Method for detecting N, N-dimethylaniline in cefazolin sodium and application

Abstract

The invention discloses a method for detecting N, N-dimethylaniline in cefazolin sodium, which comprises the following steps: respectively injecting a blank solvent, an internal standard solution, a reference substance solution and each test solution into a gas chromatograph by adopting headspace sampling, recording chromatograms, and carrying out quantitative analysis on the chromatograms by adopting an internal standard method, wherein a chromatographic column is a capillary column which takes 6 percent of cyanopropyl phenyl-94 percent of dimethyl siloxane as a stationary liquid, or a capillary chromatographic column with equivalent efficiency; a detector is a flame ionization detector (FID); the heating procedure is as follows: the column temperature is 120 DEG C, and the temperature is maintained for 30 minutes. The detection method provided by the invention has good specificity and system adaptability, good sample injection precision, extremely low detection limit and quantitation limit, good durability, good linearity, good accuracy and extremely high sensitivity, and can be widely applied to quality control of the cefazolin sodium product.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to a detection method and application of N,N-dimethylaniline in cefazolin sodium. Background Art

[0002] The chemical name of cefazolin sodium is (6R,7R)-3-[[(5-methyl-1,3,4-thiadiazol-2-yl)thio]methyl]-7-[(1H-tetrazol-1-yl)acetylamino]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid sodium salt. It is a first-generation cephalosporin with a broad antibacterial spectrum and is used to treat a variety of bacterial infections. The clinical indications for listing are: 1. It is suitable for the treatment of respiratory tract infections such as otitis media, bronchitis, pneumonia, urinary tract infections, skin and soft tissue infections, bone and joint infections, sepsis, infective endocarditis, hepatobiliary system infections, and eye, ear, nose, and throat infections caused by sensitive bacteria. 2. This product can also be used as a preventive drug before surgery. 3. This product should not be used for central nervous system infections. The efficacy is poor for chronic urinary tract infections, especially those with urinary tract anatomical abnormalities. This product should not be used to treat gonorrhea and syphilis.

[0003] Organic solvents such as N,N-dimethylaniline are used in the production of cefazolin sodium raw materials. Since residual solvents are impurities in drugs that are harmful to the human body, their content should be controlled within a limited range during the production process.

[0004] At present, the United States Pharmacopoeia and the Chinese Pharmacopoeia (2020 edition) only include the quality standards of cefazolin sodium, but neither includes the detection method and standard for the residual solvent N,N-dimethylaniline in cefazolin sodium. In order to more comprehensively detect the content of residual solvent N,N-dimethylaniline in cefazolin sodium, it is necessary to establish a detection method for N,N-dimethylaniline in cefazolin sodium. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the defect of the prior art that there is a lack of a detection method for the residual solvent of N,N-dimethylaniline contained in the raw material of cefazolin sodium, and to provide a detection method and application of N,N-dimethylaniline in cefazolin sodium. The detection method of the present invention can realize the detection of N,N-dimethylaniline in cefazolin sodium. The methodological verification shows that the detection method of the present invention has good specificity and system adaptability, good injection precision, extremely low detection limit and quantitative limit, good durability, good linearity and good accuracy, and the method has extremely high sensitivity, and can be widely used in the quality control of cefazolin sodium products.

[0006] The invention provides a method for detecting N,N-dimethylaniline in cefazolin sodium, which comprises the following steps: injecting an internal standard solution, a reference solution, and each test solution into a gas chromatograph by headspace injection, recording a chromatogram, and performing quantitative analysis on the chromatogram by an internal standard method;

[0007] Among them, the detection conditions of the chromatography are:

[0008] Instrument: Gas chromatograph;

[0009] Chromatographic column: a capillary column with 6% cyanopropylphenyl-94% dimethylsiloxane as the stationary phase, or a capillary column with equivalent performance;

[0010] Detector: hydrogen flame ionization detector (FID); carrier gas: nitrogen;

[0011] Inlet temperature: 200°C; Detector temperature: 250°C;

[0012] Temperature program: column temperature is 120°C, maintained for 30 minutes;

[0013] Flow rate: 3ml / min; split flow rate: 5ml / min;

[0014] Injection volume: 1μl; Hydrogen: 40ml / min;

[0015] Air: 400ml / min; Nitrogen: 40ml / min.

[0016] Solution preparation:

[0017] In the present invention, the blank solvent is cyclohexane.

[0018] In the present invention, the preparation method of the internal standard solution preferably comprises the following steps: weigh 50 mg of naphthalene, accurately weigh it, place it in a 50 ml volumetric bottle, add cyclohexane to dissolve and dilute it to the scale, shake it well, take 5 ml of the solution, place it in a 100 ml volumetric bottle, dilute it to the scale with cyclohexane, shake it well, and obtain it.

[0019] In the present invention, the preparation method of the reference substance stock solution preferably includes the following steps: take 50 mg of N,N-dimethylaniline, accurately weigh it, place it in a 50 ml volumetric bottle, add 25 ml of 1 mol / L hydrochloric acid and vortex to dissolve, dilute to the scale with water, shake well, accurately measure 2 ml and place it in a 100 ml volumetric bottle, dilute to the scale with water, and shake well.

[0020] In the present invention, the preparation method of the reference substance solution preferably comprises the following steps: accurately measuring 1 ml of the reference substance stock solution and placing it in a centrifuge tube, adding 5 ml of 1 mol / L sodium hydroxide solution and 1 ml of the internal standard solution, tightly capping the stopper, vigorously shaking for 1 minute, centrifuging, and taking the supernatant.

[0021] Or, in the present invention, the preparation method of the reference solution preferably includes the following steps: take 50 mg of N,N-dimethylaniline, accurately weigh it, put it in a 50 ml volumetric bottle, add 25 ml of 1 mol / L hydrochloric acid, vortex to dissolve, dilute to scale with water, shake well, accurately measure 2 ml and put it in a 100 ml volumetric bottle, dilute to scale with water, shake well. Accurately measure 1 ml and put it in a centrifuge tube, add 5 ml of 1 mol / L sodium hydroxide solution and 1 ml of internal standard solution, cover the stopper, shake vigorously for 1 minute, centrifuge, and take the supernatant.

