residual Formamide in Allopurinol_Luo Jia" discloses a method for determining residual formamide in allopurinol, which improves the sensitivity of formamide detection by increasing the concentration of the test solution. The concentration of the prepared test solution reaches 40 mg / ml, which is close to the solubility limit of the test sample, but the high concentration of the test solution will cause allopurinol to produce a large residue in the gas chromatography system, and the residual allopurinol will contaminate the chromatographic system, increase the noise in the system, but is not conducive to improving the detection sensitivity of formamide. Summary of the invention

[0006] The technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide a method for detecting formamide residues in allopurinol, which can reduce the interference of excessively high allopurinol sample concentration on a chromatographic system and improve the detection sensitivity.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0008] A method for detecting formamide residue in allopurinol comprises the following steps:

[0009] S1. Prepare the reference substance stock solution: weigh about 18 mg of formamide, place it in a 20 ml volumetric flask, dilute to the mark with dimethyl sulfoxide, and shake well;

[0010] S2. Prepare reference solution: take 1 ml of reference stock solution, place in a 100 ml volumetric flask, dilute to scale with dimethyl sulfoxide, and shake well;

[0011] S3. Prepare the test solution: weigh about 160 mg of the allopurinol sample to be tested, place it in a headspace bottle, measure 2 ml of dimethyl sulfoxide, place it in the headspace bottle, cover and seal, shake in an 80°C water bath to dissolve, then add 2 ml of non-polar solvent, let it stand in ice water for 30 minutes, centrifuge, and take the supernatant to obtain the test solution;

[0012] S4. Detection: Set the chromatographic conditions and send each solution to the gas chromatograph for detection in turn.

[0013] As a further improvement of the above technical solution: the non-polar solvent is dichloromethane.

[0014] As a further improvement of the above technical solution: the chromatographic conditions are:

[0015] Carrier gas: helium;

[0016] Detector: FID;

[0017] Flow rate: 3ml / min;

[0018] Injection volume: 2 μl;

[0019] Split ratio: 5:1;

[0020] Inlet temperature: 225°C;

[0021] Detector temperature: 260°C;

[0022] Heating program: starting temperature 45°C, maintain for 2 minutes, then increase the temperature to 55°C at a rate of 2°C per minute, then increase the temperature to 220°C at a rate of 20°C per minute, and maintain for 5 minutes;

[0023] Chromatographic column: VF-WAXms, 30m*0.25mm, 1.0μm or other chromatographic columns with equivalent performance.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The detection method of formamide residue in allopurinol disclosed in the present invention utilizes the difference in solubility of allopurinol and formamide in different solvents, selects and uses a solvent that is easily soluble in formamide, but is insoluble in allopurinol. The allopurinol sample is first dissolved in a dimethyl sulfoxide solvent at a high concentration, and then a small amount of dichloromethane solvent is added and the temperature is lowered to precipitate allopurinol. The precipitated allopurinol is removed by centrifugation, and the final obtained test solution contains only residual formamide to be tested (if any). This method can not only increase the amount of target material entering the chromatographic system, but also reduce the interference of the excessively high allopurinol sample concentration on the chromatographic system, thereby solving the problem of low sensitivity of the detection method of formamide residue in allopurinol. And the selection of helium as a carrier gas can further improve the sensitivity of the detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The figure is a spectrum of the formamide reference solution in Example 6 of the detection method for formamide residue in allopurinol of the present invention.

[0027] Figure 2 This is a spectrum of the allopurinol test solution of Example 6 in the method for detecting formamide residue in allopurinol of the present invention. DETAILED DESCRIPTION

[0028] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples, but the protection scope of the present invention is not limited thereby. The materials and instruments used in the following examples are all commercially available products.

