Method for detecting impurity E in granisetron hydrochloride injection by utilizing HPLC (High Performance Liquid Chromatography) online derivation method

Abstract

The invention discloses a method for detecting an impurity E in a granisetron hydrochloride injection by utilizing an HPLC (High Performance Liquid Chromatography) on-line derivatization method, belonging to the technical field of chemical engineering and pharmacy. According to the method, a high performance liquid chromatography method is adopted, an Ultimate AQ-C18 column is used as a chromatographic column, a mixed solution of a 0.1% phosphoric acid solution and acetonitrile is used as a mobile phase, and an o-phthalaldehyde solution is used as a derivatization reagent; quantitative detection of the impurity E in the granisetron hydrochloride injection is realized through specific instrument conditions, solution preparation and measurement recording steps. The method is simple and convenient to operate, strong in specificity, high in sensitivity and accuracy, good in linearity and capable of effectively detecting the quality of the granisetron hydrochloride injection product.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of chemical and pharmaceutical technology, and particularly relates to a method for detecting impurity E in granisetron hydrochloride injection by using HPLC online derivatization. BACKGROUND

[0002] Granisetron hydrochloride, as a first-line drug for postoperative and chemotherapy-induced nausea and vomiting, belongs to 5-HT3 receptor antagonists, and its dosage forms include injection, tablet, patch and gel, etc. In the "Chinese Expert Consensus on Drug Treatment Related Nausea and Vomiting (2022 Edition)", it is described that granisetron hydrochloride injection can be effectively used to intervene in the chemotherapy-induced vomiting symptoms of patients when it is dripped half an hour before the administration of chemotherapy drugs. Impurity E is a starting material introduced in the preparation of granisetron hydrochloride, and according to the test results of the influencing factors of the preparation, impurity E has a growth trend under light conditions. This process impurity may have toxic side effects on patients. Since there is an amino group in the molecular structure of impurity E, the HPLC online pre-column derivatization method is used to detect impurity E by referring to the amino acid OPA derivatization detection method. SUMMARY

[0003] In order to solve the technical problems in the background art, the application adopts the following technical scheme, and provides a method for detecting impurity E in granisetron hydrochloride injection by using HPLC online derivatization, which comprises the following steps:

[0004] 1. Instrument and chromatographic condition selection

[0005] 1.1 Instrument: high performance liquid chromatograph.

[0006] 1.2 Chromatographic column: Ultimate AQ-C18, column length is 100-250 mm, preferably 4.6 mm x 250 mm, 5 μm.

[0007] 1.3 Mobile phase: A is 0.1% phosphoric acid solution, B is acetonitrile, and the volume ratio of the phosphoric acid solution and the organic solvent mixture is 40:60-90:10.

[0008] 1.4 Detector: ultraviolet detector, detection wavelength is 280-400 nm, preferably 350 nm.

[0009] 1.5 Flow rate of mobile phase: 0.5-1.5 ml / min, the optimal flow rate is 1.0 ml / min.

[0010] 1.6 Column temperature: 25-45 DEG C, preferably 25 DEG C.

[0011] 1.7 Injection tray temperature: 20-30 DEG C, preferably 25 DEG C.

[0012] 1.8 Injection volume: 10 μl.

[0013] 1.9 Elution procedure is as follows:

[0014]

[0015] 2. Preparation of solutions, prepare the following solutions:

[0016] a. Blank solution: water;

[0017] b. Derivatization reagent: take o-phthaldehyde 0.5g (need to be stored in the dark) pre-dissolved in 5ml methanol, then added to 100ml 0.4mol / L boric acid buffer solution (2.48g boric acid dissolved in 100ml water), add 0.5ml 2-mercaptoethanol, adjust the pH value to 10.4 with 30% sodium hydroxide solution;

[0018] c. 0.4mol / L boric acid buffer solution: take boric acid 2.48g dissolved in 100ml water, adjust the pH value to 10.4 with 30% sodium hydroxide solution;

[0019] d. Test solution: take a suitable amount of granisetron hydrochloride injection, shake well;

[0020] e. Reference solution: take about 10mg of impurity E reference substance, accurately weigh, place in a 20ml volumetric flask, dissolve and dilute to the mark with methanol, shake well, accurately take 1ml, place in a 100ml volumetric flask, dilute to the mark with water, shake well;

[0021] f. Sensitivity solution: accurately take 1ml of the solution prepared in step e, place in a 10ml volumetric flask, dilute to the mark with water, shake well.