[0022] In the present invention, the preparation method of the test solution preferably includes the following steps: taking about 1.0 g of cefazolin sodium raw material, accurately weighing, placing in a centrifuge tube, adding 5 ml of 1 mol / L sodium hydroxide solution, vortexing to dissolve, adding 1 ml of internal standard solution, capping the stopper, vigorously shaking for 1 minute, centrifuging, and taking the supernatant.

[0023] In the present invention, the preparation method of the sample-added test solution preferably includes the following steps: taking about 1.0 g of cefazolin sodium raw material, accurately weighing, placing in a centrifuge tube, adding 4 ml of 1 mol / L sodium hydroxide solution, vortexing to dissolve, adding 1 ml of reference substance stock solution, adding 1 ml of internal standard solution, covering the stopper tightly, shaking vigorously for 1 minute, centrifuging, and taking the supernatant.

[0024] In a certain embodiment, the chromatographic column is preferably DB-624 or a capillary chromatographic column with equivalent performance. The specification of the chromatographic column is preferably 30m×0.53mm×3.00μm.

[0025] In a certain embodiment, the flow rate ranges from 2.5 ml / min to 3.5 ml / min, preferably 3.0 ml / min.

[0026] In one embodiment, the column temperature ranges from 115°C to 125°C, preferably 120°C.

[0027] In one embodiment, the detectors are both hydrogen flame ionization detectors.

[0028] in,

[0029] Internal standard method calculation formula:

[0030]

[0031]

[0032] Where: Wr: weight of N,N-dimethylaniline, mg

[0033] Pr: N,N-dimethylaniline content

[0034] Sr: dilution multiple of reference solution

[0035] S: Volume of reference stock solution added to the test solution, ml

[0036] W 内 : Weight of naphthalene, mg

[0037] P 内 :Naphthalene content

[0038] Ar: Peak area of ​​N,N-dimethylaniline in reference solution

[0039] A 内 : Peak area of ​​naphthalene in reference solution

[0040] S 内 : Dilution factor of naphthalene

[0041] As: Peak area of ​​N,N-dimethylaniline in the test solution

[0042] A S内 : Peak area of ​​naphthalene in the test solution

[0043] f: Correlation coefficient between N,N-dimethylaniline and naphthalene in the reference solution

[0044] W s : The weight of the test sample, mg.

[0045] The detection method of the present invention, the results of the method verification show that:

[0046] (1) Specificity and injection precision: The blank solvent (cyclohexane) and the internal standard solution (naphthalene) did not interfere with the determination of the main component; the peak order of the reference solution was: cyclohexane, N,N-dimethylaniline, naphthalene; the separation degree between N,N-dimethylaniline and other adjacent chromatographic peaks was 29.57 / 20.33, greater than 1.5; the reference solution was measured 6 times continuously, and the RSD of the correction factor was 0.66%, less than 3.0%. Conclusion: The method has good specificity and injection precision.

[0047] (2) Linearity and range: Concentration range: 2.013 μg / ml to 30.193 μg / ml, linear equation: y = 4224713.6x + 1662279.9, correlation coefficient r is 0.9999, ratio is 1.93%. Conclusion: The linear relationship is good.

[0048] (3) Limit of quantification: The limit of quantification is 2.013 ng, which is equivalent to 0.0002% of the test sample concentration. Six injections were made continuously, and the S / N was greater than 10. The RSD of the peak area was 3.07%.

[0049] (4) Detection limit: The detection limit is 0.604ng, which is equivalent to 0.00006% of the test sample concentration. After three consecutive injections, the S / N was greater than 3, and the RSD of the peak area was 1.94%. Conclusion: This method has high sensitivity.

[0050] (5) Accuracy: When the concentration of N,N-dimethylaniline was within the range of 10% to 150% of the limit concentration, the recovery calculated by the external standard method was within the range of 105.5% to 116.5%, with an RSD of 3.29%; the recovery calculated by the internal standard method was within the range of 103.5% to 116.4%, with an RSD of 4.09%, both less than 10.0%. Conclusion: This method has high accuracy.

[0051] (6) Precision: 1) Repeatability: The RSD of the measured amount of N,N-dimethylaniline is 0.00%; 2) Intermediate precision: The RSD of the measured amount of N,N-dimethylaniline is 0.00%. Conclusion: The method has good precision.

[0052] (7) Solution stability: After being placed at room temperature for 8 hours, the reference solution: the RSD of the N,N-dimethylaniline peak area was 3.22%, less than 10.0%; no N,N-dimethylaniline peak was detected in the test solution. Conclusion: The reference solution and the test solution are stable within 8 hours at room temperature.

[0053] (8) Durability: When the determination parameters change slightly, the peak order of the reference solution is: cyclohexane, N,N-dimethylaniline, naphthalene; the separation degree of N,N-dimethylaniline and other adjacent chromatographic peaks is greater than 1.5; the RSD of the measured amount of N,N-dimethylaniline in the test sample is: 0.00%. Conclusion: The method has good durability.

[0054] The present invention also provides an application of the above-mentioned method for detecting N,N-dimethylaniline in cefazolin sodium to detecting the content of N,N-dimethylaniline in a cefazolin sodium raw material or preparation.

[0055] In the present invention, the term "external standard method" refers to a method of using a pure product of the component to be measured as a reference substance and comparing the response signals of the reference substance and the component to be measured in the sample for quantitative analysis.

[0056] In the present invention, the term "internal standard method" refers to a method of selecting a suitable substance as a reference substance for the component to be measured, adding it to the sample in a quantitative manner, and performing quantitative analysis based on the ratio of the response values ​​(peak area or peak height) of the component to be measured and the reference substance on the detector and the amount of the reference substance added.

[0057] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0058] The reagents and raw materials used in the present invention are commercially available.

[0059] The positive and progressive effects of the present invention are:

[0060] (1) The present invention can simply, quickly and accurately detect the residual amount of N,N-dimethylaniline in cefazolin sodium.

[0061] (2) The present invention has good specificity. The blank solvents cyclohexane and naphthalene do not interfere with the determination of the main component. The peak order of the reference solution is: cyclohexane, N,N-dimethylaniline, naphthalene. The separation degree between N,N-dimethylaniline and other adjacent chromatographic peaks is 29.57 / 20.33, which is greater than 1.5. The results show that the method has good specificity. The reference solution was taken for continuous determination 6 times, and the RSD of the correction factor was 0.66%, which was less than 3.0%. The results show that the method has good injection precision.

