A method for simultaneously detecting the content of sodium pentobarbital and alcohol antibacterial agents in sodium pentobarbital solution preparations

Abstract

The present invention provides a method for simultaneously detecting the content of sodium pentobarbital and alcohol antibacterial agent in sodium pentobarbital solution preparation, belonging to the field of quality detection technology. The chromatographic conditions detected by the method are as follows: chromatographic column: octadecylsilane bonded silica gel chromatographic column; mobile phase: composed of phase A and phase B, wherein phase A is an aqueous solution containing potassium dihydrogen phosphate and tetrabutylammonium hydrogen sulfate, and the pH value of phase A is 2.8-3.2; phase B is acetonitrile; the volume ratio of phase A and phase B is (65-75): (35-25). The method of the present invention can simultaneously complete the detection of sodium pentobarbital content and benzyl alcohol content under the same chromatographic conditions, and achieve the purpose of quality control. The detection method of the present invention can reduce the running time, improve the detection efficiency, and save resources to run. The detection method of the present invention is verified by systematic inspection methodology, and the confirmation method is scientific and reasonable, with the advantages of fast, simple, high sensitivity, strong specificity, and broad application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of quality detection, and in particular relates to a method for simultaneously detecting the contents of sodium pentobarbital and an alcohol antibacterial agent in a sodium pentobarbital solution preparation. Background Art

[0002] Barbiturates have a general inhibitory effect on the central nervous system, among which sodium pentobarbital is a medium-acting barbiturate central sedative. The active ingredient of sodium pentobarbital is pentobarbital. As the dose of sodium pentobarbital increases, the central inhibitory effect changes from weak to strong, and the corresponding effects are sedation, hypnosis, anticonvulsant, anti-epileptic pain, and anesthesia. Large doses also have an inhibitory effect on the cardiovascular system. A 10-fold anesthetic dose can cause paralysis of the respiratory center and cause death. Because of its excellent ability to inhibit the central nervous system of animals, causing them to lose consciousness and relieve pain, sodium pentobarbital is widely used as the drug of choice for euthanasia of animals, especially experimental animals. For example, in the Chinese national recommended standards "GB / T 39760-2021 Guidelines for Euthanasia of Laboratory Animals" and "American Veterinary Medical Association Guidelines for Euthanasia of Animals (2020 Edition)", injection of sodium pentobarbital is the preferred method for euthanasia of experimental animals.

[0003] Considering that animal drugs are usually packaged in multiple doses (multiple dose packaged drugs refer to drugs that contain multiple dosages in the smallest sales unit), there will still be some left after opening and using for a period of time. In order to prevent microbial contamination of drugs within a period of time after the first opening of the package and to ensure the quality of the drugs, it is necessary to add antibacterial agents, such as alcohol antibacterial agents - benzyl alcohol. Currently, there are commercially available sodium pentobarbital solution preparations containing benzyl alcohol. However, benzyl alcohol has a certain toxicity and its content cannot exceed the standard. Therefore, it is necessary to strictly control and detect the content of antibacterial agents in animal drugs.

[0004] At present, some information has been reported on methods for detecting the content of pentobarbital (sodium). For example, the British Pharmacopoeia 2023 edition records a method for detecting the content of pentobarbital (sodium) using high performance liquid chromatography. The chromatographic conditions of this method are as follows: octadecylsilane bonded silica gel is used as the filler, 1.36 g / L potassium dihydrogen phosphate (the pH value is adjusted to 3.5 with phosphoric acid) and acetonitrile (65:35) are used as the mobile phase; the detection wavelength is 214 nm. However, this method is not suitable for simultaneously detecting the contents of sodium pentobarbital and benzyl alcohol in sodium pentobarbital solution preparations. When this method is used to simultaneously detect the contents of sodium pentobarbital and benzyl alcohol in sodium pentobarbital solution preparations, it does not meet the requirements of the tailing factor and repeatability of the fourth monograph 0512 High Performance Liquid Chromatography (page 63) of the Chinese Pharmacopoeia 2020 Edition: 1. When the peak height is used as a quantitative parameter, the tailing factor should be between 0.95-1.05 unless otherwise specified; 2. After 5 consecutive injections, the relative standard deviation of the peak area measurement value should not be greater than 2.0%. At present, no other methods that can simultaneously detect the contents of sodium pentobarbital and alcohol antibacterial agents in sodium pentobarbital solution preparations have been reported. Summary of the invention

[0005] The object of the present invention is to provide a method for simultaneously detecting the contents of sodium pentobarbital and alcohol antibacterial agent in a sodium pentobarbital solution preparation.