[0029] Embodiment 1:

[0030] 1. Experimental equipment

[0031] Gas chromatograph, analytical balance

[0032] 2. Chromatographic conditions

[0033] Carrier gas: nitrogen;

[0034] Detector: FID;

[0035] Flow rate: 3ml / min;

[0036] Injection volume: 2 μl;

[0037] Split ratio: 5:1;

[0038] Inlet temperature: 225°C;

[0039] Detector temperature: 260°C;

[0040] Heating program: starting temperature 45°C, maintain for 2 minutes, then increase the temperature to 55°C at a rate of 2°C per minute, then increase the temperature to 220°C at a rate of 20°C per minute, and maintain for 5 minutes;

[0041] Chromatographic column: VF-WAXms, 30m*0.25mm, 1.0μm;

[0042] 3. Solution preparation

[0043] Blank solution: dimethyl sulfoxide;

[0044] Prepare the reference stock solution: weigh about 18 mg of formamide, place in a 20 ml volumetric flask, dilute to the mark with dimethyl sulfoxide, and shake well;

[0045] Prepare the reference stock solution: measure 1 ml of the reference stock solution, place it in a 10 ml volumetric flask, dilute to the mark with dimethyl sulfoxide, and shake well; (formamide concentration is about 0.09 mg / ml)

[0046] Prepare the accuracy solution: weigh about 160 mg of pure allopurinol without formamide, place in a headspace bottle, measure 3.6 ml of dimethyl sulfoxide and 0.4 ml of reference accuracy stock solution, place in the headspace bottle, cover and seal, shake in a water bath at 80°C to dissolve, and obtain the accuracy solution. (Allopurinol concentration 40 mg / ml, formamide concentration about 0.009 mg / ml)

[0047] Set the corresponding chromatographic conditions and send each solution to the gas chromatograph for detection in turn.

[0048] Test results: The signal-to-noise ratio of the formamide peak in the accuracy solution chromatogram was 10.6.

[0049] Embodiment 2:

[0050] 1. Experimental equipment

[0051] Gas chromatograph, analytical balance

[0052] 2. Chromatographic conditions

[0053] Carrier gas: helium;

[0054] Detector: FID;

[0055] Flow rate: 3ml / min;

[0056] Injection volume: 2 μl;

[0057] Split ratio: 5:1;

[0058] Inlet temperature: 225°C;

[0059] Detector temperature: 260°C;

[0060] Heating program: starting temperature 45°C, maintain for 2 minutes, then increase the temperature to 55°C at a rate of 2°C per minute, then increase the temperature to 220°C at a rate of 20°C per minute, and maintain for 5 minutes;

[0061] Chromatographic column: VF-WAXms, 30m*0.25mm, 1.0μm;

[0062] 3. Solution preparation

[0063] Blank solution: dimethyl sulfoxide;

[0064] Prepare the reference stock solution: weigh about 18 mg of formamide, place in a 20 ml volumetric flask, dilute to the mark with dimethyl sulfoxide, and shake well;

[0065] Prepare the reference stock solution: measure 1 ml of the reference stock solution, place it in a 10 ml volumetric flask, dilute to the mark with dimethyl sulfoxide, and shake well; (formamide concentration is about 0.09 mg / ml)

[0066] Prepare the accuracy solution: weigh about 160 mg of pure allopurinol without formamide, place in a headspace bottle, measure 3.6 ml of dimethyl sulfoxide and 0.4 ml of reference accuracy stock solution, place in the headspace bottle, cover and seal, shake in a water bath at 80°C to dissolve, and obtain the accuracy solution. (Allopurinol concentration 40 mg / ml, formamide concentration about 0.009 mg / ml)

[0067] Set the corresponding chromatographic conditions and send each solution to the gas chromatograph for detection in turn.

[0068] Test results: The signal-to-noise ratio of the formamide peak in the accuracy solution chromatogram was 15.3.

[0069] The results of Example 1 and Example 2 show that Example 2 uses helium as a carrier gas, which improves the sensitivity and specificity of formamide detection.

