Impurity detection method for promethazine hydrochloride

Abstract

The invention provides an impurity detection method for promethazine hydrochloride, and relates to the field of medicine detection. According to the impurity detection method for the promethazine hydrochloride, a high performance liquid chromatography method is adopted for carrying out impurity detection on a promethazine hydrochloride raw material medicine, and in the chromatographic condition, a mobile phase takes phosphate buffer solution-acetonitrile as a mobile phase A and takes phosphate buffer solution-acetonitrile-methanol as a mobile phase B; the phosphate buffer solution contains 0.8 to 1.2 g / L of monopotassium phosphate and 0.08 to 0.12 wt% of triethylamine; the volume ratio of the phosphate buffer solution to the acetonitrile in the mobile phase A is (75-85): (15-25); the volume ratio of the phosphate buffer solution to the acetonitrile to the methanol in the mobile phase B is 10: (48-52): (38-42); linear gradient elution is adopted as an elution mode in the chromatographic conditions, the concentration of a mobile phase A is adjusted within the range of 90% v / v-30% v / v, and the concentration of a mobile phase B is adjusted within the range of 10% v / v-70% v / v. The promethazine hydrochloride impurity detection method disclosed by the invention is not only efficient, but also accurate and reliable.

Description

Technical Field

[0001] The invention relates to the field of drug detection, and in particular to a method for detecting impurities of promethazine hydrochloride. Background Art

[0002] Promethazine hydrochloride, molecular weight: 320.89, this product is (±)-N, N, a-trimethyl-10H-phenothiazine-10-ethylamine hydrochloride. Calculated on the basis of dry product, it contains C 17 H 21 C1N 2 S shall not be less than 99.0%. This product is white or off-white powder or granules; almost odorless; it deteriorates over time in the air and turns blue. This product is very soluble in water, easily soluble in ethanol or chloroform, and almost insoluble in acetone or ether. Promethazine hydrochloride, promethazine is a phenothiazine derivative, an antihistamine, can be used for antiemetic, anti-dizziness, motion sickness, and sedative hypnotic effects, and has antihistamine, anti-emetic, anti-motion sickness, and sedative hypnotic effects.

[0003] The 2020 edition of the Chinese Pharmacopoeia standard records the detection method for promethazine hydrochloride related substances: Determine according to high performance liquid chromatography (General Rule 0512). Operate in the dark. Test solution: Take an appropriate amount of this crystal, dissolve it in 0.1mol / L hydrochloric acid solution and dilute it to make a solution containing about 0.2mg per 1ml. Control solution Accurately measure 1ml of the test solution, place it in a 100ml volumetric flask, dilute it to the scale with 0.1mol / L hydrochloric acid solution, and shake it well. Chromatographic conditions: Use octadecylsilane bonded silica gel as filler; water (adjust pH to 2.3 with glacial acetic acid)-methanol (55; 45) as mobile phase; detection wavelength is 254nm; injection volume is 20ul. System suitability requirements: The number of theoretical plates calculated based on the promethazine peak is not less than 3000, and the separation between the promethazine peak and the impurity peak with a relative retention time of 1.1 to 1.2 should be greater than 2.0. Determination method: Accurately measure the test solution and the control solution, inject them into the liquid chromatograph respectively, and record the chromatogram to 3 times the retention time of the main component chromatographic peak. Limit: If there are impurity peaks in the chromatogram of the test solution, the sum of the areas of the impurity peaks shall not be greater than the main peak area of ​​the control solution (1.0%).

[0004] On the basis of the pharmacopoeia method, the present invention further develops and optimizes the detection method, more accurately detects impurities in promethazine hydrochloride, improves the sensitivity and specificity of detection, can further improve the quality control level of promethazine hydrochloride, and better ensure the safety of patients' medication. Summary of the invention

[0005] In view of this, the present invention proposes a method for detecting impurities of promethazine hydrochloride, which is efficient, accurate and reliable.