[0006] The present invention provides a method for simultaneously detecting the contents of sodium pentobarbital and alcohol antibacterial agent in a sodium pentobarbital solution preparation, the method comprising the following steps:

[0007] (1) Preparation of reference solution: Take pentobarbital and alcohol antibacterial agent, add diluent to dilute, and obtain the reference solution;

[0008] (2) preparing a test solution: taking a sodium pentobarbital solution preparation and diluting it with a diluent to obtain a test solution; the sodium pentobarbital solution preparation contains an alcohol antibacterial agent;

[0009] (3) The test solution and the reference solution are respectively drawn and injected into a liquid chromatograph for detection to obtain a chromatogram, and then the contents of sodium pentobarbital and alcohol antibacterial agent in the sodium pentobarbital solution preparation are calculated; the chromatographic conditions for the detection are as follows:

[0010] Chromatographic column: octadecylsilane bonded silica gel chromatographic column;

[0011] Mobile phase: composed of phase A and phase B, wherein phase A is an aqueous solution containing potassium dihydrogen phosphate and tetrabutylammonium hydrogen sulfate, and the pH value of phase A is 2.8-3.2; phase B is acetonitrile; the volume ratio of phase A to phase B is (65-75):(35-25).

[0012] Furthermore, the calculation method is an external standard method.

[0013] Furthermore, the alcohol antibacterial agent is benzyl alcohol.

[0014] Furthermore, the diluent is a mobile phase.

[0015] Furthermore, the chromatographic column is a Gemini C18 chromatographic column.

[0016] Furthermore, in the phase A, the concentration of potassium dihydrogen phosphate is 0.005-0.02M, and the concentration of tetrabutylammonium hydrogen sulfate is 0.001-0.01M.

[0017] Furthermore, in the phase A, the concentration of potassium dihydrogen phosphate is 0.01M, and the concentration of tetrabutylammonium hydrogen sulfate is 0.005M.

[0018] Furthermore, the pH value of phase A is 3.0, and the volume ratio of phase A to phase B is 70:30.

[0019] Furthermore, in the chromatographic conditions, the injection volume is 5-20 μl and the flow rate is 0.9-1.1 ml / min.

[0020] Furthermore, in the chromatographic conditions, the injection volume is 10 μl and the flow rate is 1.0 ml / min.

[0021] Furthermore, in the chromatographic conditions, the column temperature is 35-45°C.

[0022] Furthermore, in the chromatographic conditions, the column temperature is 40°C.

[0023] The present invention can use one detection under the same chromatographic conditions to simultaneously complete the detection of pentobarbital content and benzyl alcohol content by high performance liquid chromatography external standard method, so as to achieve the purpose of quality control. In the detection method of the present invention, the elution time of sodium pentobarbital is about 11.1 minutes, the elution time of benzyl alcohol is about 5.6 minutes, and the separation degree is as high as 22.114; the number of theoretical plates of the main peak of pentobarbital is 18568, the tailing factor is 0.95, and the number of theoretical plates of the main peak of benzyl alcohol is 15929, and the tailing factor is 1.0. Compared with multiple detections to obtain the contents of both, the detection method of the present invention can reduce the running time, improve the detection efficiency, and save resources to run. The detection method of the present invention is verified by systematic inspection methodology, and the confirmation method is scientific and reasonable, with the advantages of fast, simple, high sensitivity, strong specificity, and broad application prospects.

[0024] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0025] The above contents of the present invention are further described in detail below through specific implementation methods in the form of examples. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The chromatogram of the test solution of Example 1 is shown in FIG.

[0027] Figure 2 This is the chromatogram of the test solution of Control Example 5.

[0028] Figure 3 This is the linear regression equation and linear relationship diagram of pentobarbital content.

[0029] Figure 4 This is the linear regression equation and linear relationship diagram of benzyl alcohol content. DETAILED DESCRIPTION

[0030] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0031] The preparation method of the sodium pentobarbital solution preparation adopted in the following examples, control examples and experimental examples is as follows: 30g of powdered sodium pentobarbital (converted according to the raw material content determination and moisture determination) is placed in a conical flask, 10ml of ethanol and 40ml of propylene glycol are added, and the mixture is heated in a water bath to 65°C, and stirred thoroughly to dissolve all the powders, and then 2g of benzyl alcohol is added. While stirring thoroughly, the pH is adjusted to about 10.6 with 0.5mol / L sodium hydroxide solution, and then an appropriate amount of water is added to a total amount of 100ml. After stirring evenly, the mixture is filtered through a 0.22um pharmaceutical grade hydrophilic polytetrafluoroethylene filter, and then packaged in a packaging container at 10ml / bottle to obtain the sodium pentobarbital solution preparation.

[0032] Pentobarbital reference substance was purchased from Shanghai Yuansi Biaowu Technology Co., Ltd., batch number 030007082BA00220230901; benzyl alcohol reference substance was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., batch number C15091444.

[0033] Example 1: Method for simultaneously detecting the contents of sodium pentobarbital and alcohol antibacterial agents in sodium pentobarbital solution preparations

[0034] (1) Preparation of reference solution

[0035] Take pentobarbital (C 11 H 18 N2O3,M W226.27) and benzyl alcohol reference substance in appropriate amounts, accurately weighed, added to the mobile phase to dissolve and dilute to prepare a solution containing 0.1 mg of pentobarbital and 0.008 mg of benzyl alcohol per 1 ml as the reference solution.