[0070] Embodiment three:

[0071] 1. Experimental equipment

[0072] Gas chromatograph, analytical balance

[0073] 2. Chromatographic conditions

[0074] Carrier gas: helium;

[0075] Detector: FID;

[0076] Flow rate: 3ml / min;

[0077] Injection volume: 2 μl;

[0078] Split ratio: 5:1;

[0079] Inlet temperature: 225°C;

[0080] Detector temperature: 260°C;

[0081] Heating program: starting temperature 45°C, maintain for 2 minutes, then increase the temperature to 55°C at a rate of 2°C per minute, then increase the temperature to 220°C at a rate of 20°C per minute, and maintain for 5 minutes;

[0082] Chromatographic column: VF-WAXms, 30m*0.25mm, 1.0μm;

[0083] 3. Solution preparation

[0084] Blank solution: dimethyl sulfoxide;

[0085] Prepare the reference stock solution: weigh about 18 mg of formamide, place in a 20 ml volumetric flask, dilute to the mark with dimethyl sulfoxide, and shake well;

[0086] Prepare the reference stock solution: measure 1 ml of the reference stock solution, place it in a 10 ml volumetric flask, dilute to the mark with dimethyl sulfoxide, and shake well; (formamide concentration is about 0.09 mg / ml)

[0087] Prepare the accuracy solution: weigh about 160 mg of pure allopurinol without formamide, place in a headspace bottle, measure 1.6 ml of dimethyl sulfoxide and 0.4 ml of reference stock solution, place in the headspace bottle, cover and seal, shake in an 80°C water bath to dissolve, then add 2 ml of methanol, let stand in ice water for 30 minutes, centrifuge, and take the supernatant to obtain the accuracy solution. (Formamide concentration is about 0.009 mg / ml)

[0088] Set the corresponding chromatographic conditions and send each solution to the gas chromatograph for detection in turn.

[0089] Test results: The signal-to-noise ratio of the formamide peak in the accuracy solution chromatogram was 12.1.

[0090] Embodiment 4:

[0091] 1. Experimental equipment

[0092] Gas chromatograph, analytical balance

[0093] 2. Chromatographic conditions

[0094] Carrier gas: helium;

[0095] Detector: FID;

[0096] Flow rate: 3ml / min;

[0097] Injection volume: 2 μl;

[0098] Split ratio: 5:1;

[0099] Inlet temperature: 225°C;

[0100] Detector temperature: 260°C;

[0101] Heating program: starting temperature 45°C, maintain for 2 minutes, then increase the temperature to 55°C at a rate of 2°C per minute, then increase the temperature to 220°C at a rate of 20°C per minute, and maintain for 5 minutes;

[0102] Chromatographic column: VF-WAXms, 30m*0.25mm, 1.0μm;

[0103] 3. Solution preparation

[0104] Blank solution: dimethyl sulfoxide;

[0105] Prepare the reference stock solution: weigh about 18 mg of formamide, place in a 20 ml volumetric flask, dilute to the mark with dimethyl sulfoxide, and shake well;

[0106] Prepare the reference stock solution: measure 1 ml of the reference stock solution, place it in a 10 ml volumetric flask, dilute to the mark with dimethyl sulfoxide, and shake well; (formamide concentration is about 0.09 mg / ml)

[0107] Prepare the accuracy solution: weigh about 160 mg of pure allopurinol without formamide, place in a headspace bottle, measure 1.6 ml of dimethyl sulfoxide and 0.4 ml of reference accuracy stock solution, place in the headspace bottle, cover and seal, shake in an 80°C water bath to dissolve, then add 2 ml of ethyl acetate, let stand in ice water for 30 minutes, centrifuge, and take the supernatant to obtain the accuracy solution. (Formamide concentration is about 0.009 mg / ml)

[0108] Set the corresponding chromatographic conditions and send each solution to the gas chromatograph for detection in turn.

[0109] Test results: The signal-to-noise ratio of the formamide peak in the accuracy solution chromatogram was 22.5.