[0062] (3) The present invention has good system adaptability, and the RSD and tailing factor of the peak area meet the requirements.

[0063] (4) The method described in the present invention has high sensitivity and extremely low detection limit and quantification limit. The detection limit is 0.604 ng, which is equivalent to 0.00006% of the test sample concentration. The quantification limit is 2.013 ng, which is equivalent to 0.0002% of the test sample concentration.

[0064] (5) The method described in the present invention has good linearity.

[0065] (6) The method of the present invention has good accuracy. When the concentration of N,N-dimethylaniline is within the range of 10% to 150% of the limit concentration, the recovery rate calculated by the external standard method is within the range of 105.5% to 116.5%, and the RSD is 3.29%; the recovery rate calculated by the internal standard method is within the range of 103.5% to 116.4%, and the RSD is 4.09%, both of which are less than 10.0%.

[0066] (8) The method described in the present invention has good durability. When the determination parameters change slightly, the peak order of the reference solution is: cyclohexane, N,N-dimethylaniline, naphthalene; the separation degree of N,N-dimethylaniline and other adjacent chromatographic peaks is greater than 1.5; the RSD of the measured amount of N,N-dimethylaniline in the test sample is: 0.00%. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 This is the GC spectrum of the sample solution added in Example 4 (entering the first needle).

[0068] Figure 2 This is the GC spectrum of the sample solution added in Example 4 (entering the fourth needle).

[0069] Figure 3This is the GC spectrum of the test solution in Example 5.

[0070] Figure 4 This is the GC spectrum of the test solution added in Example 5.

[0071] Figure 5 This is the linear correlation diagram of N,N-dimethylaniline in Example 6.

[0072] Figure 6 This is the GC spectrum of N,N-dimethylaniline-method verification-durability (flow rate: 2.5 ml / min)-test solution in Example 6.

[0073] Figure 7 This is the GC spectrum of N,N-dimethylaniline - method verification - durability (flow rate: 3.5 ml / min) - test solution in Example 6.

[0074] Figure 8 This is the GC spectrum of N,N-dimethylaniline-method verification-durability (column temperature: 115°C)-test solution in Example 6.

[0075] Fig. 9 This is the GC spectrum of N,N-dimethylaniline-method verification-durability (column temperature: 125°C)-test solution in Example 6.

[0076] Fig.10 This is the GC spectrum of the N,N-dimethylaniline-sample measurement-reference solution in Example 6.

[0077] Fig.11 This is the GC spectrum of the N,N-dimethylaniline-sample determination-test sample (Batch K2502103012) solution in Example 6.

[0078] Fig.12 This is the GC spectrum of the N,N-dimethylaniline-sample determination-test sample (Batch K2502103022) solution in Example 6.

[0079] Fig.13 This is the GC spectrum of the N,N-dimethylaniline-sample determination-test sample (K2501905012 batch) solution in Example 6. DETAILED DESCRIPTION

[0080] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0081] Test sample: Cefazolin sodium API (production location: North China Pharmaceutical Hebei Huamin Pharmaceutical Co., Ltd.; batch number: K2502103022 (production date 2021.03.18), K2502103012 (production date 2021.03.17), K2501905012 (production date 2019.04.27)). Reference substance: N,N-dimethylaniline (batch number: ZVMDALS, content: 99.0%, source manufacturer: TGI), naphthalene (batch number: AL180926-17, content: 99.48%, source manufacturer: Shenzhen Botaitong).

[0082] Instruments: gas chromatograph (model Trace1300, manufacturer: Thermo), electronic balance (model ME204T and model XS105DU, both manufactured by: Mettler-Toledo Instrument Co., Ltd.).

[0083] The reagents in the present invention are all analytically pure: cyclohexane (manufacturer: Tianjin Fuyu), sodium hydroxide (manufacturer: Tianjin Guangfu), hydrochloric acid (manufacturer: Beijing Chemical Plant), purified water (self-made).

[0084] Example 1 Establishment of the detection method - first method development

[0085] Determined by reference to gas chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Rules 0521).

[0086] Chromatographic column: a capillary column with 6% cyanopropylphenyl-94% dimethylsiloxane as the stationary phase (DB-624, 30m×0.53mm×3.00μm, or a chromatographic column with similar polarity);

[0087] Detector: FID; Carrier gas: nitrogen;

[0088] Inlet temperature: 200°C; Detector temperature: 250°C;

[0089] Temperature program: column temperature is 120°C, maintained for 20 minutes;

[0090] Flow rate: 2ml / min; split flow rate: 10ml / min;

[0091] Injection volume: 1μl; Hydrogen: 40ml / min;

[0092] Air: 400ml / min; Nitrogen: 40ml / min;

[0093] Solution preparation:

[0094] Blank solvent 1: Take 5 ml of anhydrous ethanol and place it in a 50 ml volumetric flask. Dilute it to the mark with methanol-water (60:40) and shake well.

[0095] Blank solvent 2: methanol-water (60:40).

[0096] Reference substance positioning solution: Weigh about 0.02g of N,N-dimethylaniline, accurately weigh it, place it in a 20ml volumetric flask, add anhydrous ethanol to dissolve and dilute to the scale, shake well, and obtain.

[0097] Reference solution: Take 5 ml of reference solution and place it in a 50 ml volumetric flask. Add methanol-water (60:40) to dilute to the scale and shake well.

[0098] Take 1 μl of blank solvent 1, blank solvent 2 and control solution, respectively, inject them into the gas chromatograph, and record the chromatogram.

[0099] Conclusion: The peaks of blank solvent 1 and blank solvent 2 were both between 0 and 4 min, and the control solution did not have a peak. The subsequent experiment of extending the column temperature maintenance time was carried out.

[0100] Example 2 Establishment of the detection method - Development of the second method

[0101] Referring to the experimental method of Example 1, only the temperature rising program was modified: the column temperature was 120° C., maintained for 60 minutes, and other conditions remained unchanged.

[0102] Solution: Prepare the solution using the experimental method of Example 1.

[0103] Take 1 μl of the reference solution and reference solution, respectively, and inject them into the gas chromatograph to record the chromatogram. The results are shown in Table 1.