[0036] (2) Preparation of test solution

[0037] Take the sodium pentobarbital solution preparation, accurately measure an appropriate amount of the product with a volumetric pipette, wash the inner wall of the pipette several times with the mobile phase, add the washing liquid into a 100ml volumetric flask, dilute to the scale with the mobile phase, and shake well; then accurately measure an appropriate amount and dilute to the scale with the mobile phase, shake well, as the test solution (the converted pentobarbital is 0.1mg / ml).

[0038] (3) Detection

[0039] The reference solution and the test solution were injected into the high performance liquid chromatograph for detection respectively. The chromatographic conditions were as follows:

[0040] Gemini C18 (4.6 mm*25 cm, 5 μm) chromatographic column (Fenomi, USA);

[0041] The mobile phase consists of phase A and phase B, phase A is an aqueous solution containing 0.01M potassium dihydrogen phosphate and 0.005M tetrabutylammonium hydrogen sulfate, and the pH value of the aqueous solution is adjusted to 3.0 with phosphoric acid; phase B is acetonitrile; the volume ratio of phase A to phase B is 70:30; isocratic elution;

[0042] The detection wavelength is 214nm;

[0043] The injection volume was 10 μl;

[0044] Flow rate: 1.0 ml / min;

[0045] The column temperature was 40°C.

[0046] The chromatogram of the test solution is as follows Figure 1 Among them, the elution time of sodium pentobarbital is about 11.1 minutes, the elution time of benzyl alcohol is about 5.6 minutes, and the separation degree is as high as 22.114; the theoretical plate number of the main peak of pentobarbital is 18568, the tailing factor is 0.95, and the theoretical plate number of the main peak of benzyl alcohol is 15929, and the tailing factor is 1.0.

[0047] Pentobarbital (C 11 H 18 N2O3,M W 226.27) and benzyl alcohol content, and then converted to obtain the content of pentobarbital sodium (C 11 H 17 N2NaO3,M W248.25) and benzyl alcohol content: sodium pentobarbital 309 mg / ml (100.3% of the labeled amount), benzyl alcohol 23.16 mg / ml (115.8% of the labeled amount).

[0048] The following is a control test method.

[0049] Comparative Examples 1-4:

[0050] The detection method of Example 1 is referred to, except that the mobile phase is adjusted according to Table 1, including adjusting the pH value of phase A, controlling the ratio of phase A to phase B, and whether tetrabutylammonium hydrogen sulfate is added. The chromatogram analysis results of the obtained test solution are shown in Table 1.

[0051] Table 1: Comparison of test results under chromatographic conditions of Example 1 and Comparative Examples 1-4

[0052]

[0053] It can be seen that compared with Control Examples 1-4, Example 1 of the present invention significantly improves the peak shape of pentobarbital (tailing factor 0.95) by adding tetrabutylammonium hydrogen sulfate to the mobile phase and controlling the pH value of the phase A aqueous solution (3.0), and can delay the elution time of benzyl alcohol to an appropriate time (5.6 min), thereby improving the column efficiency (theoretical plate number 15929).

[0054] Comparative Example 5:

[0055] The preparation methods of the reference solution and the test solution are the same as those in Example 1;

[0056] The detection was carried out according to the chromatographic conditions recorded in the British Pharmacopoeia 2023 edition: octadecylsilane bonded silica gel was used as the filler, 1.36 g / L potassium dihydrogen phosphate (the pH value was adjusted to 3.5 with phosphoric acid) and acetonitrile (65:35) as the mobile phase; the detection wavelength was 214 nm.

[0057] The chromatogram of the test solution is as follows Figure 2 The retention time of the main peak of pentobarbital is 10.5min, with obvious tailing (tailing factor 1.2), and the peak of benzyl alcohol is early (retention time 3.5min), and the column efficiency is not high (theoretical plate number 8393).

[0058] According to the requirements for tailing factor and repeatability in the 2020 edition of the Chinese Pharmacopoeia, Part IV, Monograph 0512, High Performance Liquid Chromatography (page 63): 1. When peak height is used as a quantitative parameter, unless otherwise specified, the tailing factor should be between 0.95 and 1.05; 2. After 5 consecutive injections, the relative standard deviation of the peak area measurement value should not be greater than 2.0%. It can be seen that the tailing factor of the main peak of pentobarbital obtained by the detection method recorded in the 2023 edition of the British Pharmacopoeia does not meet the above requirements and is not suitable for the simultaneous detection of the content of sodium pentobarbital and benzyl alcohol in sodium pentobarbital solution preparations.

[0059] Compared with the detection method recorded in the British Pharmacopoeia 2023 edition, the detection method of Example 1 of the present invention not only significantly improved the peak shape of the main peak of pentobarbital (tailing factor 0.95), but also significantly improved the column efficiency (theoretical plate number 15929).

[0060] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0061] Experimental Example 1: Evaluation of the detection effect of the detection method of the present invention on the content of pentobarbital in pentobarbital sodium solution preparation

[0062] 1.1 Specificity and system suitability

[0063] The chromatographic conditions used in this experiment are the same as those in Example 1.

