A quality detection method for diclofenac sodium and lidocaine hydrochloride injection

Abstract

The present invention relates to a quality detection method for diclofenac sodium and lidocaine hydrochloride injection, belonging to the field of drug detection. The present invention provides a quality detection method for diclofenac sodium and lidocaine hydrochloride injection. Through a large number of screenings and developments on the basis of the legal standards, the present invention conducts the specificity investigation of known impurities and the specificity investigation of unknown impurities, realizing the accurate and specific detection of related substances in diclofenac sodium and lidocaine hydrochloride injection, providing a reasonable and scientific detection method for the release of manufacturers of diclofenac sodium and lidocaine hydrochloride injection, providing a practical basis for the improvement of the quality standard of related substances in diclofenac sodium and lidocaine hydrochloride injection, and providing a guarantee for the safety of clinical medication. The detection efficiency, specificity, operability, low detection cost and quality control of the method are significantly better than the literature methods and legal standards reported at present.

Description

Technical Field

[0001] The present invention relates to a quality detection method for diclofenac sodium and lidocaine hydrochloride injection, belonging to the field of drug detection. Background Art

[0002] The dosage form of diclofenac sodium and lidocaine injection is injection, and the specification is 2 ml: diclofenac sodium 75 mg and lidocaine hydrochloride 20 mg. Diclofenac sodium is a non-steroidal anti-inflammatory drug (NSAIDs), and it is one of the analgesic drugs with the most sufficient clinical evidence and the largest prescription volume at present. Lidocaine belongs to amide local anesthetics, and its compound preparation is a commonly used therapeutic drug for musculoskeletal pain in clinic, mainly used for severe inflammation or degenerative rheumatic diseases.

[0003] The structural formula of diclofenac sodium is as follows:

[0004]

[0005] The structural formula of lidocaine hydrochloride is as follows:

[0006]

[0007] According to the queried prescription information, in addition to the two clear main components, there are corresponding other excipients in diclofenac sodium and lidocaine hydrochloride injection: propylene glycol, polyethylene glycol, disodium edetate, and acetylcysteine.

[0008] There are safety problems with diclofenac sodium and lidocaine injection. There are literature reports that the adverse reactions of diclofenac sodium and lidocaine hydrochloride injection include allergic reactions, laryngeal edema, hyperthermia reactions, and anaphylactic shock (Li Wei, et al., Literature overview of adverse reactions of diclofenac sodium and lidocaine hydrochloride injection, Chinese Journal of Drug Abuse Prevention and Treatment, Vol. 25, No. 1, 2019). For quality control and ensuring clinical safety, it is particularly important. The quality standard information of diclofenac sodium and lidocaine injection queried is as follows: ① The Chinese Pharmacopoeia (ChP), European Pharmacopoeia (EP), and United States Pharmacopoeia (USP) have published the standards for diclofenac sodium and lidocaine hydrochloride raw materials; ② No single-component diclofenac sodium injection standards have been published in each country and region; ③ The Chinese Pharmacopoeia has published the standards for single-component lidocaine hydrochloride injection, and no relevant standards have been published in other countries and regions; ④ The import registration standard has published the standards for diclofenac sodium and lidocaine hydrochloride injection.

[0009] Among the published legal standards, according to the current standards and the actual requirements of quality control, there are: ① In diclofenac sodium, there are impurities A / B / C / D / E / F published by EP, a total of 6. Among them, the degradation impurity that needs to be focused on is impurity A, which will continuously increase during the production, storage of raw materials and preparations; ② In lidocaine hydrochloride, there are impurities A / B / C / D / E / F / G / H / I / J published by EP, a total of 10. Among them, the impurity that needs to be focused on is impurity A. According to the "Scientific Review of the Risk of Nitrosamine Impurities in Human Medicinal Products" issued by EMA, this impurity contains a warning structure that needs attention and can be considered as a general teratogenic impurity, which needs to be focused on and controlled.

[0010] The current literature on the quality detection of diclofenac sodium and lidocaine hydrochloride injection includes: Zhang Li, et al., Determination of the contents of diclofenac sodium and lidocaine hydrochloride in compound diclofenac sodium injection by HPLC. Chromatographic column: Shim-pack CLC-ODS(M) column, 4.6 mm × 150 mm. Mobile phase: acetate buffer solution (5.2 g of ammonium acetate and 7.4 mL of triethylamine added to water to 1000 mL, adjusted to pH 4.0 with glacial acetic acid)-methanol (38:62), flow rate is 1.0 mL·min -1 , detection wavelength 235 nm, injection volume 20 μL. This literature is for the detection of the contents of diclofenac sodium and lidocaine hydrochloride, without the detection of impurities therein.