[0006] The technical solution of the present invention is achieved as follows: a method for detecting impurities of promethazine hydrochloride, using high performance liquid chromatography to detect impurities in the promethazine hydrochloride raw material, and the mobile phase in the chromatographic conditions is: phosphate buffer-acetonitrile as mobile phase A, and phosphate buffer-acetonitrile-methanol as mobile phase B.

[0007] Furthermore, the phosphate buffer contains 0.8-1.2 g / L potassium dihydrogen phosphate and 0.08%-0.12% wt triethylamine, and the pH value is adjusted to 6.9-7.1 with 15%-25% wt phosphoric acid solution.

[0008] Furthermore, the phosphate buffer contains 1.0 g / L potassium dihydrogen phosphate and 0.1%wt triethylamine, and the pH value is adjusted to 7.0 with 20%wt phosphoric acid solution.

[0009] Furthermore, the volume ratio of phosphate buffer to acetonitrile in the mobile phase A is 75-85:15-25.

[0010] Furthermore, the volume ratio of phosphate buffer, acetonitrile and methanol in the mobile phase B is 10:48-52:38-42.

[0011] Furthermore, the volume ratio of phosphate buffer to acetonitrile in the mobile phase A is 80:20; the volume ratio of phosphate buffer, acetonitrile and methanol in the mobile phase B is 10:50:40.

[0012] Furthermore, the injection volume was 8-12 μl, the flow rate was 0.9-1.2 ml / min, the column temperature was 32-38° C., and the detection wavelength was 252-256 nm.

[0013] Furthermore, the elution method in the chromatographic conditions adopts linear gradient elution, the concentration of mobile phase A is adjusted within the range of 90% v / v-30% v / v, the concentration of mobile phase B is adjusted within the range of 10% v / v-70% v / v, and the elution time is 60-70 min.

[0014] Furthermore, the linear gradient elution program of the chromatographic conditions is:

[0015]

[0016] Further, the solution of the present invention is prepared:

[0017] Prepare a test solution, a reference solution, and a system suitability solution. The test solution is as follows: take promethazine hydrochloride raw material as the test sample, dissolve it in a solvent, and dilute it to prepare a solution containing 0.8-1.2 mg per 1 ml;

[0018] Reference solution: Take the test solution and dilute it with solvent to make a solution containing 8-12 μg of isopropyl hydrochloride per 1 ml;

[0019] System suitability solution: Take promethazine hydrochloride, impurity A, impurity B, impurity C, impurity D, impurity E and impurity F reference substances, dissolve in solvent and dilute to make each ml contain promethazine hydrochloride 0.8-1.2 mg, impurity A 0.8-1.2 μg, impurity B 7.5-8.5 μg, impurity C 1.8-2.2 μg, impurity D 0.8-1.2 μg, impurity E 0.8-1.2 μg and impurity F 0.8-1.2 μg of solution; the impurity A is phenothiazine; the impurity B is N,N-dimethyl-2-(10H-phenothiazine-10-yl)propane-1-amine; the impurity C is N-methyl-1-phenothiazine-10-ylpropan-2-amine; the impurity D is 10-(2-(dimethylamino)propyl)-10H-phenothiazine 5-oxide; the impurity E is N-dimethyl-1-(5-oxo-10H-phenothiazine-10-yl)propan-2-amine oxide; the impurity F is 10-(2-(dimethylamino)propyl)-10H-phenothiazine 5-dioxide.