[0104] Table-1 Method development results of Example 2

[0105] name Rt(min) Peak area (A) Tailing Factor Theoretical plate number Reference solution 23.804 2003470892 0.97 37574 Reference solution 23.802 199697361 1.02 28858

[0106] Conclusion: The results show that the peak time of N,N-dimethylaniline in this method is relatively late and the peak shape is good. The method will be initially tested for its robustness in the future.

[0107] Example 3 Establishment of the detection method - Development of the third method

[0108] Preliminary durability: refer to the experimental method of Example 1, and modify some of the conditions:

[0109] (1) Modify the heating program: the column temperature is 125°C for 30 minutes, and other conditions remain unchanged.

[0110] (2) Modify the heating program: the column temperature is 130°C, maintained for 30 min, and other conditions remain unchanged.

[0111] (3) Change the split flow rate to 20 ml / min; maintain the column temperature for 30 minutes, and keep other conditions unchanged.

[0112] (4) Change the flow rate to 3 ml / min; maintain the column temperature for 30 min, and keep other conditions unchanged.

[0113] Solution: Prepare the solution using the experimental method of Example 1.

[0114] Take 1 μl of the reference solution and inject it into the gas chromatograph under different chromatographic conditions. Record the chromatogram. The results are shown in Table 2.

[0115] Table-2 Method development results of Example 3

[0116] condition Rt(min) Peak area (A) Tailing Factor Theoretical plate number Column temperature: 125°C 20.449 198540821 1.03 25436 Column temperature: 130°C 17.715 201929200 0.98 23641 Split flow rate: 20ml / min 23.799 101874707 1.02 27694 Flow rate: 3ml / min 16.108 284789532 1.01 17667

[0117] Conclusion: The results show that the peak shape of each condition is good, and the peak time is more suitable when the flow rate is 3ml / min, so this condition is temporarily selected for preliminary verification.

[0118] Example 4 Establishment of the detection method - fourth method development

[0119] Analysis method: Determined by gas chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Rules 0521).

[0120] Chromatographic column: a capillary column with 6% cyanopropylphenyl-94% dimethylsiloxane as the stationary phase (DB-624, 30m×0.53mm×3.00μm, or a chromatographic column with similar polarity);

[0121] Detector: FID; Carrier gas: nitrogen;

[0122] Inlet temperature: 200°C; Detector temperature: 250°C;

[0123] Temperature program: column temperature is 120°C, maintained for 30 minutes;

[0124] Flow rate: 3ml / min; split flow rate: 10ml / min;

[0125] Injection volume: 1μl; Hydrogen: 40ml / min;

[0126] Air: 400ml / min; Nitrogen: 40ml / min;

[0127] Solution preparation: The solution was prepared using the experimental method of Example 1.

[0128] Test solution: Take 1.0 g of this product (Batch K2501905012), place it in a 20 ml volumetric flask, add methanol-water (60:40) to dissolve and dilute to the scale, shake well, and obtain.

[0129] Low-concentration test solution: Take 1.0 g of this product (Batch K2501905012), place it in a 20 ml volumetric flask, add an appropriate amount of methanol-water (60:40) to dissolve, add 1 ml of reference solution, add methanol-water (60:40) and dilute to the scale, shake well, and obtain.

[0130] Medium concentration test solution: Take 1.0g of this product (Batch K2501905012), place it in a 20ml volumetric flask, add an appropriate amount of methanol-water (60:40) to dissolve, add 2ml of reference solution, add methanol-water (60:40) and dilute to the scale, shake well, and obtain.

[0131] High concentration test solution: Take 1.0g of this product (Batch K2501905012), place it in a 20ml volumetric flask, add an appropriate amount of methanol-water (60:40) to dissolve, add 3ml of reference solution, add methanol-water (60:40) and dilute to the scale, shake well, and obtain.

[0132] Take 1 μl of each test solution and inject it into the gas chromatograph, record the chromatogram, and the results are shown in Table 3.

[0133] Table-3 Method development results of Example 4

[0134]

[0135] Conclusion: The results show that N,N-dimethylaniline was not detected in this product, and the sample recovery rate RSD was 4.2%, which met the requirements. However, observation of the spectrum revealed that the baseline of the test solution fluctuated after continuous injection, and it was suspected that the sample solution was not completely vaporized, which may affect the subsequent sample determination. Therefore, repeated injection experiments of the test solution are required.

[0136] Considering that the limit of N,N-dimethylaniline is relatively low, the split flow rate in the method is adjusted to 5 ml / min; injection volume: 2 μl; other conditions remain unchanged.

[0137] Add sample to test solution: Take 1.0g of this product (Batch K2501905012), place it in a 10ml volumetric flask, add appropriate amount of methanol-water (60:40) to dissolve, add 1ml of reference solution, add methanol-water (60:40) and dilute to the scale, shake well, and obtain.

[0138] Take 1 μl of the test solution and the sample solution, inject them into the gas chromatograph, and record the chromatogram. Figure 1-2 .

[0139] Conclusion: N,N-dimethylaniline was not detected in the test solution, and the baseline was stable. After continuous sampling of the test solution, it was found that there were impurity peaks before and after the N,N-dimethylaniline peak, and bulging appeared. When the fourth injection was made, the abnormal peak had affected N,N-dimethylaniline. This method is not feasible and further exploration is needed.

[0140] Example 5 Establishment of the detection method - Fifth method development

[0141] Analysis method: Determined by gas chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Rules 0521).

[0142] Chromatographic column: a capillary column with 6% cyanopropylphenyl-94% dimethylsiloxane as the stationary phase (DB-624, 30m×0.53mm×3.00μm, or a chromatographic column with similar polarity);

[0143] Detector: FID; Carrier gas: nitrogen;

[0144] Inlet temperature: 200°C; Detector temperature: 250°C;

[0145] Temperature program: column temperature is 120°C, maintained for 30 minutes;

[0146] Flow rate: 3ml / min; split flow rate: 5ml / min;

[0147] Injection volume: 1μl; Hydrogen: 40ml / min;

[0148] Air: 400ml / min; Nitrogen: 40ml / min;

[0149] Solution preparation:

[0150] Blank solvent 1: cyclohexane.