[0110] Embodiment five:

[0111] 1. Experimental equipment

[0112] Gas chromatograph, analytical balance

[0113] 2. Chromatographic conditions

[0114] Carrier gas: helium;

[0115] Detector: FID;

[0116] Flow rate: 3ml / min;

[0117] Injection volume: 2 μl;

[0118] Split ratio: 5:1;

[0119] Inlet temperature: 225°C;

[0120] Detector temperature: 260°C;

[0121] Heating program: starting temperature 45°C, maintain for 2 minutes, then increase the temperature to 55°C at a rate of 2°C per minute, then increase the temperature to 220°C at a rate of 20°C per minute, and maintain for 5 minutes;

[0122] Chromatographic column: VF-WAXms, 30m*0.25mm, 1.0μm;

[0123] 3. Solution preparation

[0124] Blank solution: dimethyl sulfoxide;

[0125] Prepare the reference stock solution: weigh about 18 mg of formamide, place in a 20 ml volumetric flask, dilute to the mark with dimethyl sulfoxide, and shake well;

[0126] Prepare the reference stock solution: measure 1 ml of the reference stock solution, place it in a 10 ml volumetric flask, dilute to the mark with dimethyl sulfoxide, and shake well; (formamide concentration is about 0.09 mg / ml)

[0127] Prepare the accuracy solution: weigh about 160 mg of pure allopurinol without formamide, place in a headspace bottle, measure 1.6 ml of dimethyl sulfoxide and 0.4 ml of reference stock solution, place in the headspace bottle, cover and seal, shake in an 80°C water bath to dissolve, then add 2 ml of dichloromethane, let stand in ice water for 30 minutes, centrifuge, and take the supernatant to obtain the accuracy solution. (Formamide concentration is about 0.009 mg / ml)

[0128] Set the corresponding chromatographic conditions and send each solution to the gas chromatograph for detection in turn.

[0129] Test results: The signal-to-noise ratio of the formamide peak in the accuracy solution chromatogram was 41.3.

[0130] The results of Examples 3 to 5 show that in Example 5, allopurinol is precipitated by adding dichloromethane solvent and lowering the temperature, and the precipitated allopurinol is removed by centrifugation. The final solution contains only formamide, thereby increasing the amount of target substance entering the chromatographic system and reducing the interference of excessive allopurinol sample concentration on the chromatographic system, thereby improving the sensitivity of formamide detection.

[0131] According to Example 5, a method for detecting formamide residue in allopurinol is obtained, comprising the following steps:

[0132] S1. Prepare the reference substance stock solution: weigh about 18 mg of formamide, place it in a 20 ml volumetric flask, dilute to the mark with dimethyl sulfoxide, and shake well;

[0133] S2. Prepare reference solution: Take 1 ml of reference stock solution, place in a 100 ml volumetric flask, dilute to scale with dimethyl sulfoxide, and shake well; (formamide concentration is about 0.009 mg / ml)

[0134] S3. Prepare the test solution: weigh about 160 mg of the allopurinol sample to be tested, place it in a headspace bottle, measure 2 ml of dimethyl sulfoxide, place it in the headspace bottle, cover and seal, shake in an 80°C water bath to dissolve, then add 2 ml of dichloromethane, let it stand in ice water for 30 minutes, centrifuge, and take the supernatant to obtain the test solution;

[0135] S4. Detection: Set the chromatographic conditions and send each solution to the gas chromatograph for detection in turn.

[0136] Further, the chromatographic conditions in step S4 are:

[0137] Carrier gas: helium;

[0138] Detector: FID;

[0139] Flow rate: 3ml / min;

[0140] Injection volume: 2 μl;

[0141] Split ratio: 5:1;

[0142] Inlet temperature: 225°C;

[0143] Detector temperature: 260°C;

[0144] Heating program: starting temperature 45°C, maintain for 2 minutes, then increase the temperature to 55°C at a rate of 2°C per minute, then increase the temperature to 220°C at a rate of 20°C per minute, and maintain for 5 minutes;

[0145] Chromatographic column: VF-WAXms, 30m*0.25mm, 1.0μm or other chromatographic columns with equivalent performance.