[0020] The solvent is composed of methanol and triethylamine in a volume ratio of 950-1050:0.9-1.1.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention provides an efficient, accurate and reliable method for detecting impurities of promethazine hydrochloride. The method has good specificity, and the separation between impurities E, impurity D, impurity F, impurity C, impurity A, promethazine and impurity B is good (separation 3.34-17.69); the linear relationship between the content and peak area of ​​impurities E, impurity D, impurity F, impurity C, impurity A, promethazine and impurity B is good, the quantitative limit and detection limit meet the requirements, and the accuracy is good at low, medium and high concentrations. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 . High performance liquid chromatogram of blank solvent of Example 1 of the present invention;

[0024] Figure 2 . High performance liquid chromatogram of impurity A in the specificity test of Example 1 of the present invention;

[0025] Figure 3 . High performance liquid chromatogram of impurity B in the specificity test of Example 1 of the present invention;

[0026] Figure 4 . High performance liquid chromatogram of impurity C in the specificity test of Example 1 of the present invention;

[0027] Figure 5 . High performance liquid chromatogram of impurity D in the specificity test of Example 1 of the present invention;

[0028] Figure 6 . High performance liquid chromatogram of impurity E in the specificity test of Example 1 of the present invention;

[0029] Figure 7 . High performance liquid chromatogram of impurity F in the specificity test of Example 1 of the present invention;

[0030] Figure 8 . High performance liquid chromatogram of the mixed solution in the specificity test of Example 1 of the present invention;

[0031] Fig. 9 . Linear regression curve of peak area and concentration of impurity E in Example 1 of the present invention;

[0032] Fig.10 . Linear regression curve of peak area and concentration of impurity D in Example 1 of the present invention;

[0033] Fig.11 . Linear regression curve of peak area and concentration of impurity F in Example 1 of the present invention;

[0034] Fig.12 . Linear regression curve of peak area and concentration of impurity C in Example 1 of the present invention;

[0035] Fig.13 . Linear regression curve of peak area and concentration of impurity A in Example 1 of the present invention;

[0036] Fig.14 . Linear regression curve of promethazine peak area and concentration in Example 1 of the present invention;

[0037] Fig.15 . Linear regression curve of peak area and concentration of impurity B in Example 1 of the present invention;

[0038] Fig.16 The specific HPLC chromatogram of Reference Example 1 (Chinese Pharmacopoeia method);

[0039] Fig.17 Specific HPLC chromatogram of Control Example 2 (United States Pharmacopeia method). DETAILED DESCRIPTION

[0040] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0041] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0042] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0043] The chemical name and structural formula of impurity AF are shown in Table 1 below:

[0044]

[0045] Example 1 - Promethazine Hydrochloride Impurity Detection Method

[0046] The operation of the embodiment of the present invention complies with high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition Part Four General Rules 0512). Operate in dark and prepare freshly before use.

[0047] 1. Detection method:

[0048] 1.1 Solution preparation

[0049] 1.1.1 Solvent: methanol-triethylamine (1000:1, v / v)

[0050] 1.1.2 Test solution: Take an appropriate amount of the product, dissolve it in solvent and dilute it to make a solution containing about 1 mg per 1 ml.

[0051] 1.1.3 Control solution: Take an appropriate amount of the test sample solution and quantitatively dilute it with solvent to make a solution containing approximately 10 μg of promethazine hydrochloride per 1 ml.

[0052] 1.1.4 System suitability solution: Take appropriate amount of promethazine hydrochloride, impurity A, impurity B, impurity C, impurity D, impurity E and impurity F reference substances, dissolve in solvent and dilute to make a solution containing approximately 1 mg of promethazine hydrochloride, 1 μg of impurity A, 8 μg of impurity B, 2 μg of impurity C, 1 μg of impurity D, 1 μg of impurity El μg and 1 μg of impurity F per 1 ml.

[0053] 1.2 Chromatographic conditions: octadecylsilane bonded silica gel was used as filler (Agilent Eclipse XDB-C18, 150 mm × 4.6 mm, 5 μm or a chromatographic column with similar performance); a ghost trapping column (Ghost Trapping Column II 50 × 4.0 mm or a ghost trapping column with similar performance) was connected; phosphate buffer (containing 1.0 g / L potassium dihydrogen phosphate, 0.1% wt triethylamine, and the pH value was adjusted to 7.0 with 20% wt phosphoric acid solution)-acetonitrile (80:20, v / v) was used as mobile phase A, and phosphate buffer (containing 1.0 g / L potassium dihydrogen phosphate, 0.1% wt triethylamine, and the pH value was adjusted to 7.0 with 20% wt phosphoric acid solution)-acetonitrile-methanol (10:50:40, v / v / v) was used as mobile phase B; the detection wavelength was 254 nm; the injection volume was 10 μl, the flow rate was 1.0 ml / min, and the column temperature was 35°C. Perform linear gradient elution according to the program in Table 3:

[0054] Table 3 Elution program

[0055]

[0056] 1.3 Determination method: Accurately measure the test solution, control solution and system suitability solution, inject them into the liquid chromatograph respectively, and record the chromatogram.

[0057] 1.4 Requirements

[0058] System suitability requirements: In the system suitability solution chromatogram, the order of peaks is impurity E, impurity D, impurity F, impurity C, impurity A, promethazine, and impurity B. The separation between each peak should meet the requirements.

[0059] Limits: If there are impurity peaks in the chromatogram of the test solution, calculate according to the above formula: impurity A shall not exceed 0.1%, impurity B shall not exceed 0.8%, impurity C shall not exceed 0.2%, impurity D shall not exceed 0.1%, impurity E shall not exceed 0.1%, impurity F shall not exceed 0.1%, other single unknown impurities shall not exceed 0.1%, and total impurities (impurity B is not included in the total impurities) shall not exceed 1.0%. Impurities less than 0.01% are ignored.

[0060] Calculation formula

[0061]

[0062] Ag: peak area of ​​promethazine hydrochloride in control solution;

[0063] A 供 : Peak area of ​​impurities in the test solution;

[0064] f: Relative correction factor (impurity A is 0.6, impurity D is 3.8, impurity E is 4.8, impurity F is

[0065] 6.6, other impurities are calculated as 1.0.)

[0066] 2 The verification results of the above-mentioned promethazine hydrochloride impurity detection method are as follows:

[0067] 2.1 Exclusivity

[0068] Table 4-1 Specificity - Impurity Reference Solution

[0069] Solution name Impurity Name Retention time (min) Peak area Impurity E reference solution Impurity E 3.217 12447 Impurity D Reference Solution Impurity D 5.600 16141 Impurity F Reference Solution Impurity F 9.716 9659 Impurity C Reference Solution Impurity C 16.303 120249 Impurity A reference solution Impurity A 21.392 136465 Impurity B reference solution Impurity B 35.436 568546

[0070] Table 4-2 Specificity - System Suitability Solutions

[0071]

[0072] Specific HPLC chromatograms, such as Figure 1-8 shown.

[0073] The results show that the impurity detection method of promethazine hydrochloride of the present invention has good specificity, and the separation between impurity E, impurity D, impurity F, impurity C, impurity A, promethazine, and impurity B is good (separation 3.34-17.69).

[0074] 2.2 Linearity

[0075] Table 5-1 Linearity results - Impurity E

[0076]

[0077] Table 5-2 Linearity results - Impurity D

[0078]

[0079]

[0080] Table 5-3 Linearity results - Impurity F

[0081]

[0082] Table 5-4 Linearity results - Impurity C

[0083]

[0084]

[0085] Table 5-5 Linearity results - Impurity A

[0086]

[0087] Table 5-6 Linearity results - Promethazine

[0088]

[0089]

[0090] Table 5-7 Linearity results - Impurity B

[0091]

[0092] The above results show that there is a good linear relationship between the content and peak area of ​​impurity E, impurity D, impurity F, impurity C, impurity A, promethazine and impurity B.

[0093] 2.3 Limit of quantitation

[0094] Table 6-1 Limit of Quantitation - Impurity B

[0095]

[0096] Table 6-2 Limit of Quantitation - Impurity D

[0097]

[0098] Table 6-3 Limit of Quantitation - Impurity F

[0099]

[0100] Table 6-4 Limit of Quantitation - Impurity C

[0101]

[0102]

[0103] Table 6-5 Limit of Quantitation - Impurity A

[0104]

[0105] Table 6-5 Limit of Quantitation - Promethazine

[0106]

[0107] Table 6-6 Limit of Quantitation - Impurity B

[0108]

[0109] The above results show that the quantitative limits of impurity E, impurity D, impurity F, impurity C, impurity A, promethazine and impurity B are good.