[0151] Blank solvent 2: Weigh 50 mg of naphthalene accurately, place in a 50 ml volumetric flask, add cyclohexane to dissolve and dilute to the scale, shake well, take 5 ml of the solution, place in a 100 ml volumetric flask, dilute to the scale with cyclohexane, shake well, and obtain.

[0152] Reference substance stock solution: Take 50 mg of N,N-dimethylaniline, accurately weigh it, place it in a 50 ml volumetric flask, add 25 ml of 1 mol / L hydrochloric acid and vortex to dissolve, dilute to the scale with water, shake well, accurately measure 2 ml and place it in a 100 ml volumetric flask, dilute to the scale with water, and shake well.

[0153] Reference substance solution: Accurately measure 1 ml of reference substance stock solution and place it in a centrifuge tube. Add 5 ml of 1 mol / L sodium hydroxide solution and 1 ml of internal standard solution. Close the stopper tightly, shake vigorously for 1 minute, centrifuge, and take the supernatant.

[0154] Test solution: Take about 1.0 g of the product, weigh accurately, place in a centrifuge tube, add 5 ml of 1 mol / L sodium hydroxide solution, vortex to dissolve, add 1 ml of internal standard solution, cover the stopper tightly, shake vigorously for 1 minute, centrifuge, and take the supernatant.

[0155] Add test solution: Take about 1.0g of the product, weigh accurately, put it in a centrifuge tube, add 4ml of 1mol / L sodium hydroxide solution, vortex to dissolve, add 1ml of reference stock solution, add 1ml of internal standard solution, cover the stopper tightly, shake vigorously for 1 minute, centrifuge, and take the supernatant.

[0156] Take 1 μl of blank solvent 1, blank solvent 2, control solution, test solution and sampled test solution, inject them into the gas chromatograph and record the chromatogram. The results are shown in Table 4. Figure 3-4 .

[0157] Table-4 Method development results of Example 5

[0158]

[0159]

[0160] Conclusion: The results show that N,N-dimethylaniline was not detected in this product. The RSD of the peak area of ​​N,N-dimethylaniline in the sample was 1.68%, which met the requirements. No unknown impurity peaks were found in the continuous injection of the sample, and the results were good. This method was used for subsequent verification.

[0161] Example 6 Verification Example - Methodology Verification

[0162] The specific methodological validation contents are shown in Table 5.

[0163] Table-5 Summary of N,N-dimethylaniline method validation results

[0164]

[0165]

[0166] Method Validation:

[0167] (1) Specificity and injection precision

[0168] Blank solvent: cyclohexane.

[0169] Internal standard solution: Weigh 50 mg of naphthalene, accurately weigh it, place it in a 50 ml volumetric flask, add cyclohexane to dissolve and dilute to the scale, shake well, take 5 ml of the solution, place it in a 100 ml volumetric flask, dilute to the scale with cyclohexane, shake well, and you have it.

[0170] Reference solution: Take 50 mg of N,N-dimethylaniline, weigh accurately, place in a 50 ml volumetric flask, add 25 ml of 1 mol / L hydrochloric acid, vortex to dissolve, dilute to scale with water, shake well, accurately measure 2 ml and place in a 100 ml volumetric flask, dilute to scale with water, shake well. Accurately measure 1 ml and place in a centrifuge tube, add 5 ml of 1 mol / L sodium hydroxide solution and 1 ml of internal standard solution, cover the stopper, shake vigorously for 1 minute, centrifuge, and take the supernatant.

[0171] Test solution: Take about 1.0 g of the product, weigh accurately, place in a centrifuge tube, add 5 ml of 1 mol / L sodium hydroxide solution, vortex to dissolve, add 1 ml of internal standard solution, cover the stopper tightly, shake vigorously for 1 minute, centrifuge, and take the supernatant.

[0172] Take 1 μl of blank solvent, internal standard solution, reference solution, and test solution respectively, inject into gas chromatograph, and record chromatogram. Take reference solution as injection precision solution, inject 6 injections continuously, and record chromatogram. The results are shown in Table-6 to Table-7.

[0173] Table-6 N,N-Dimethylaniline method validation-specificity test results

[0174]

[0175] Table-7 N, N-dimethylaniline method validation - injection precision test results

[0176]

[0177] (2) Linearity and range

[0178] Internal standard solution: Weigh 50 mg of naphthalene, accurately weigh it, place it in a 50 ml volumetric flask, add cyclohexane to dissolve and dilute to the scale, shake well, take 5 ml of the solution, place it in a 100 ml volumetric flask, dilute to the scale with cyclohexane, shake well, and you have it.

[0179] Linear stock solution: Take 50 mg of N,N-dimethylaniline, accurately weigh it, place it in a 50 ml volumetric flask, add 25 ml of 1 mol / L hydrochloric acid, vortex to dissolve, add water to dilute to the scale, and shake well.

[0180] ① Accurately measure 2 ml of 100% linear solution and place it in a 20 ml volumetric flask, then add water to dilute to the mark as 10% linear solution;

[0181] ② Accurately measure 5 ml of 100% linear solution and place it in a 25 ml volumetric flask, add water to dilute to the mark, and use it as the 20% linear solution;

[0182] ③ Accurately measure 1 ml of the linear stock solution and place it in a 100 ml volumetric flask, then add water to dilute to the mark as the 50% linear solution;

[0183] ④ Accurately measure 2 ml of the linear stock solution and place it in a 100 ml volumetric flask, add water to dilute to the scale, and use it as the 100% linear solution;

[0184] ⑤ Accurately measure 3 ml of the linear stock solution and place it in a 100 ml volumetric flask. Add water to dilute to the scale as the 150% linear solution.

[0185] Accurately measure 1 ml of each of the above linear solutions and place them in different centrifuge tubes, add 5 ml of 1 mol / L sodium hydroxide solution and 1 ml of internal standard solution to each tube, cover the stopper tightly, shake vigorously for 1 minute, centrifuge, and take the supernatant as the linear solution of each concentration.

[0186] Take 1 μl of each concentration linear solution, inject it into the gas chromatograph, and record the chromatogram. The results are shown in Table 8.