[0064] a. Solution Preparation

[0065] Diluent: Mobile phase.

[0066] System suitability solution: Accurately weigh 108.3 mg of pentobarbital reference substance, place in a 100 ml volumetric flask, dissolve and dilute to the scale with diluent, shake well, then accurately measure 10 ml, place in a 100 ml volumetric flask, dilute to the scale with diluent, shake well.

[0067] Test solution: Take sodium pentobarbital solution, accurately measure 1 ml of the product, place it in a 50 ml volumetric flask, dilute to the scale with diluent, shake well, accurately measure 1 ml again, place it in a 50 ml volumetric flask, dilute to the scale with diluent, shake well, and obtain.

[0068] Accurately measure 10 μl of each of the above solutions, inject into the liquid chromatograph, and record the chromatogram. The results are shown in the table below.

[0069] b. Acceptable limits

[0070] The diluent shows no chromatographic peak or does not interfere with the chromatographic peaks of known components;

[0071] In the system suitability solution, the number of theoretical plates, calculated based on the pentobarbital peak, should be no less than 15,000.

[0072] In the system suitability solution, the tailing factor should be 0.9-1.1 based on the pentobarbital peak.

[0073] In the system suitability solution, the reference solution is injected 6 times continuously, and the RSD of the pentobarbital peak area shall not exceed 2.0%.

[0074] In the test solution, the peak purity of the pentobarbital chromatographic peak should meet the requirements (if applicable, the peak purity in Labsolution should be not less than 0.980).

[0075] Table 2: System suitability and specificity validation results for pentobarbital content method validation

[0076] Thinner No chromatographic peaks were shown for diluent System Suitability Solutions The number of theoretical plates is 18568 based on the pentobarbital peak. System Suitability Solutions The tailing factor is based on the pentobarbital peak and is 0.949 System Suitability Solutions The reference solution was injected 6 times continuously, and the RSD of the peak area was 0 Test solution In the test solution, the peak purity of the pentobarbital chromatographic peak is 0.999998

[0077] Conclusion: The system suitability and specificity validation results meet the requirements.

[0078] 1.2 Linearity and range

[0079] The chromatographic conditions used in this experiment are the same as those in Example 1, and the linear relationship of at least 5 points within the range of 10% to 200% (i.e. 2.0%) of pentobarbital is investigated.

[0080] The linear regression equation was calculated with concentration as the abscissa and peak area as the ordinate. The correlation coefficient R should be no less than 0.995, and the RSD of three injections of each concentration linear solution should be no more than 2.0%.

[0081] Pentobarbital reference substance stock solution: Accurately weigh 113.2 mg of pentobarbital reference substance, place in a 100 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and obtain.

[0082] Accurately measure an appropriate amount of "pentobarbital reference stock solution" and dilute it with a diluent to prepare a series of reference solutions in the range of limit concentration 10% to limit concentration 200%.

[0083] Table 3: Linearization solution preparation

[0084] Linear solution Stock solution Volume measured (mL) Dilution volume (mL) Linear solution 1 Linear solution 3 1.0 10 Linear solution 2 Reference stock solution 0.5 10 Linear solution 3 Reference stock solution 1.0 10 Linear solution 4 Reference stock solution 1.5 10 Linear solution 5 Reference stock solution 2.0 10

[0085] Accurately measure 10 μl of each of the above solutions, inject into the liquid chromatograph, and record the chromatogram. The results are shown in the table below.

[0086] Table 4: Pentobarbital linearity results

[0087]

[0088] The linear regression equation and linear relationship diagram of pentobarbital content are shown in Figure 3 The verification result meets the requirements.

[0089] 1.3 Precision (repeatability)

[0090] The chromatographic conditions used in this experiment were the same as those in Example 1. The same batch of preparations was used and the same person repeated the operation 6 times to examine the %RSD of the pentobarbital content. Six test sample solutions were prepared in parallel according to the method, and each solution was injected with one injection for testing.

[0091] a. Solution Preparation

[0092] Pentobarbital reference solution 1: Accurately weigh 108.3 mg of pentobarbital reference solution, place in a 100 ml volumetric flask, dissolve and dilute to the mark with diluent, shake well, then accurately measure 10 ml, place in a 100 ml volumetric flask, dilute to the mark with diluent, shake well, and obtain.

[0093] Pentobarbital reference solution 2: Accurately weigh 114.3 mg of pentobarbital reference solution, place in a 100 ml volumetric flask, dissolve and dilute to the scale with diluent, shake well, then accurately measure 10 ml, place in a 100 ml volumetric flask, dilute to the scale with diluent, shake well, and obtain.

[0094] Test solution: Take sodium pentobarbital solution, accurately measure 1 ml of this product, place it in a 50 ml volumetric flask, dilute it to the mark with diluent, shake well, accurately measure 1 ml again, place it in a 50 ml volumetric flask, dilute it to the mark with diluent, shake well, and you have it. Prepare 6 copies in parallel.

[0095] b. Acceptable limits

[0096] The %RSD (n=6) of the pentobarbital content in the 6 test samples should not be greater than 2.0%. The results are shown in the table below.