[0022] The following sequence is injected:

[0023] Name Injection needle number Blank solution ≥ 1 needle Sensitivity solution 1 needle Reference solution 5 needles Test solution 1 needle

[0024] Impurity E in the test solution of granisetron hydrochloride injection is calculated by peak area according to the external standard method.

[0025] ① The conditions of the high performance liquid chromatography method include: using ultraviolet detector, detection wavelength is preferably 350nm; using 0.1% phosphoric acid solution-acetonitrile as mobile phase, the optimal flow rate of mobile phase is 1.0ml / min; column temperature is 25℃; the chromatographic column is preferably Ultimate AQ-C18, 4.6mm×250mm, 5μm; column length is 250mm.

[0026] ② The HPLC online derivatization injection volume is: 5μl of 0.4mol / L boric acid buffer solution + 10μl of test solution + 10μl of derivatization reagent.

[0027] ③ The inspection method is impurity quantitative detection of impurity E.

[0028] The beneficial effects of the present application are as follows:

[0029] The blank solution and the test product solution of the present application have no interference at the peak position of impurity E. By adjusting the derivation method, the precision of the impurity E sample is ensured to meet the requirements, thereby providing a method for quantitatively detecting the content of impurity E in granisetron hydrochloride injection. The method is simple in operation, strong in specificity, high in sensitivity and accuracy, thereby ensuring the controllable quality of granisetron hydrochloride injection. The present application can provide a reliable detection method for the quality control of granisetron hydrochloride injection. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 A linear test result graph of the embodiment of the present application;

[0031] Figure 2 A blank solution spectrum of the embodiment of the present application;

[0032] Figure 3 A sensitivity solution spectrum of the embodiment of the present application;

[0033] Figure 4 A reference solution spectrum of the embodiment of the present application;

[0034] Figure 5 A test product solution spectrum of the embodiment of the present application. DETAILED DESCRIPTION

[0035] The above will be further supplemented by the embodiments (drawings), but should not be understood as limiting the scope of the present application to the following examples.

[0036] Example 1, specificity and precision investigation:

[0037] 1.1 Instruments and chromatographic conditions: the instrument is high performance liquid chromatograph; the chromatographic column is Ultimate AQ-C18, 4.6mmx250mm, 5um; the mobile phase is a mixed solution of 0.1% phosphoric acid solution and acetonitrile; the detection wavelength is 350nm; the column temperature is 25℃; the flow rate is 1.0ml / min; the injection amount is 10ul; the injection disc temperature is 25℃; the gradient elution program is as follows:

[0038]

[0039] 1.2 Solution preparation:

[0040] 1.2.1 Derivative reagent: Take o-phthaldehyde 0.5020 g (avoid light storage) and dissolve in 5 ml of methanol, then add to 100 ml of 0.4 mol / L boric acid buffer solution (2.4763 g of boric acid dissolved in 100 ml of water), add 0.5 ml of 2-mercaptoethanol, and adjust the pH value to 10.4 with 30% sodium hydroxide solution.

[0041] 1.2.2.4 mol / L boric acid buffer solution: Take boric acid 2.4985 g and dissolve in 100 ml of water, and adjust the pH value to 10.4 with 30% sodium hydroxide solution.

[0042] 1.2.3 Blank solution: water.

[0043] 1.2.4 Control stock solution: Take impurity E control 10.07 mg, accurately weigh, place in a 20 ml volumetric flask, dissolve and dilute to the mark with methanol, and shake well.

[0044] 1.2.5 Control solution: Accurately take 1 ml of control stock solution, place in a 100 ml volumetric flask, dilute to the mark with water, shake well.

[0045] 1.2.6 Test solution: Take an appropriate amount of granisetron hydrochloride injection, shake well.