[0187] Table-8 N,N-Dimethylaniline method validation-linearity and range test results

[0188]

[0189] (3) Limit of quantification and detection limit

[0190] Take 10% linear solution under the linearity and range items, operate according to the method, and dilute step by step until the S / N is not less than 10, which is the quantitative limit solution, and the S / N is not less than 3, which is the detection limit solution. Take 1μl of the quantitative limit solution and the detection limit solution respectively, inject them into the gas chromatograph, record the chromatogram, and examine the signal-to-noise ratio. The results are shown in Table-9 to Table-10.

[0191] Table-9 N,N-dimethylaniline method validation-detection limit experimental results

[0192]

[0193]

[0194] Table-10N,N-Dimethylaniline Method Verification-Quantitative Limit Experimental Results

[0195]

[0196] (4) Accuracy

[0197] Internal standard solution: Weigh 50 mg of naphthalene, accurately weigh it, place it in a 50 ml volumetric flask, add cyclohexane to dissolve and dilute to the scale, shake well, take 5 ml of the solution, place it in a 100 ml volumetric flask, dilute to the scale with cyclohexane, shake well, and you have it.

[0198] Test solution: Take about 1.0 g of the product, weigh accurately, place in a centrifuge tube, add 5 ml of 1 mol / L sodium hydroxide solution, vortex to dissolve, add 1 ml of internal standard solution, cover the stopper tightly, shake vigorously for 1 minute, centrifuge, and take the supernatant.

[0199] Reference substance stock solution: Take 50 mg of N,N-dimethylaniline, weigh accurately, place in a 50 ml volumetric flask, add 25 ml of 1 mol / L hydrochloric acid, vortex to dissolve, add water to dilute to the scale, and shake well.

[0200] Reference solution: Take 2 ml of the reference stock solution, place it in a 100 ml volumetric flask, dilute to the mark with water, and shake well. Accurately measure 1 ml and place it in a centrifuge tube, add 5 ml of 1 mol / L sodium hydroxide solution and 1 ml of internal standard solution, cover the stopper tightly, shake vigorously for 1 minute, centrifuge, and take the supernatant.

[0201] Reference substance dilution 1: Take reference substance dilution 2: 5 ml, place in a 50 ml volumetric flask, add water to dilute to the scale, shake well, and obtain.

[0202] Reference substance dilution solution 2: Take 2 ml of reference substance stock solution, place in a 100 ml volumetric flask, add water to dilute to the scale, shake well, and obtain.

[0203] Reference substance dilution solution 3: Take 3 ml of reference substance stock solution, place in a 100 ml volumetric flask, add water to dilute to the scale, shake well, and obtain.

[0204] Low-concentration test solution: Take about 1.0g of the product, weigh accurately, place in a centrifuge tube, add 4ml of 1mol / L sodium hydroxide solution, accurately add 1ml of reference diluent 1, vortex to dissolve, add 1ml of internal standard solution, cover the stopper tightly, shake vigorously for 1 minute, centrifuge, and take the supernatant. Prepare 3 copies in parallel.

[0205] Medium concentration test solution: Take about 1.0g of the product, weigh accurately, place in a centrifuge tube, add 4ml of 1mol / L sodium hydroxide solution, accurately add 1ml of reference diluent 2, vortex to dissolve, add 1ml of internal standard solution, cover the stopper tightly, shake vigorously for 1 minute, centrifuge, and take the supernatant. Prepare 3 copies in parallel.

[0206] High concentration test solution: Take about 1.0g of the product, weigh accurately, place in a centrifuge tube, add 4ml of 1mol / L sodium hydroxide solution, accurately add 1ml of reference diluent 3, vortex to dissolve, add 1ml of internal standard solution, cover the stopper tightly, shake vigorously for 1 minute, centrifuge, and take the supernatant. Prepare 3 copies in parallel.

[0207] Take 1 μl of each of the internal standard solution, reference solution, and low / medium / high concentration test solution, inject into the gas chromatograph, record the chromatogram, and calculate the recovery rate. The accuracy test results are shown in Tables 11 to 13.

[0208] Table-11N,N-Dimethylaniline method verification-accuracy test results (external standard method)

[0209]

[0210]

[0211] Table-12 N,N-dimethylaniline method verification-accuracy test results (internal standard method)

[0212]

[0213] Note: (1) Calculation formula of external standard method:

[0214]

[0215]

[0216]

[0217] Where: As: Peak area of ​​N,N-dimethylaniline in the test solution

[0218] Wr: weight of N,N-dimethylaniline, mg

[0219] Ar: Peak area of ​​N,N-dimethylaniline in reference solution

[0220] Ws: weight of the test sample, mg

[0221] Sr: dilution multiple of reference solution

[0222] Ss: dilution factor of the test solution

[0223] S: Volume of reference stock solution added to the test solution, ml

[0224] Pr: N,N-dimethylaniline content

[0225] (2) Calculation formula of internal standard method:

[0226]

[0227]

[0228]

[0229]

[0230] Where: Wr: weight of N,N-dimethylaniline, mg

[0231] Pr: N,N-dimethylaniline content

[0232] Sr: dilution multiple of reference solution

[0233] S: Volume of reference stock solution added to the test solution, ml

[0234] W 内 : Weight of naphthalene, mg

[0235] P 内 :Naphthalene content

[0236] Ar: Peak area of ​​N,N-dimethylaniline in reference solution

[0237] A 内 : Peak area of ​​naphthalene in reference solution

[0238] S 内 : Dilution factor of naphthalene

[0239] As: Peak area of ​​N,N-dimethylaniline in the test solution

[0240] A S内 : Peak area of ​​naphthalene in the test solution

[0241] f: Correlation coefficient table of N,N-dimethylaniline and naphthalene in reference solution - 13N,N-dimethylaniline method verification - recovery rate experimental results summary table

[0242]

[0243] (5) Repeatability

[0244] Internal standard solution: Weigh 50 mg of naphthalene, accurately weigh it, place it in a 50 ml volumetric flask, add cyclohexane to dissolve and dilute to the scale, shake well, take 5 ml of the solution, place it in a 100 ml volumetric flask, dilute to the scale with cyclohexane, shake well, and you have it.

[0245] Reference substance stock solution: Take 50 mg of N,N-dimethylaniline, weigh accurately, place in a 50 ml volumetric flask, add 25 ml of 1 mol / L hydrochloric acid and vortex to dissolve, dilute to the scale with water, shake well, take 2 ml, place in a 100 ml volumetric flask, dilute to the scale with water, and shake well.