[0011] Wu Liangyong, et al., Discussion on the research ideas of impurity control of diclofenac sodium and lidocaine hydrochloride injection. Chinese Pharmaceutical Standards, 2022, 23(6). An HPLC method for the determination of impurities in diclofenac sodium and lidocaine hydrochloride injection was established, and the key points of impurity control in compound preparations were analyzed. Methods: Using a Kromasil C18 (4.6 mm × 250 mm, 5 μm) chromatographic column, with methanol-4% glacial acetic acid solution (65:35) as the mobile phase, detection wavelength 254 nm, for the determination of related substances of diclofenac sodium; using a CNW Athena C 18 (4.6 mm × 250 mm, 5 μm) as the chromatographic column, with phosphate buffer solution (taking 1.3 mL of 1 mol·L -1 sodium dihydrogen phosphate solution and 32.5 mL of 0.5 mol·L -1 disodium hydrogen phosphate solution, diluted with water to 1000 mL, adjusted to pH 8.0 with 10% phosphoric acid)-acetonitrile (50:50) as the mobile phase, detection wavelength 230 nm, for the determination of the known impurity 2,6-dimethylaniline of lidocaine hydrochloride.

[0012] Li Chunying, et al., Determination of Related Substances in Diclofenac Sodium and Lidocaine Hydrochloride Injection by HPLC, North Pharmacy, Vol. 13, No. 9, 2016. An HPLC method was established for the determination of related substances in the compound preparation of diclofenac sodium and lidocaine hydrochloride injection. Method: The reversed-phase high-performance liquid chromatography method was adopted, and the amount of related substances in diclofenac sodium and lidocaine hydrochloride injection was calculated by the self-control method.

[0013] According to the relevant regulations of current laws, the key points (or difficulties) of the standard quality control of diclofenac sodium and lidocaine hydrochloride injection are as follows: ① The method has considerable specificity, can locate 16 impurities included in the standard, and accurately quantify two of the special impurities; ② The injection matrix is complex and contains a large amount of organic excipients, and the method needs to have considerable durability to ensure a large number of detections of samples and detection efficiency; ③ Diclofenac sodium and lidocaine hydrochloride may produce other unknown degradation impurities with other co-existing excipients under the conditions of heat, humidity and light. The established method can not only control the known impurities, but also have a certain ability to distinguish unknown impurities.

[0014] Furthermore, although the imported registration standard has formulated the quality standard of diclofenac sodium and lidocaine hydrochloride injection, the included standard is relatively early. Three sets of methods are adopted to control the impurity detection in the injection, and the control requirements are low, which can no longer meet the quality and safety requirements of the current national relevant departments for the market registration of injections. Therefore, it has no practical reference value and the quality detection standard of the injection needs to be developed. Summary of the Invention

[0015] The present invention provides a quality detection method for diclofenac sodium and lidocaine hydrochloride injection.

[0016] The present invention provides a quality detection method for diclofenac sodium and lidocaine hydrochloride injection, which detects the impurities in diclofenac sodium and lidocaine hydrochloride injection by HPLC method. The impurities include diclofenac sodium impurity A, diclofenac sodium impurity B, diclofenac sodium impurity C, diclofenac sodium impurity D, diclofenac sodium impurity E, diclofenac sodium impurity F, lidocaine hydrochloride impurity A, lidocaine hydrochloride impurity B, lidocaine hydrochloride impurity C, lidocaine hydrochloride impurity D, lidocaine hydrochloride impurity E, lidocaine hydrochloride impurity F, lidocaine hydrochloride impurity G, lidocaine hydrochloride impurity H, lidocaine hydrochloride impurity I, lidocaine hydrochloride impurity J;

[0017] The chromatographic conditions are as follows:

[0018] Chromatographic column: Thermo GOLD C18 4.6*250mm 5μm;

[0019] Mobile phase: Mobile phase A is acetonitrile-phosphate buffer solution; Mobile phase B is acetonitrile-aqueous solution;

[0020] Column temperature: 40 °C;

[0021] Flow rate: 1.2 ml / min;

[0022] Detection wavelength: 230 nm;

[0023] Sample injection volume: 20 μl;

[0024] Elution method: gradient elution, and the elution conditions are as follows:

[0025] Time min A% B% 0 90 10 45 50 50 55 20 80 56 90 10 65 90 10

[0026] Among them, it includes the following steps:

[0027] a. Preparation of test solution: Accurately measure 1 ml of diclofenac sodium and lidocaine hydrochloride injection, place it in a 10-ml volumetric flask, and dilute it to the mark with 10% acetonitrile;

[0028] b. Preparation of impurity reference: Weigh impurity A / B / C / D / E / F / G / H / I / J of lidocaine hydrochloride, dissolve it in a small amount of acetonitrile, and prepare an impurity reference mother liquor with 10% acetonitrile; Weigh impurity A / B / C / D / E / F of diclofenac sodium, dissolve it in a small amount of acetonitrile, and prepare an impurity reference mother liquor with 10% acetonitrile; Weigh the raw materials of lidocaine hydrochloride and diclofenac sodium into the same volumetric flask, add an appropriate amount of the impurity reference mother liquor to prepare a mixed solution with the relative content of each impurity to lidocaine hydrochloride being 0.1%, in which the concentration of lidocaine hydrochloride is 2.5 mg / ml, and a mixed solution with the relative content of each impurity to diclofenac sodium being 0.1%, in which the concentration of diclofenac sodium is 7.5 mg / ml;

[0029] c. Preparation of control solution: Accurately measure 1 ml of the test solution and dilute it to 100 ml with 10% acetonitrile;

[0030] d. Detect according to the described HPLC chromatographic conditions.

[0031] Among them, the pH of the acetonitrile-phosphate buffer solution is adjusted to 8.0 with sodium hydroxide.

[0032] Among them, the retention times of the detected impurities are as follows: Diclofenac impurity E: 8.778 min, Lidocaine hydrochloride impurity C: 10.057 min, Lidocaine hydrochloride impurity B: 10.832 min, Lidocaine hydrochloride impurity D: 15.329 min, Lidocaine hydrochloride impurity H: 18.3 min, Lidocaine hydrochloride impurity A: 20.557 min, Lidocaine hydrochloride impurity G: 22.094 min, Diclofenac impurity D: 25.694 min, Lidocaine hydrochloride impurity E: 28.923 min, Diclofenac impurity A: 41.13 min, Lidocaine hydrochloride impurity F: 43.995 min, Diclofenac impurity C: 44.584 min, Lidocaine hydrochloride impurity I: 45.982 min, Lidocaine hydrochloride impurity J: 46.619 min, Diclofenac impurity F: 49.393 min, Diclofenac impurity B: 52.031 min, with the retention time fluctuating up and down by 0.4 min.

[0033] The beneficial effects of the present invention are as follows:

[0034] Through a large number of screenings and developments on the basis of the legal standards, the present invention conducts specificity investigations on known impurities and unknown impurities, realizes the accurate and specific detection of related substances in the injection of lidocaine hydrochloride and diclofenac sodium, provides a reasonable and scientific detection method for the release of manufacturers of the injection of lidocaine hydrochloride and diclofenac sodium, provides a practical basis for the improvement of the quality standard of related substances in the injection of lidocaine hydrochloride and diclofenac sodium, and provides a guarantee for the safety of clinical medication. The detection efficiency, specificity, operability, low detection cost and quality control of the method are significantly better than the literature methods and legal standards reported at present. Description of the Drawings

[0035] Figure 1 It is a typical chromatogram for the quality detection of the injection of lidocaine hydrochloride and diclofenac sodium of the present invention;

[0036] Figure 2 It is a chromatogram of the blank solution;

[0037] Figure 3 It is a chromatogram of the blank excipient solution;

[0038] Figure 4 It is a typical chromatogram for the method reproducibility of EP lidocaine hydrochloride raw material;

[0039] Figure 5 It is a typical chromatogram for the development of the related substances gradient method;

[0040] Figure 6 It is a typical chromatogram of gradient elution 2;

[0041] Figure 7It is a typical chromatogram of gradient elution 3;

[0042] Figure 8 It is a typical chromatogram of gradient elution 4;

[0043] Figure 9 It is a typical chromatogram of gradient elution 5;

[0044] Figure 10 It is a typical chromatogram of gradient elution 6;

[0045] Figure 11 It is a typical chromatogram of gradient elution 7. Specific implementation mode

[0046] Example 1 Quality detection method of diclofenac sodium and lidocaine hydrochloride injection of the present invention

[0047] The impurities detected by the quality detection method of the present invention are as follows:

[0048]

[0049] Diclofenac sodium impurity A (National Institutes for Food and Drug Control)

[0050]

[0051] Diclofenac sodium impurity B (National Institutes for Food and Drug Control)

[0052]