[0046] 1.2.7 Test solution (light for 5 days): Take an appropriate amount of test solution lighted for 5 days.

[0047] 1.2.8 Impurity mixture solution: Take impurities A, B, C, D control 1.91 mg, 1.96 mg, 2.01 mg, 2.00 mg respectively, place in the same 20 ml volumetric flask, dissolve with methanol-water (25:75), accurately take 5 ml, place in a 100 ml volumetric flask, dilute to the mark with water, shake well.

[0048] 1.2.9 Precision solution: Same as control solution.

[0049] 1.3 Experimental process: Accurately take 10 μL of blank solution, test solution, control solution, and precision solution, inject into the liquid chromatograph, record the liquid chromatogram, see Figures 2-5 .

[0050] 1.4 Results:

[0051] 1.4.1 The system suitability results are shown in Table 1, the RSD of the peak area of the 5 control solution is 1.7%.

[0052]

[0053] Table 1

[0054] Name Retention time (min) Peak area Resolution Previous peak of impurity E 12.700 0.0960 7.5 Impurity E 13.967 0.6239 6.9 Next peak of impurity E 15.325 2.2321 N / A

[0055] Table 2

[0056] Name 1 2 3 4 5 6 Average RSD % Impurity E 0.6253 0.6110 0.6068 0.6057 0.5975 0.5989 0.6075 1.7

[0057] Table 3

[0058] Example 2, limit of quantification investigation:

[0059] 2.1 Solution preparation: 1 ml of impurity E reference solution was accurately measured into a 10 ml volumetric flask, diluted to the mark with water, and shaken to prepare a limit of quantification solution.

[0060] 2.2 Experimental process: 10 μL of the limit of quantification solution was injected continuously for 6 times, and the chromatogram was recorded.

[0061] 2.3 Results: The specific results are shown in Table 4, the ratio of the limit of quantification concentration of impurity E to the test sample concentration is 0.05%, the RSD of the peak area of 6 limit of quantification solutions is 4.2%, which is less than 10%. The detection sensitivity of impurity E is high, which can meet the requirement of limit detection of impurity E in granisetron hydrochloride injection.

[0062] Name 1 2 3 4 5 6 Average RSD % Impurity E 0.0317 0.0337 0.0310 0.0330 0.0342 0.0312 0.0325 4.2

[0063] Table 4

[0064] Example 3, linearity investigation:

[0065] 3.1 Solution preparation:

[0066] 3.1.1 Linear stock solution: 5 ml of impurity E reference stock solution was accurately measured into a 50 ml volumetric flask, diluted to the mark with water, and shaken to prepare a linear stock solution.

[0067] 3.1.2 Linear solutions: 1 ml, 1 ml, 2 ml, 3 ml, and 2 ml of the linear stock solution were accurately measured respectively into 50 ml, 20 ml, 20 ml, 20 ml, and 10 ml volumetric flasks, diluted to the mark with water, and shaken to prepare 20%, 50%, 100%, 150%, and 200% linear solutions.

[0068] 3.2 Experimental process: 10 μL of each of the above linear solutions and the limit of quantification solution was injected into the liquid chromatograph, and the chromatogram was recorded. The concentration was taken as the abscissa, and the peak area was taken as the ordinate for linear regression analysis.

[0069] 3.3 Results: The linearity was good, and the correlation coefficient was 0.9997. The specific results are shown in Table 5. Figure 1

[0070] Example 4, accuracy investigation:

[0071] 4.1 Solution preparation:

[0072] 4.1.1 The impurity E reference substance was precisely measured at 1 ml, and was diluted to the mark with granisetron hydrochloride injection in a 10-ml volumetric flask, and was shaken well.

[0073] 4.1.2 The above solution was precisely measured at 1 ml, and was diluted to the mark with granisetron hydrochloride injection in a 10-ml volumetric flask, and was shaken well.

[0074] 4.2 Experimental process: sample injection analysis, recording chromatogram, and calculating recovery. The impurity E reference substance was precisely measured at 1 ml, and was diluted to the mark with granisetron hydrochloride injection in a 10-ml volumetric flask, and was shaken well.