[0110] 2.4 Detection limit

[0111] Table 7 Detection Limits

[0112]

[0113] The results showed that the detection limits of impurity E, impurity D, impurity F, impurity C, impurity A, promethazine and impurity B were good.

[0114] 2.5 Accuracy

[0115] Table 8 Accuracy

[0116]

[0117] The results showed that the detection method of the present invention had good accuracy at low, medium and high concentrations.

[0118] In summary, the present invention provides an impurity detection method for promethazine hydrochloride, which has good specificity, good separation between impurity E, impurity D, impurity F, impurity C, impurity A, promethazine, and impurity B (separation 3.34-17.69); impurity E, impurity D, impurity F, impurity C, impurity A, promethazine, and impurity B have a good linear relationship with the peak area, and the quantitative limit and detection limit meet the requirements, and the accuracy at low, medium, and high concentrations is good. It shows that the present invention provides an efficient, accurate, and reliable method for detecting impurities of promethazine hydrochloride.

[0119] Durability test

[0120] Determination method: Under different chromatographic conditions, observe the changes in retention time, theoretical plate number, tailing factor, separation degree, impurity content of promethazine and each impurity, and whether the blank solvent interferes with the detection.

[0121] Table 9 Durability verification scheme

[0122] project Chromatographic conditions Initial conditions The chromatographic conditions of Example 1 are the initial conditions wavelength 252nm、256nm Column temperature 32℃、38℃ Flow rate 1.2ml / min、0.9ml / min Injection volume 8μl、12μl Solvents 950:1, 1050:1 (methanol:triethylamine, v / v) Mobile phase A ratio 75:25, 85:15 (phosphate buffer:acetonitrile, v / v) Mobile phase B ratio 10:48:42, 10:52:38 (phosphate buffer:acetonitrile:methanol, v / v / v) Phosphate buffer pH 7.1、6.9

[0123] Acceptable criteria: When the chromatographic conditions change slightly, the blank solvent should have no interference, the separation between adjacent chromatographic peaks should meet the requirements, and the impurity content RSD ≤ 15%.

[0124] Results: The factors of mobile phase A, mobile phase B, pH value of mobile phase A, column temperature, detection wavelength, and flow rate were investigated, and the results all met the durability requirements. Adjusting the above chromatographic conditions within a certain range did not affect the accuracy of the determination, and the durability test results were good.

[0125] Comparative Example 1-Reproduction of Chinese Pharmacopoeia Method

[0126] The test was carried out according to the "Related Substances" method for promethazine hydrochloride in the 2020 edition of the Chinese Pharmacopoeia.

[0127] 1 Chromatographic conditions

[0128] Table 10 Chromatographic conditions - Chinese Pharmacopoeia

[0129]

[0130] 2 Experimental results

[0131] Table 11 Experimental results-Chinese Pharmacopoeia

[0132]

[0133] Conclusion: The Chinese Pharmacopoeia method was reproduced, and the retention time of the main peak was short, which was 5.367min; impurities B and C overlapped, and impurities D, E, and F were not completely separated.

[0134] Control Example 2 - Reproduction of the USP Method

[0135] 1 Chromatographic conditions

[0136] Table 13 Chromatographic conditions - United States Pharmacopoeia

[0137]

[0138]

[0139] 2 Experimental results

[0140] Table 14 Experimental results - United States Pharmacopoeia

[0141]

[0142] Conclusion: The USP method was reproduced, impurity E overlapped with the main peak, and impurities A and B were not completely separated.