[0053] Diclofenac sodium impurity C (National Institutes for Food and Drug Control)

[0054]

[0055] Diclofenac sodium impurity D (National Institutes for Food and Drug Control)

[0056]

[0057] Diclofenac sodium impurity E (National Institutes for Food and Drug Control)

[0058]

[0059] Diclofenac sodium impurity F (National Institutes for Food and Drug Control)

[0060]

[0061] Lidocaine hydrochloride impurity A (QCS Standard Substance R & D Center)

[0062]

[0063] Lidocaine Hydrochloride Impurity B (QCS Reference Substance R & D Center)

[0064]

[0065] Lidocaine Hydrochloride Impurity C (QCS Reference Substance R & D Center)

[0066]

[0067] Lidocaine Hydrochloride Impurity D (QCS Reference Substance R & D Center)

[0068]

[0069] Lidocaine Hydrochloride Impurity E (QCS Reference Substance R & D Center)

[0070]

[0071] Lidocaine Hydrochloride Impurity F (QCS Reference Substance R & D Center)

[0072]

[0073] Lidocaine Hydrochloride Impurity G (QCS Reference Substance R & D Center)

[0074]

[0075] Lidocaine Hydrochloride Impurity H (QCS Reference Substance R & D Center)

[0076]

[0077] Lidocaine Hydrochloride Impurity I (QCS Reference Substance R & D Center)

[0078]

[0079] Lidocaine Hydrochloride Impurity J (QCS Reference Substance R & D Center)

[0080] Test solution: Accurately pipette 1 mL of the preparation solution (manufacturer: Teva, marketed country: Czech Republic), add 10% acetonitrile, and dilute to 10 mL.

[0081] Reference solution (for calculating the impurity content in the finished preparation): Pipette 1 mL of the test solution, add 10% acetonitrile, and dilute to 100 mL.

[0082] Spiked impurity solution: Weigh impurities A / B / C / D / E / F / G / H / I / J of lidocaine hydrochloride, dissolve them in a small amount of acetonitrile, and prepare a stock solution of impurity reference substances by adding 10% acetonitrile; weigh impurities A / B / C / D / E / F of diclofenac sodium, dissolve them in a small amount of acetonitrile, and prepare a stock solution of impurity reference substances by adding 10% acetonitrile; weigh the lidocaine hydrochloride raw material and diclofenac sodium raw material into the same volumetric flask, add an appropriate amount of the stock solution of impurity reference substances to prepare a mixed solution in which the content of each impurity relative to lidocaine hydrochloride is 0.1%, with the concentration of lidocaine hydrochloride being 2.5 mg / ml, and a mixed solution in which the content of each impurity relative to diclofenac sodium is 0.1%, with the concentration of diclofenac sodium being 7.5 mg / ml.

[0083] Chromatographic conditions:

[0084] Chromatographic column: Thermo GOLD C18 4.6*250mm 5μm;

[0085] Mobile phase: Mobile phase A is acetonitrile - phosphate buffer solution; mobile phase B is acetonitrile - aqueous solution; mobile phase A: 4.85 g / L potassium dihydrogen phosphate buffer solution (adjust the pH to 8.0 with sodium hydroxide) - acetonitrile (97:3); mobile phase B: Take 970 ml of acetonitrile and add 30 ml of water, mix well.

[0086] Elution mode: Gradient elution, and the elution conditions are shown in Table 1:

[0087] Column temperature: 40 °C;

[0088] Flow rate: 1.2 ml / min;

[0089] Detection wavelength: 230 nm;

[0090] Injection volume: 20 μl;

[0091] Sample solution used: Spiked impurity solution;

[0092] Blank solution and diluent: 10% acetonitrile.

[0093] Table 1 Gradient elution conditions

[0094] T / min A% B% 0 90 10 45 50 50 55 20 80 56 90 10 65 90 10

[0095] By injecting the spiked impurity sample and the humidity - degraded sample simultaneously, the specificity of the method for known and unknown impurities was investigated.

[0096] Results: ① For the blank solution and blank excipient solution, there was no interference in the detection. The resolution between known impurities was good, the peak shapes of each peak were good, and the tailing was low, meeting the requirements for accurate determination of specific impurities and the localization of general impurities. The results are shown in Table 2, and the typical chromatogram is shown in Figure 1 , and the chromatogram of the blank solution is shown inFigure 2 , the chromatogram of the blank excipient solution is shown in Figure 3 ; ② For the moderately degraded samples, the possible degradation impurities did not interfere with the detection of the specific impurities and the main peak.