[0075] 4.3 Results: the recovery of the impurity E determination results in 6 samples was between 100.42% and 105.70%, the average recovery was 102.2%, and all were within the range of 90% to 108%, and the results were shown in Table 5.

[0076]

[0077]

[0078] Table 5

[0079] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, several improvements can be made without departing from the principles of the present application, and these improvements should also be considered as the protection of the present application.

Claims

1. A method for detecting impurity E in granisetron hydrochloride injection using HPLC online derivatization method, characterized in that: The following steps are involved: 1) Instrument selection: High performance liquid chromatography; Chromatographic column: Ultimate AQ-C18; mobile phase A: 0.1% phosphoric acid solution, mobile phase B: acetonitrile; UV detector, detection wavelength: 280-400 nm; mobile phase flow rate: 0.5-1.5 ml / min; column temperature: 25-45°C; column length: 100-250 mm; injection plate temperature: 20-30°C; injection volume: 10 μl; elution program: 2) Prepare the following solutions: a. Blank solution: water; b. Derivatization reagent: Dissolve 0.5 g of o-phthalaldehyde in 5 ml of methanol, add to 100 ml of 0.4 mol / L boric acid buffer solution, add 0.5 ml of 2-mercaptoethanol, and adjust the pH to 10.4 with 30% sodium hydroxide solution. c. 0.4 mol / L boric acid buffer solution: dissolve 2.48 g of boric acid in 100 ml of water and adjust the pH to 10.4 with 30% sodium hydroxide solution; d. Test solution: Measure an appropriate amount of granisetron hydrochloride injection and shake well; e. Reference solution: Take about 10 mg of impurity E reference substance, accurately weigh it, place it in a 20 ml volumetric flask, dissolve it in methanol and dilute it to the mark, shake well, accurately measure 1 ml, place it in a 100 ml volumetric flask, dilute it to the mark with water, and shake well; f. Sensitivity solution: Accurately measure 1 ml of the solution prepared in step e, place it in a 10 ml volumetric flask, dilute to the mark with water, and shake well; 3) Measurement record: Accurately measure 10 μL each of the blank solution prepared in step a, the sensitivity solution prepared in step f, the reference solution prepared in step e, and the test solution prepared in step d, and inject them into the high performance liquid chromatograph described in step 1 respectively. Record the chromatogram and calculate the content of impurity E by peak area according to the external standard method.

2. The method for detecting impurity E in granisetron hydrochloride injection by HPLC online derivatization according to claim 1, characterized in that: The derivatization conditions are as follows: accurately draw 1μl~15μl of air, 5μl~15μl of 0.4mol / L boric acid buffer solution, 5μl~15μl of test solution, mix 5~20 times, wash the needle 1~10 times, accurately draw 1μl~15μl of air, 5μl~15μl of derivatization reagent, mix 5~20 times, derivatize for 0~10 minutes, wash the needle 1~10 times, and inject into the liquid chromatograph.

3. The method for detecting impurity E in granisetron hydrochloride injection by HPLC online derivatization according to claim 1, characterized in that: The column length of the chromatographic column is 100 to 250 mm.

4. The method for detecting impurity E in granisetron hydrochloride injection by HPLC online derivatization according to claim 1, characterized in that The volume ratio of the phosphoric acid solution to the organic solvent is 40:60 to 90:

10.

5. The method for detecting impurity E in granisetron hydrochloride injection by HPLC online derivatization according to claim 1, characterized in that The flow rate of the mobile phase is 0.5-1.5 ml / min.

6. The method for detecting impurity E in granisetron hydrochloride injection by HPLC online derivatization according to claim 1, characterized in that: The temperature of the sample injection plate is 20-30°C.

7. The method for detecting impurity E in granisetron hydrochloride injection by HPLC online derivatization according to claim 2, characterized in that: The pH value of the 0.4 mol / L boric acid buffer solution is adjusted to 9.0-12.0 with 30% sodium hydroxide solution.

8. The method for detecting impurity E in granisetron hydrochloride injection by HPLC online derivatization according to claim 2, characterized in that: The pH value of the derivatization reagent is adjusted to 9.0-12.0 with 30% sodium hydroxide solution.

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