[0097] Table 5: Pentobarbital content repeatability verification results

[0098]

[0099] Conclusion: The precision (repeatability) verification results meet the requirements.

[0100] 1.4 Accuracy

[0101] The chromatographic conditions used in this experiment were the same as those in Example 1. The accuracy was determined by adding three different concentrations of pentobarbital reference stock solutions of 80%, 100%, and 120% of the limit to the test sample, then measuring the amount of pentobarbital in the added sample, and calculating the ratio (recovery rate) between the measured amount and the theoretical value, expressed as a percentage.

[0102] a. Preparation of solution:

[0103] Pentobarbital reference solution 1: Accurately weigh 109.2 mg of pentobarbital reference, place in a 100 ml volumetric flask, dissolve and dilute to the mark with diluent, then accurately measure 10 ml, place in a 100 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0104] Pentobarbital reference solution 2: Accurately weigh 108.8 mg of pentobarbital reference, place in a 100 ml volumetric flask, dissolve and dilute to the mark with diluent, then accurately measure 10 ml, place in a 100 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0105] Pentobarbital reference substance stock solution: Accurately weigh 119.2 mg of pentobarbital reference substance, place it in a 10 ml volumetric flask, dissolve it with diluent and dilute to the scale, and shake well.

[0106] Preparation of 80% limit concentration solution: accurately measure 1 ml of this product, place it in a 50 ml volumetric flask, dilute it to the mark with diluent, shake well, accurately measure 1 ml again, place it in a 100 ml volumetric flask, accurately add 0.3 ml of pentobarbital reference stock solution to this volumetric flask, dilute it to the mark with diluent, shake well, and you have it. Prepare 3 copies in parallel.

[0107] Preparation of 100% limit concentration solution: accurately measure 1 ml of this product, place it in a 50 ml volumetric flask, dilute it to the mark with diluent, shake well, accurately measure 1 ml again, place it in a 100 ml volumetric flask, accurately add 0.5 ml of pentobarbital reference stock solution to this volumetric flask, dilute it to the mark with diluent, shake well, and you have it. Prepare 3 copies in parallel.

[0108] Preparation of 120% limit concentration solution: accurately measure 1 ml of this product, place it in a 50 ml volumetric flask, dilute it to the mark with diluent, shake well, accurately measure 1 ml again, place it in a 100 ml volumetric flask, accurately add 0.5 ml of pentobarbital reference stock solution to this volumetric flask, dilute it to the mark with diluent, shake well, and you have it. Prepare 3 copies in parallel.

[0109] Preparation of test solution: Accurately measure 1 ml of the product, place it in a 50 ml volumetric flask, dilute it to the scale with diluent, shake well, then accurately measure 1 ml, place it in a 100 ml volumetric flask, dilute it to the scale with diluent, shake well, and obtain.

[0110] b. Acceptable limits:

[0111] The recoveries of the 9 sample solutions should be within the range of 98.0%-102.0%, and the cumulative RSD (n=9) of the recoveries at different concentrations should not be greater than 2.0%.

[0112] Accurately measure 10 μl of each of the above solutions, inject them into the liquid chromatograph, and record the chromatogram. The results are shown in the table below.

[0113] Table 6: Pentobarbital accuracy results

[0114]

[0115] Conclusion: The accuracy verification results meet the requirements.

[0116] 1.5 Solution stability

[0117] The chromatographic conditions used in this experiment are the same as those in Example 1.

[0118] a. Solution Preparation

[0119] Pentobarbital reference solution: Accurately weigh 108.3 mg of pentobarbital reference solution, place in a 100 ml volumetric flask, dissolve and dilute to the scale with diluent, shake well, then accurately measure 10 ml, place in a 100 ml volumetric flask, dilute to the scale with diluent, shake well, and obtain.

[0120] Test solution: Accurately measure 1 ml of the product, place it in a 50 ml volumetric flask, dilute it to the scale with diluent, shake well, then accurately measure 1 ml, place it in a 50 ml volumetric flask, dilute it to the scale with diluent, shake well, and you have it.

[0121] b. Acceptable limits

[0122] The %RSD of the main component peak area in the test solution and the reference solution at each time point should not be greater than 2.0%.

[0123] Place the reference solution and the test solution at room temperature, and accurately measure 10 μl at 0, 4, 13, 17, and 26 hours, inject it into the liquid chromatograph, and record the chromatogram. The results are shown in the table below.

[0124] Table 7: Pentobarbital solution stability test sample verification results

[0125] name 0h 4h 13h 17h 26h RSD% Reference 1955826 1958798 1957007 1956307 1957436 0.1 Test product 2014156 2019255 2017027 2018323 2016878 0.1

[0126] Conclusion: The test solution is stable within 26 hours at room temperature.

[0127] 1.6 Durability

[0128] On the basis of the chromatographic conditions of Example 1, the flow rate, mobile phase ratio, mobile phase pH value and column temperature were changed to evaluate the tolerance level of the pentobarbital assay results not being affected by slight changes in the assay conditions.