[0097] Table 2 Results of the specificity study of known impurities

[0098] Impurity name Retention time min Resolution Number of theoretical plates Tailing factor SL-E 8.778 / 29281 0.9 LD-C 10.057 5.73 27811 1.18 LD-B 10.832 2.06 7196 1.09 LD-D 15.329 11.38 48373 1.06 LD-H 18.3 9.94 52463 1 LD-A 20.557 6.87 59035 0.99 LD-G 22.094 4.71 80241 1.04 Diclofenac sodium 24.664 7.86 82801 1.64 SL-D 25.694 3.49 175766 0.98 LD-E 28.923 11.99 155607 1 Unknown impurity 35.248 20.84 201708 1.03 Lidocaine 37.568 8.72 474297 1.3 SL-A 41.13 12.54 219974 0.96 LD-F 43.995 7.86 217705 1.02 SL-C 44.584 1.55 212374 1.16 LD-I 45.982 3.62 231014 1 LD-J 46.619 1.67 237947 1 SL-F 49.393 8.12 430495 1 SL-B 52.031 9.6 700407 1.01

[0099] An HPLC method was established to accurately, efficiently, reliably and stably determine the related substances in diclofenac sodium and lidocaine hydrochloride injection.

[0100] Example 2 Screening test of the conditions of the detection method of the present invention

[0101] During the early development by the inventors, first, the official standards of various pharmacopoeias were compared. Then, in combination with the properties of diclofenac sodium and lidocaine hydrochloride compounds and the information on the known impurities related to the main components, it was inferred that the method for related substances of lidocaine hydrochloride raw material drug included in EP had more potential for development.

[0102] 1. Reproducibility of the method for lidocaine hydrochloride raw material drug in EP

[0103] The inventors planned to develop the method using a DAD detector and a Waters C18 column (4.6 mm * 250 mm * 5 μm). The chromatographic conditions for this test are shown in Table 3.

[0104] Table 3 Chromatographic conditions

[0105] Item Content Chromatographic column Waters C18, 4.6mm * 150mm * 5μm Mobile phase Phosphate buffer - acetonitrile = 70 - 30 Elution mode Isocratic elution Column temperature (℃) 30 Detection wavelength (nm) 230 Injection volume (μl) 20 Sample tray temperature (℃) 8 Samples to be investigated Spiked solution of related impurities of lidocaine hydrochloride

[0106] In this test example, the solutions were prepared as follows:

[0107] Mobile phase: 4.85 g / L potassium dihydrogen phosphate buffer solution (adjusted to pH 8.0 with sodium hydroxide) - acetonitrile (70:30)

[0108] Blank solution and diluent: 30% acetonitrile.

[0109] Test samples: Lidocaine hydrochloride impurities A / B / C / D / E / F / G / H / I / J were weighed, dissolved in a small amount of acetonitrile, and then diluted with 30% acetonitrile to prepare a mother solution of the impurity reference substance. Lidocaine hydrochloride raw material drug was weighed into a volumetric flask, and an appropriate amount of the mother solution of the impurity reference substance was added to prepare a mixed solution in which the content of each impurity relative to lidocaine hydrochloride was 0.1%, and the concentration of lidocaine hydrochloride was 5 mg / ml.

[0110] The experimental process and results are shown in Figure 4: ① The elution times of the impurities of lidocaine are between 2 min and 12 min, and the elution time of the main peak is about 25 min. ② The system of this method is a phosphate buffer system with a pH of 8.0. According to the chemical properties of diclofenac sodium (it belongs to a pH-dependent API, insoluble in acidic environment and soluble in alkaline environment), this system can be compatible with the related impurities of diclofenac sodium. ③ The elution times of the peaks in this method are relatively dispersed, indicating that the elution selectivity of this method is strong, providing possible time and space conditions for the elution separation and localization of a total of 16 impurities. ④ The method for detecting the related substances of lidocaine and diclofenac injection can be optimized based on this method.

[0111] 2. Development of the gradient method for the related substances of the injection

[0112] Based on the analysis of the impurity properties of diclofenac sodium and lidocaine hydrochloride, a gradient development of the isocratic method is carried out on the basis of item 1 of this example.

[0113] Chromatographic conditions:

[0114] Chromatographic column: Waters C18, 4.6 mm * 150 mm * 5 μm;

[0115] Mobile phase: Mobile phase A is acetonitrile - phosphate buffer; Mobile phase B is acetonitrile - aqueous solution

[0116] Column temperature: 30 °C;

[0117] Detection wavelength: 230 nm;

[0118] Injection volume: 20 μl;

[0119] Sample tray temperature: 8 °C;

[0120] Concentration of the test sample: 0.1 mg / ml;

[0121] Sample under investigation: Impurity spike solution.