[0143] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting impurities of promethazine hydrochloride, characterized in that: The impurities of the promethazine hydrochloride raw material drug are detected by high performance liquid chromatography. The mobile phases in the chromatographic conditions are as follows: phosphate buffer-acetonitrile is used as mobile phase A, and phosphate buffer-acetonitrile-methanol is used as mobile phase B; the phosphate buffer contains 0.8-1.2 g / L potassium dihydrogen phosphate and 0.08%-0.12%wt triethylamine; the volume ratio of phosphate buffer and acetonitrile in the mobile phase A is 75-85:15-25; the volume ratio of phosphate buffer, acetonitrile and methanol in the mobile phase B is 10:48-52:38-42; the elution mode in the chromatographic conditions adopts linear gradient elution, the concentration of mobile phase A is adjusted within the range of 90% v / v-30% v / v, and the concentration of mobile phase B is adjusted within the range of 10% v / v-70% v / v.

2. The impurity detection method of promethazine hydrochloride according to claim 1, characterized in that: The pH value of the phosphate buffer is adjusted to 6.9-7.1 using 15%-25% wt phosphoric acid solution.

3. The impurity detection method of promethazine hydrochloride according to claim 2, characterized in that: The phosphate buffer contains 1.0 g / L potassium dihydrogen phosphate and 0.1%wt triethylamine, and the pH value is adjusted to 7.0 with 20%wt phosphoric acid solution.

4. The impurity detection method of promethazine hydrochloride according to claim 1, characterized in that: The volume ratio of phosphate buffer to acetonitrile in the mobile phase A is 80:20; the volume ratio of phosphate buffer to acetonitrile to methanol in the mobile phase B is 10:50:

40.

5. The impurity detection method of promethazine hydrochloride according to claim 1, characterized in that: The injection volume was 8-12 μl, the flow rate was 0.9-1.2 ml / min, the column temperature was 32-38°C, and the detection wavelength was 252-256 nm.

6. The impurity detection method of promethazine hydrochloride according to claim 1, characterized in that: The elution time is 60-70 minutes.

7. The impurity detection method of promethazine hydrochloride according to claim 1, characterized in that: The linear gradient elution program of the chromatographic conditions is: 。 8. The impurity detection method of promethazine hydrochloride according to claim 1, characterized in that: Prepare the test solution and the reference solution. The test solution is as follows: take the promethazine hydrochloride raw material as the test sample, dissolve it in a solvent and dilute it to prepare a solution containing 0.8-1.2 mg per 1 ml; the reference solution is as follows: take the test solution and dilute it with a solvent to prepare a solution containing 8-12 μg of isopropyl hydrochloride per 1 ml.

9. The impurity detection method of promethazine hydrochloride according to claim 1, characterized in that: Prepare the system suitability solution: take promethazine hydrochloride, impurity A, impurity B, impurity C, impurity D, impurity E and impurity F reference substances, dissolve and dilute with solvent to make each 1 ml contain promethazine hydrochloride 0.8-1.2 mg, impurity A 0.8-1.2 μg, impurity B 7.5-8.5 μg, impurity C 1.8-2.2 μg, impurity D 0.8-1.2 μg, impurity E 0.8-1.2 μg and impurity F 0.8-1.2 μg of solution; the impurity A is phenothiazine; the impurity B is N,N-dimethyl-2-(10H-phenothiazine-10-yl)propane-1-amine; the impurity C is N-methyl-1-phenothiazine-10-ylpropan-2-amine; the impurity D is 10-(2-(dimethylamino)propyl)-10H-phenothiazine 5-oxide; the impurity E is N-dimethyl-1-(5-oxo-10H-phenothiazine-10-yl)propan-2-amine oxide; the impurity F is 10-(2-(dimethylamino)propyl)-10H-phenothiazine 5-dioxide.

10. The impurity detection method of promethazine hydrochloride according to claim 8 or 9, characterized in that: The solvent is methanol and triethylamine in a volume ratio of 950-1050: 0.9-1.1 composition.

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