[0129] Table 8: Chromatographic conditions of pentobarbital content

[0130]

[0131] a. Solution Preparation

[0132] Pentobarbital reference solution 1: Accurately weigh 108.3 mg of pentobarbital reference, place in a 100 ml volumetric flask, dissolve and dilute to the mark with diluent, then accurately measure 10 ml, place in a 100 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0133] Pentobarbital reference solution 2: Accurately weigh 114.3 mg of pentobarbital reference, place in a 100 ml volumetric flask, dissolve and dilute to the mark with diluent, then accurately measure 10 ml, place in a 100 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0134] Test solution: Accurately measure 1 ml of the product, place it in a 50 ml volumetric flask, dilute it to the scale with diluent, shake well, then accurately measure 1 ml, place it in a 50 ml volumetric flask, dilute it to the scale with diluent, shake well, and you have it.

[0135] b. Acceptable limits

[0136] The applicability of each parameter adjustment system meets the requirements;

[0137] The relative difference between the pentobarbital content in the test solution after adjustment of each parameter and the result under standard conditions should not be greater than 2.0%.

[0138] Accurately measure 10 μl of the reference solution and the test solution and inject them into the liquid chromatograph to record the chromatogram. The results are shown in the table below.

[0139] Table 9: Pentobarbital content durability system suitability verification results

[0140]

[0141] Table 10: Pentobarbital content test sample durability verification results

[0142]

[0143] Conclusion: The durability verification results meet the requirements.

[0144] Experimental Example 2: Evaluation of the detection effect of the detection method of the present invention on the content of benzyl alcohol in pentobarbital sodium solution preparation

[0145] 2.1 Exclusivity

[0146] The chromatographic conditions used in this experiment are the same as those in Example 1.

[0147] a. Solution Preparation

[0148] Diluent: Mobile phase

[0149] System suitability solution: Accurately weigh 85.2 mg of benzyl alcohol reference substance, place in a 100 ml volumetric flask, dilute to the mark with diluent, shake well, then accurately measure 1 ml, place in a 100 ml volumetric flask, dilute to the mark with diluent, shake well, and obtain.

[0150] Test solution: Accurately measure 1 ml of the product, place it in a 50 ml volumetric flask, dilute it to the scale with diluent, shake well, then accurately measure 1 ml, place it in a 50 ml volumetric flask, dilute it to the scale with diluent, shake well, and you have it.

[0151] Accurately measure 10 μl of each of the above solutions, inject into the liquid chromatograph, and record the chromatogram. The results are shown in the table below.

[0152] b. Acceptable limits

[0153] The diluent shows no chromatographic peak or does not interfere with the chromatographic peaks of known components;

[0154] In the system suitability solution, the number of theoretical plates, calculated based on the benzyl alcohol peak, should be no less than 10,000.

[0155] In the system suitability solution, the tailing factor is based on the benzyl alcohol peak and the tailing factor is 0.9-1.1.

[0156] In the system suitability solution, the reference solution is injected 6 times continuously, and the RSD of the benzyl alcohol peak area shall not exceed 2.0%.

[0157] In the test solution, the peak purity of the benzyl alcohol chromatographic peak should meet the requirements (if applicable, the peak purity in Labsolution should be not less than 0.980).

[0158] Table 11: System suitability and specificity validation results for benzyl alcohol content method validation

[0159] Thinner No chromatographic peaks were shown for diluent System Suitability Solutions The number of theoretical plates is 15929 based on the benzyl alcohol peak. System Suitability Solutions The tailing factor is based on the benzyl alcohol peak and is 1.039 System Suitability Solutions The reference solution was injected 6 times continuously, and the RSD of the peak area was 0.1% Test solution The peak purity of the benzyl alcohol chromatographic peak is 0.999958

[0160] Conclusion: The system suitability and specificity validation results meet the requirements.

[0161] 2.2 Linearity

[0162] The chromatographic conditions used in this experiment were the same as those in Example 1, and the linear relationship of at least 5 points in the range of 10% to 200% of benzyl alcohol was examined.

[0163] a. Solution Preparation

[0164] Benzyl alcohol reference stock solution: Accurately weigh 85.2 mg of benzyl alcohol reference, place in a 100 ml volumetric flask, add diluent to dilute to the scale, shake well, and obtain.

[0165] Accurately measure an appropriate amount of "benzyl alcohol reference stock solution" and dilute it with a diluent to prepare a series of reference solutions in the range of limit concentration 10% to limit concentration 200%.

[0166] Table 12: Linearity solution preparation

[0167] Linear solution Stock solution Volume measured (mL) Dilution volume (mL) Linear solution 1 Linear solution 3 1.0 10 Linear solution 2 Reference stock solution 0.5 10 Linear solution 3 Reference stock solution 1.0 10 Linear solution 4 Reference stock solution 1.5 10 Linear solution 5 Reference stock solution 2.0 10

[0168] b. Acceptable limits

[0169] Linearity: With concentration as the abscissa and peak area as the ordinate, calculate the linear regression equation of linear solutions 1 to 5. The correlation coefficient R should be no less than 0.995, and the RSD of three injections of each concentration of linear solution should be no more than 2.0%.