[0122] Table 4 Gradient elution conditions

[0123] T / min A% B% 0 70 30 15 50 50 25 50 50 25.1 70 30 35 70 30

[0124] In this test example, the solutions are prepared as follows:

[0125] Mobile phase A: 4.85 g / L potassium dihydrogen phosphate buffer (adjusted to pH 8.0 with sodium hydroxide) - acetonitrile (97:3); Mobile phase B: 97% acetonitrile.

[0126] Blank solution and diluent: 10% acetonitrile.

[0127] Samples for investigation: Weigh lidocaine hydrochloride impurities A / B / C / D / E / F / G / H / I / J, dissolve them in a small amount of acetonitrile, and then prepare a stock solution of impurity reference substances by adding 30% acetonitrile; weigh diclofenac sodium impurities A / B / C / D / E / F, dissolve them in a small amount of acetonitrile, and then prepare a stock solution of impurity reference substances by adding 30% acetonitrile; weigh lidocaine hydrochloride raw material and diclofenac sodium raw material into the same volumetric flask, add an appropriate amount of the stock solution of impurity reference substances to prepare a mixed solution in which the content of each impurity relative to lidocaine hydrochloride is 0.1%, with the concentration of lidocaine hydrochloride being 2.5 mg / ml, and a mixed solution in which the content of each impurity relative to diclofenac sodium is 0.1%, with the concentration of diclofenac sodium being 7.5 mg / ml.

[0128] The experimental process and results are shown in Figure 5 : ① By using the relevant method for lidocaine in EP, the isocratic elution was changed to gradient elution, and the maximum proportion of the organic phase was about 50% acetonitrile. A total of 12 impurities were located, and it was judged that 4 impurities overlapped or failed to elute in the system; ② In this method, diclofenac sodium eluted at 12 min and lidocaine at 18 min. Based on the properties of the main components and impurities, it was judged that there was sufficient space for separation of the impurities introduced by the two main components. Therefore, it could be further optimized under this system; ④ Under this condition, the main interference existed between 2 min and 10 min. Therefore, the method of optimizing the gradient was to reduce the initial proportion of the mobile phase and investigate the elution conditions of 16 impurities and two main components at different elution rates.

[0129] 3. Further development of the gradient method

[0130] Based on the judgment in item 2, the elution program was further optimized.

[0131] Table 5 Chromatographic conditions

[0132] Item Content Chromatographic column Waters C18, 4.6mm * 150mm * 5μm Mobile phase Mobile phase A is acetonitrile - phosphate buffer solution; mobile phase B is acetonitrile - aqueous solution Elution mode Gradient elution Column temperature (℃) 30 Detection wavelength (nm) 230 Injection volume (μl) 20 Flow rate (ml / min) 1.0 Sample tray temperature (℃) 8 Concentration of test sample (mg / ml) 0.1 Samples to be investigated Spiked solution of impurities

[0133] Table 6 Gradient elution conditions

[0134]

[0135] In this test example, the mobile phase and solution were the same as those shown in item 2.

[0136] The experimental process and results are shown in Figure 6 - Figure 11 :

[0137] ① Gradient condition 2: Based on item 2, the initial proportion was reduced, and the retention time of each peak was delayed; each peak had a separation trend, and the reduction of the initial proportion or the reduction of the gradient change rate was continued to be investigated;

[0138] ② Gradient condition 3: Compared with gradient condition 2, there was almost no difference in the elution of each peak;

[0139] ③Gradient condition 4: Further reduce the initial ratio, and the elution time of the main peak is delayed to 14 min, and the main peak is delayed to 20 min;

[0140] ④Gradient condition 5: Perform segmented elution on the basis of gradient 2. There is an interfering peak before the main peak of diclofenac sodium, and the retention time of lidocaine is delayed to about 20 min;

[0141] ⑤Gradient condition 6: Further optimize on the basis of gradient 4. The detected known peaks have good peak shapes; the resolution of each peak is good; through positioning, only diclo impurity E overlaps with the main peak of lidocaine, and the rest of the impurities can be located and accurately detected; further optimization is carried out by selecting a chromatographic column; when the initial organic phase ratio is increased from 10% to 50% within 45 min, the impurities have good separation effects.