[0146] Further, the injection procedure and system suitability in step S4 are shown in Table 1, wherein the blank solution is dimethyl sulfoxide; the program control solution is the reference solution;

[0147] Table 1: Injection procedures and system suitability requirements

[0148] name Injection times System suitability requirements blank solution At least 1 time The blank solution has no interference with the determination of the target solvent peak Reference solution 6 times The RSD of the target solvent peak area in the chromatogram of the reference solution injected 6 times continuously is ≤10.0% Test solution 1 1 time - Test solution 2 1 time - … 1 time - Test solution n 1 time - Program Control Solution 1 time The reference solution and all program control solutions were injected 6 times continuously, and the RSD of the target solvent peak area was ≤15.0%

[0149] Embodiment six:

[0150] According to the above-mentioned method for detecting formamide residues in allopurinol, the experiment was carried out. The spectrum of the formamide reference solution is shown in the attached figure. Figure 1 The spectrum of the allopurinol test solution is shown in the attached Figure 2 As shown, attached Figure 1 It shows that formamide in the formamide reference solution was detected. Figure 2 This indicates that formamide was not detected in the allopurinol test solution.

[0151] The detection method of formamide residue in allopurinol disclosed in the present invention utilizes the difference in solubility of allopurinol and formamide in different solvents, selects and uses a solvent that is easily soluble in formamide, but is insoluble in allopurinol. The allopurinol sample is first dissolved in a dimethyl sulfoxide solvent at a high concentration, and then a small amount of dichloromethane solvent is added and the temperature is lowered to precipitate allopurinol. The precipitated allopurinol is removed by centrifugation, and the final obtained test solution contains only residual formamide to be tested (if any). This method can not only increase the amount of target material entering the chromatographic system, but also reduce the interference of the excessively high allopurinol sample concentration on the chromatographic system, thereby solving the problem of low sensitivity of the detection method of formamide residue in allopurinol. And the selection of helium as a carrier gas can further improve the sensitivity of the detection.

[0152] Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the technical content disclosed above without departing from the scope of the technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention should fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for detecting formamide residue in allopurinol, characterized in that: The following steps are involved: Prepare the reference stock solution: weigh about 18 mg of formamide, place in a 20 ml volumetric flask, dilute to the mark with dimethyl sulfoxide, and shake well; Prepare reference solution: take 1 ml of reference stock solution, place in a 100 ml volumetric flask, dilute to scale with dimethyl sulfoxide, and shake well; Prepare the test solution: weigh about 160 mg of the allopurinol sample to be tested, place it in a headspace bottle, measure 2 ml of dimethyl sulfoxide, place it in the headspace bottle, cover and seal, shake in an 80°C water bath to dissolve, then add 2 ml of non-polar solvent, let it stand in ice water for 30 minutes, centrifuge, and take the supernatant to obtain the test solution; Detection: Set the chromatographic conditions and send each solution into the gas chromatograph for detection in turn.

2. The method for detecting residual formamide in allopurinol according to claim 1, wherein: The non-polar solvent is dichloromethane.

3. The method for detecting residual formamide in allopurinol according to claim 1, wherein: The chromatographic conditions are: Carrier gas: helium; Detector: FID; Flow rate: 3ml / min; Injection volume: 2 μl; Split ratio: 5:1; Inlet temperature: 225°C; Detector temperature: 260°C; Heating program: starting temperature 45°C, maintain for 2 minutes, then increase the temperature to 55°C at a rate of 2°C per minute, then increase the temperature to 220°C at a rate of 20°C per minute, and maintain for 5 minutes; Chromatographic column: VF-WAXms, 30m*0.25mm, 1.0μm or other chromatographic columns with equivalent performance.

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