[0246] Reference substance solution: Accurately measure 1 ml of reference substance stock solution and place it in a centrifuge tube. Add 5 ml of 1 mol / L sodium hydroxide solution and 1 ml of internal standard solution. Close the stopper tightly, shake vigorously for 1 minute, centrifuge, and take the supernatant.

[0247] Test solution: Take about 1.0g of this product, weigh accurately, place in a centrifuge tube, add 4ml of 1mol / L sodium hydroxide solution, accurately add 1ml of reference stock solution, vortex to dissolve, add 1ml of internal standard solution, cover the stopper tightly, shake vigorously for 1 minute, centrifuge, and take the supernatant. Prepare 6 portions in parallel.

[0248] Take 1 μl of each of the internal standard solution, reference solution and test solution, inject them into the gas chromatograph, record the chromatogram, and the repeatability test results are shown in Table 14.

[0249] Table-14 N,N-dimethylaniline method validation-repeatability test results

[0250]

[0251] Note: Calculation formula of internal standard method:

[0252]

[0253]

[0254] Where: Wr: weight of N,N-dimethylaniline, mg

[0255] Pr: N,N-dimethylaniline content

[0256] Sr: dilution multiple of reference solution

[0257] S: Volume of reference stock solution added to the test solution, ml

[0258] W 内 : Weight of naphthalene, mg

[0259] P 内 :Naphthalene content

[0260] Ar: Peak area of ​​N,N-dimethylaniline in reference solution

[0261] A 内 : Peak area of ​​naphthalene in reference solution

[0262] S 内 : Dilution factor of naphthalene

[0263] As: Peak area of ​​N,N-dimethylaniline in the test solution

[0264] A S内 : Peak area of ​​naphthalene in the test solution

[0265] f: Correlation coefficient between N,N-dimethylaniline and naphthalene in the reference solution

[0266] W s : Weight of the test sample, mg

[0267] (6) Intermediate precision

[0268] The same experimental method was used under the repeatability item, and different people performed the test at different times. The results are shown in Table 15.

[0269] Table-15N,N-Dimethylaniline Method Verification-Intermediate Precision Experiment Results

[0270]

[0271] (7) Solution stability

[0272] Internal standard solution: Weigh 50 mg of naphthalene, accurately weigh it, place it in a 50 ml volumetric flask, add cyclohexane to dissolve and dilute to the scale, shake well, take 5 ml of the solution, place it in a 100 ml volumetric flask, dilute to the scale with cyclohexane, shake well, and you have it.

[0273] Reference solution: Take 50 mg of N,N-dimethylaniline, weigh accurately, place in a 50 ml volumetric flask, add 25 ml of 1 mol / L hydrochloric acid, vortex to dissolve, dilute to scale with water, shake well, accurately measure 2 ml and place in a 100 ml volumetric flask, dilute to scale with water, shake well. Accurately measure 1 ml and place in a centrifuge tube, add 5 ml of 1 mol / L sodium hydroxide solution and 1 ml of internal standard solution, cover the stopper, shake vigorously for 1 minute, centrifuge, and take the supernatant.

[0274] Test solution: Take about 1.0 g of the product, weigh accurately, place in a centrifuge tube, add 5 ml of 1 mol / L sodium hydroxide solution, vortex to dissolve, add 1 ml of internal standard solution, cover the stopper tightly, shake vigorously for 1 minute, centrifuge, and take the supernatant.

[0275] Take 1 μl of each of the internal standard solution, reference solution, and test solution at different time points (0h, 2h, 4h, 8h) at room temperature, inject into the gas chromatograph, record the chromatogram, and examine the stability of the solution. The results are shown in Table 16.

[0276] Table-16 N, N-dimethylaniline method verification - solution stability test results (N, N-dimethylaniline peak area)

[0277]

[0278] (8) Durability

[0279] According to the guidance principles for validation of analytical methods for drug quality standards in Part IV 9101 of the 2020 edition of the Chinese Pharmacopoeia, the durability test was conducted. For the conditions of this product, the durability test was mainly conducted from the aspects of flow rate and column temperature to examine the chromatographic conditions for the determination of this product. The inspection conditions are shown in Table 17.

[0280] Table-17N,N-Dimethylaniline Method Verification-Durability Inspection Items

[0281] Durability research project Proposed conditions Investigate changing conditions Flow rate 3ml / min 2.5ml / min、3.5ml / min Column temperature 120℃ 115℃、125℃

[0282] Internal standard solution: Weigh 50 mg of naphthalene, accurately weigh it, place it in a 50 ml volumetric flask, add cyclohexane to dissolve and dilute to the scale, shake well, take 5 ml of the solution, place it in a 100 ml volumetric flask, dilute to the scale with cyclohexane, shake well, and you have it.

[0283] Reference substance stock solution: Take 50 mg of N,N-dimethylaniline, weigh accurately, place in a 50 ml volumetric flask, add 25 ml of 1 mol / L hydrochloric acid and vortex to dissolve, dilute to the scale with water, shake well, take 2 ml, place in a 100 ml volumetric flask, dilute to the scale with water, and shake well.

[0284] Reference substance solution: Accurately measure 1 ml of reference substance stock solution and place it in a centrifuge tube. Add 5 ml of 1 mol / L sodium hydroxide solution and 1 ml of internal standard solution. Close the stopper tightly, shake vigorously for 1 minute, centrifuge, and take the supernatant.

[0285] Test solution: Take about 1.0 g of the product, accurately weigh it, put it in a centrifuge tube, add 4 ml of 1 mol / L sodium hydroxide solution, accurately add 1 ml of the reference stock solution, vortex to dissolve, add 1 ml of the internal standard solution, cover the stopper tightly, shake vigorously for 1 minute, centrifuge, and take the supernatant.

[0286] Take 1 μl of each of the internal standard solution, reference solution, and test solution according to the above conditions, inject them into the gas chromatograph, and record the chromatogram. The durability results are shown in Table 18. Figure 6 to Figure 9 .

[0287] Table-18 N,N-Dimethylaniline Method Verification-Durability Test Results

[0288]

[0289] Note: "*" indicates proposed conditions. The proposed conditions adopt the experimental results under the repeatability item, the separation adopts the result of control 1, and the N,N-dimethylaniline content takes the average value.