[0142] ⑥Gradient condition 7: Targeted optimization is carried out on the segments with impurity interference in gradient condition 6. After optimization, 16 related substances of diclofenac sodium and lidocaine hydrochloride are all located. Among them, the worst resolution between impurities and between impurities and the main peak is 1.2 (lidocaine impurity A and diclo impurity G), which basically meets the relevant requirements of specificity. However, lidocaine impurity A is a basic toxic impurity, and the resolution needs to be improved for accurate quantification.

[0143] In summary, through the gradient screening test, by using 10%, 20%, and 30% initial organic phase ratios and adjusting the increasing rate of the organic phase gradient, the separation of diclo and lidocaine impurities can be completed completely. However, after gradient screening using the chromatographic column, the column efficiency decreases significantly, which is presumably directly related to the complex sample components and high concentration. To further improve the durability, a series of screenings of the chromatographic column of the method were carried out, and the screening target was to use a C18 chromatographic column with a longer specification.

[0144] 4. Durability investigation - Chromatographic column screening

[0145] On the basis of item 3 "Gradient elution 7", further screen chromatographic columns with better durability and lower prices. Currently, the commercially available Waters C 18 column costs about 8000 yuan per piece. Select the Thermo GOLD column (price about 4000 yuan / piece). When the column length of the chromatographic column increases, the elution time of each peak will necessarily be extended, and the selectivity is theoretically better. However, at the same time, there may be strongly retained substances that cannot be eluted from the column. Therefore, on the basis of "Gradient elution 7", the flow rate is increased to 1.2 ml / min, and at the same time, during the high proportion organic phase flushing stage after 45 min, the organic phase ratio is further increased, in order to elute the strongly retained substances in the column without changing the elution situation before 45 min, extend the service life of the chromatographic column, enhance the durability of the method, and significantly reduce the detection cost.

[0146] The chromatographic conditions are as follows:

[0147] Chromatographic column: Thermo GOLD C18 4.6*250mm 5μm;

[0148] Mobile phase: Mobile phase A is acetonitrile - phosphate buffer solution; Mobile phase B is acetonitrile - aqueous solution;

[0149] Elution method: Gradient elution, see Table 7;

[0150] Column temperature: 40°C;

[0151] Flow rate: 1.2 ml / min;

[0152] Detection wavelength: 230 nm;

[0153] Sample injection volume: 20 μl;

[0154] Solution used: Impurity spike solution;

[0155] See Table 7 below.

[0156] Table 7 Gradient elution conditions

[0157] T / min A% B% 0 90 10 45 50 50 55 20 80 56 90 10 65 90 10

[0158] In this test example, the solutions are the same as in item 2.

[0159] The experimental process and results are shown in Figure 1 : ① 16 impurities and 2 main components were completely detected; ② The worst resolution between adjacent impurities and between impurities and the main peak was 1.03 (lidocaine impurity F and diclofenac impurity C); ③ The resolutions of lidocaine impurity A from the adjacent impurities before and after were 7.07 and 3.54 respectively; The resolutions of diclofenac impurity A from the adjacent peaks before and after were 9.2 and 8.02 respectively; ④ This chromatographic condition fully meets the requirements for the localization of known impurities and the accurate quantification of special impurities; ⑤ It is planned to further reduce the concentration of the test sample under this chromatographic condition to reduce matrix interference without affecting the normal detection of impurities.

[0160] 5. Durability investigation - Concentration of test sample

[0161] The chromatographic conditions are the same as in item 4. During sample injection, 10 / 20 / 50 μl of the sample was injected respectively.

[0162] Sample used: 1 self - made stability sample (2 ml, 25 mg / 75 mg), diluted 10 times with 10% acetonitrile.

[0163] Results: ① When injecting 10 μl compared with injecting 20 μl, the number of detected impurities did not change significantly; ② When injecting 50 μl, the peak overloading was serious; ③ The sample injection volume of 20 μl was selected.

[0164] 6. Durability investigation - Detection wavelength

[0165] The chromatographic conditions are the same as item 4.

[0166] Sample used: 1 self-made stability sample (2 ml, 25 mg / 75 mg), diluted 10 times with 30% acetonitrile.

[0167] Different detection wavelengths have obvious effects on the impurity detection results. The detection wavelength of diclofenac sodium is selected as 254 nm in the legal standard, and that of lidocaine hydrochloride is selected as 230 nm, showing a large difference. Therefore, first, a full-wavelength scan was performed on each known impurity, and the maximum absorption wavelengths of the known impurities are shown in Table 8 below. It was determined that there are overlapping parts in the absorption spectra of the impurities of lidocaine and those of diclofenac sodium.