[0170] Range: Precision, accuracy and linearity should be good within the range of 10% to 200%.

[0171] Accurately measure 10 μl of each of the above solutions, inject into the liquid chromatograph, and record the chromatogram. The results are shown in the table below.

[0172] Table 13: Benzyl alcohol linearity results

[0173]

[0174] The linear relationship diagram and linear equation of benzyl alcohol content are as follows: Figure 4 shown.

[0175] Conclusion: The linear verification results meet the requirements.

[0176] 2.3 Precision (repeatability)

[0177] The chromatographic conditions used in this experiment were the same as those in Example 1. The same batch of preparations was used and the same person repeated the operation 6 times to examine the %RSD of the benzyl alcohol content. Six test sample solutions were prepared in parallel according to the method, and each solution was injected with one injection for testing.

[0178] a. Solution Preparation

[0179] Benzyl alcohol reference solution 1: Accurately weigh 85.2 mg of benzyl alcohol reference, place in a 100 ml volumetric flask, dilute to the mark with diluent, then accurately measure 1 ml, place in a 100 ml volumetric flask, dilute to the mark with diluent, shake well, and obtain.

[0180] Benzyl alcohol reference solution 2: Accurately weigh 84.4 mg of benzyl alcohol reference, place in a 100 ml volumetric flask, dilute to the mark with diluent, then accurately measure 1 ml, place in a 100 ml volumetric flask, dilute to the mark with diluent, shake well, and obtain.

[0181] Test solution: accurately measure 1 ml of the product, place it in a 50 ml volumetric flask, dilute to the mark with diluent, shake well, accurately measure 1 ml again, place it in a 50 ml volumetric flask, dilute to the mark with diluent, shake well, and the solution is ready. Prepare 6 copies in parallel.

[0182] b. Acceptable limits

[0183] The %RSD (n=6) of the benzyl alcohol content in the 6 test samples should not be greater than 2.0%.

[0184] Accurately measure 10 μl of each of the above solutions, inject into the liquid chromatograph, and record the chromatogram. The results are shown in the table below.

[0185] Table 14: Benzyl alcohol repeatability verification results

[0186]

[0187] Conclusion: The precision (repeatability) verification results meet the requirements.

[0188] 2.4 Accuracy

[0189] The chromatographic conditions used in this experiment were the same as those in Example 1. The accuracy was determined by adding three different concentrations of benzyl alcohol reference stock solutions of 80%, 100%, and 120% of the limit to the test sample, then measuring the amount of benzyl alcohol in the added sample, and calculating the ratio between the measured amount and the theoretical value (recovery rate), expressed as a percentage.

[0190] a. Solution Preparation

[0191] Benzyl alcohol reference solution 1: Accurately weigh 78.0 mg of benzyl alcohol reference, place in a 100 ml volumetric flask, dilute to the mark with diluent, then accurately measure 1 ml, place in a 100 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0192] Benzyl alcohol reference solution 2: Accurately weigh 84.2 mg of benzyl alcohol reference, place in a 100 ml volumetric flask, dilute to the mark with diluent, then accurately measure 1 ml, place in a 100 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0193] Benzyl alcohol reference stock solution: Accurately weigh 87.4 mg of benzyl alcohol reference, place in a 100 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0194] Preparation of 80% limit concentration solution: accurately measure 1 ml of this product, place it in a 50 ml volumetric flask, dilute it to the mark with diluent, shake well, accurately measure 1 ml again, place it in a 100 ml volumetric flask, accurately add 0.3 ml of benzyl alcohol reference stock solution to this volumetric flask, dilute it to the mark with diluent, shake well, and the solution is ready. Prepare 3 copies in parallel.

[0195] Preparation of 100% limit concentration solution: accurately measure 1 ml of this product, place it in a 50 ml volumetric flask, dilute it to the mark with diluent, shake well, accurately measure 1 ml again, place it in a 100 ml volumetric flask, accurately add 0.5 ml of benzyl alcohol reference stock solution to this volumetric flask, dilute it to the mark with diluent, shake well, and the solution is ready. Prepare 3 copies in parallel.

[0196] Preparation of 120% limit concentration solution: accurately measure 1 ml of this product, place it in a 50 ml volumetric flask, dilute it to the mark with diluent, shake well, accurately measure 1 ml again, place it in a 100 ml volumetric flask, accurately add 0.7 ml of benzyl alcohol reference stock solution to this volumetric flask, dilute it to the mark with diluent, shake well, and the solution is ready. Prepare 3 copies in parallel.

[0197] Preparation of test solution: Accurately measure 1 ml of the product, place it in a 50 ml volumetric flask, dilute it to the scale with diluent, shake well, then accurately measure 1 ml, place it in a 100 ml volumetric flask, dilute it to the scale with diluent, shake well, and obtain.