[0290] (9) Sample measurement

[0291] Blank solvent: cyclohexane.

[0292] Internal standard solution: Weigh 50 mg of naphthalene, accurately weigh it, place it in a 50 ml volumetric flask, add cyclohexane to dissolve and dilute to the scale, shake well, take 5 ml of the solution, place it in a 100 ml volumetric flask, dilute to the scale with cyclohexane, shake well, and you have it.

[0293] Reference solution: Take 50 mg of N,N-dimethylaniline, weigh accurately, place in a 50 ml volumetric flask, add 25 ml of 1 mol / L hydrochloric acid, vortex to dissolve, dilute to scale with water, shake well, accurately measure 2 ml and place in a 100 ml volumetric flask, dilute to scale with water, shake well. Accurately measure 1 ml and place in a centrifuge tube, add 5 ml of 1 mol / L sodium hydroxide solution and 1 ml of internal standard solution, cover the stopper, shake vigorously for 1 minute, centrifuge, and take the supernatant.

[0294] Test solution: Take about 1.0 g of each batch of raw material drug, accurately weigh, place in a centrifuge tube, add 5 ml of 1 mol / L sodium hydroxide solution, vortex to dissolve, add 1 ml of internal standard solution, cover the stopper tightly, shake vigorously for 1 minute, centrifuge, and take the supernatant.

[0295] Take 1 μl of each of the internal standard solution, reference solution, and test solution according to the above conditions, inject them into the gas chromatograph, and record the chromatogram. The results are shown in Table 19. Figure 10 to Figure 13 .

[0296] Table-19 Sample testing results

[0297] Batch No. / Condition N,N-Dimethylaniline content (%) K2502103012 batch Not detected K2502103022 batch Not detected K2501905012 batch Not detected

Claims

1. A method for detecting N,N-dimethylaniline in cefazolin sodium, comprising the following steps: The blank solvent, internal standard solution, reference solution, and each test solution were injected into the gas chromatograph by headspace injection, and the chromatograms were recorded and quantitatively analyzed by the internal standard method; Among them, the detection conditions of the chromatography are: Instrument: Gas chromatograph; Chromatographic column: a capillary column with 6% cyanopropylphenyl-94% dimethylsiloxane as the stationary phase, or a capillary column with equivalent performance; Detector: hydrogen flame ionization detector (FID); carrier gas: nitrogen; Inlet temperature: 200°C; Detector temperature: 250°C; Temperature program: column temperature is 120°C, maintained for 30 minutes; Injection volume: 1μl; Hydrogen: 40ml / min; Air: 400ml / min; Nitrogen: 40ml / min.

2. The method for detecting N,N-dimethylaniline in cefazolin sodium according to claim 1, characterized in that: The chromatographic column is DB-624 or a capillary chromatographic column with equivalent performance; And / or, the specification of the chromatographic column is preferably 30m×0.53mm×3.00μm.

3. The method for detecting N,N-dimethylaniline in cefazolin sodium according to claim 1, characterized in that: The flow rate ranges from 2.5 ml / min to 3.5 ml / min.

4. The method for detecting N,N-dimethylaniline in cefazolin sodium according to claim 3, characterized in that: The flow rate is 3.0 ml / min.

5. The method for detecting N,N-dimethylaniline in cefazolin sodium according to claim 1, characterized in that: Split flow rate: 5ml / min.

6. The method for detecting N,N-dimethylaniline in cefazolin sodium according to claim 1, characterized in that: The column temperature ranges from 115°C to 125°C.

7. The method for detecting N,N-dimethylaniline in cefazolin sodium according to claim 6, characterized in that: The column temperature was 120°C.

8. The method for detecting N,N-dimethylaniline in cefazolin sodium according to claim 1, characterized in that: The solution is prepared as follows: The blank solvent is cyclohexane; The preparation method of the internal standard solution preferably comprises the following steps: weigh 50 mg of naphthalene, accurately weigh it, place it in a 50 ml measuring bottle, add cyclohexane to dissolve and dilute it to the scale, shake it well, take 5 ml of the solution, place it in a 100 ml measuring bottle, dilute it to the scale with cyclohexane, shake it well, and obtain; The preparation method of the reference solution preferably includes the following steps: take 50 mg of N,N-dimethylaniline, accurately weigh it, place it in a 50 ml volumetric bottle, add 25 ml of 1 mol / L hydrochloric acid, vortex to dissolve, dilute to scale with water, shake well, accurately measure 2 ml and place it in a 100 ml volumetric bottle, dilute to scale with water, shake well. Accurately measure 1 ml and place it in a centrifuge tube, add 5 ml of 1 mol / L sodium hydroxide solution and 1 ml of internal standard solution, cover the stopper, shake vigorously for 1 minute, centrifuge, and take the supernatant; The preparation method of the test solution preferably includes the following steps: taking about 1.0 g of cefazolin sodium raw material, accurately weighing, placing in a centrifuge tube, adding 5 ml of 1 mol / L sodium hydroxide solution, vortexing to dissolve, adding 1 ml of internal standard solution, capping the stopper, vigorously shaking for 1 minute, centrifuging, and taking the supernatant.

9. The method for detecting N,N-dimethylaniline in cefazolin sodium according to claim 1, characterized in that: The calculation formula of the internal standard method is: Where: Wr: weight of N,N-dimethylaniline, mg Pr: N,N-dimethylaniline content Sr: dilution multiple of reference solution S: Volume of reference stock solution added to the test solution, ml W 内 : Weight of naphthalene, mg P 内 :Naphthalene content Ar: Peak area of ​​N,N-dimethylaniline in reference solution A 内 : Peak area of ​​naphthalene in reference solution S 内 : Dilution factor of naphthalene As: Peak area of ​​N,N-dimethylaniline in the test solution A S内 : Peak area of ​​naphthalene in the test solution f: Correlation coefficient between N,N-dimethylaniline and naphthalene in the reference solution W s : The weight of the test sample, mg.

10. A method for detecting N,N-dimethylaniline in cefazolin sodium according to any one of claims 1 to 9, and use thereof in detecting the content of N,N-dimethylaniline in a cefazolin sodium bulk drug or preparation.

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