[0168] Table 8 Maximum absorption wavelengths of known impurities

[0169]

[0170] Secondly, the stability sample (the test solution with more impurities) was detected, and the impurity contents at different wavelengths were calculated.

[0171] Test results: The amount and number of detected impurities at 230 nm wavelength are more than those at 254 nm wavelength, and the amount of impurities has an obvious increase; it is speculated that at 230 nm wavelength, more lidocaine impurities are mainly detected. Therefore, the final selected wavelength is 230 nm.

Claims

1. A quality detection method for diclofenac sodium and lidocaine hydrochloride injection, characterized in that: The method detects impurities in diclofenac sodium and lidocaine hydrochloride injection by HPLC, wherein the impurities include diclofenac sodium impurity A, diclofenac sodium impurity B, diclofenac sodium impurity C, diclofenac sodium impurity D, diclofenac sodium impurity E, diclofenac sodium impurity F, lidocaine hydrochloride impurity A, lidocaine hydrochloride impurity B, lidocaine hydrochloride impurity C, lidocaine hydrochloride impurity D, lidocaine hydrochloride impurity E, lidocaine hydrochloride impurity F, lidocaine hydrochloride impurity G, lidocaine hydrochloride impurity H, lidocaine hydrochloride impurity I and lidocaine hydrochloride impurity J; The chromatographic conditions are: Chromatographic column: Thermo GOLD C18 4.6*250mm 5μm; Mobile phase: Mobile phase A is acetonitrile-phosphate buffer; mobile phase B is acetonitrile-water solution; Column temperature: 40°C; Flow rate: 1.2 ml / min; Detection wavelength: 230nm; Injection volume: 20 μl; Elution method: gradient elution, elution conditions are as follows: The diclofenac sodium and lidocaine hydrochloride injection is from the manufacturer: Teva.

2. The quality detection method of diclofenac sodium and lidocaine hydrochloride injection according to claim 1, characterized in that: The steps include: a. Preparation of test solution: Accurately measure 1 ml of diclofenac sodium and lidocaine hydrochloride injection, place in a 10 ml volumetric flask, and dilute to the mark with 10% acetonitrile; b. Preparation of impurity control: Weigh lidocaine hydrochloride impurities A / B / C / D / E / F / G / H / I / J, add acetonitrile to dissolve, and then add 10% acetonitrile to prepare an impurity reference substance mother solution; weigh diclofenac sodium impurities A / B / C / D / E / F, add acetonitrile to dissolve, and then add 10% acetonitrile to prepare an impurity reference substance mother solution; weigh lidocaine hydrochloride bulk drug and diclofenac sodium bulk drug into the same volumetric flask, add the impurity reference substance mother solution to prepare a mixed solution containing 0.1% of each impurity relative to lidocaine hydrochloride, wherein the concentration of lidocaine hydrochloride is 2.5 mg / ml, and a mixed solution containing 0.1% of each impurity relative to diclofenac sodium, wherein the concentration of diclofenac sodium is 7.5 mg / ml; c. Preparation of control solution: Accurately measure 1 ml of the test solution and dilute it to 100 ml with 10% acetonitrile; d. Detection according to the HPLC chromatographic conditions of claim 1.

3. The quality detection method of diclofenac sodium and lidocaine hydrochloride injection according to claim 1, characterized in that: The pH of the acetonitrile-phosphate buffer solution was adjusted to 8.0 with sodium hydroxide.

4. The quality detection method of diclofenac sodium and lidocaine hydrochloride injection according to any one of claims 1 to 3, characterized in that: The retention times of the detected impurities are: Diclofenac Impurity E: 8.778 min, Lidocaine Hydrochloride Impurity C: 10.057 min, Lidocaine Hydrochloride Impurity B: 10.832 min, Lidocaine Hydrochloride Impurity D: 15.329 min, Lidocaine Hydrochloride Impurity H: 18.3 min, Lidocaine Hydrochloride Impurity A: 20.557 min, Lidocaine Hydrochloride Impurity G: 22.094 min, Diclofenac Impurity D: 25.694 min, Lidocaine Hydrochloride Impurity E: 28.923 min, Diclofenac Impurity A: 41.13 min, Lidocaine Hydrochloride Impurity F: 43.995 min, Diclofenac Impurity C: 44.584 min, Lidocaine Hydrochloride Impurity I: 45.982 min, Lidocaine Hydrochloride Impurity J: 46.619 min. min, diclofenac impurity F: 49.393 min, diclofenac impurity B: 52.031 min, and the retention time fluctuated by 0.4 min.

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