[0198] b. Acceptable limits

[0199] Calculate the content of benzyl alcohol in each added test solution, and compare it with the actual added content to calculate the recovery rate. The recovery rate should be in the range of 98.0% to 102.0%; the cumulative RSD (n=9) of the recovery rate at different concentrations is not greater than 2.0%.

[0200] Accurately measure 10 μl of each of the above solutions, inject into the liquid chromatograph, and record the chromatogram. The results are shown in the table below.

[0201] Table 15: Benzyl alcohol accuracy verification results

[0202]

[0203] Conclusion: The accuracy verification results meet the requirements.

[0204] 2.5 Solution stability

[0205] The chromatographic conditions used in this experiment are the same as those in Example 1.

[0206] a. Solution Preparation

[0207] Benzyl alcohol reference solution: Accurately weigh 85.2 mg of benzyl alcohol reference, place in a 100 ml volumetric flask, dilute to the mark with diluent, then accurately measure 1 ml, place in a 100 ml volumetric flask, dilute to the mark with diluent, shake well, and obtain.

[0208] Test solution: Accurately measure 1 ml of the product, place it in a 50 ml volumetric flask, dilute it to the scale with diluent, shake well, then accurately measure 1 ml, place it in a 50 ml volumetric flask, dilute it to the scale with diluent, shake well, and you have it.

[0209] b. Acceptable limits

[0210] The %RSD of the main component peak area in the test solution and the reference solution at each time point should not be greater than 2.0%.

[0211] Place the reference solution and the test solution at room temperature, and accurately measure 10 μl at 0, 4, 13, 17, and 26 hours, inject it into the liquid chromatograph, and record the chromatogram. The results are shown in the table below.

[0212] Table 16: Benzyl alcohol solution stability verification results

[0213] name 0h 4h 13h 17h 26h RSD% Reference 213177 213440 213508 213589 213633 0.1 Test product 245176 245514 245459 245637 245662 0.1

[0214] Conclusion: The test solution and the reference solution are stable within 24 hours at room temperature.

[0215] 2.6 Durability

[0216] On the basis of the chromatographic conditions of Example 1, the flow rate, mobile phase ratio, mobile phase pH value and column temperature were changed to evaluate the tolerance of the content determination results not being affected when the determination conditions were slightly changed.

[0217] Table 17: Parameters of chromatographic conditions for benzyl alcohol content

[0218]

[0219] a. Solution Preparation

[0220] Benzyl alcohol reference solution 1: Accurately weigh 85.2 mg of benzyl alcohol reference, place in a 100 ml volumetric flask, dilute to the mark with diluent, then accurately measure 1 ml, place in a 100 ml volumetric flask, dilute to the mark with diluent, shake well, and obtain.

[0221] Benzyl alcohol reference solution 2: Accurately weigh 84.4 mg of benzyl alcohol reference, place in a 100 ml volumetric flask, dilute to the mark with diluent, then accurately measure 1 ml, place in a 100 ml volumetric flask, dilute to the mark with diluent, shake well, and obtain.

[0222] Test solution: Accurately measure 1 ml of the product, place it in a 50 ml volumetric flask, dilute it to the scale with diluent, shake well, then accurately measure 1 ml, place it in a 50 ml volumetric flask, dilute it to the scale with diluent, shake well, and you have it.

[0223] b. Acceptable limits

[0224] The applicability of each parameter adjustment system meets the requirements;

[0225] The relative difference between the benzyl alcohol content in the test solution after adjustment of each parameter and the result under standard conditions should not be greater than 2.0%.

[0226] Accurately measure 10 μl of the reference solution and the test solution and inject them into the liquid chromatograph to record the chromatogram. The results are shown in the table below.

[0227] Table 18: Benzyl alcohol content durability verification results

[0228]

[0229]

[0230] Table 19: Benzyl alcohol content durability verification results

[0231]

[0232] Conclusion: The durability verification results meet the requirements.

[0233] In summary, the results of the verification of the detection effect of detecting the content of pentobarbital in the sodium pentobarbital solution preparation using the method of the present invention are summarized in Table 20, and the results of the verification of the detection effect of detecting the content of benzyl alcohol in the sodium pentobarbital solution preparation are summarized in Table 21. The detection method is scientific and reasonable, has the advantages of being fast, simple, highly accurate, and highly specific, and is suitable for simultaneously detecting the contents of sodium pentobarbital and benzyl alcohol in the sodium pentobarbital solution preparation.

[0234] Table 20: Summary of the results of the test results for pentobarbital content in pentobarbital sodium solution preparations

[0235]

[0236]

[0237] Note: The system suitability standards in the table are self-proposed, and the other standards are general standards for guiding principles of drug quality standard research.

[0238] Table 21: Summary of the results of the test results for the content of benzyl alcohol in sodium pentobarbital solution preparations

[0239]

[0240]

[0241] Note: The system suitability standards in the table are self-proposed, and the other standards are general standards for guiding principles of drug quality standard research.

Claims

1. A method for simultaneously detecting the contents of sodium pentobarbital and alcohol antibacterial agent in a sodium pentobarbital solution preparation, the method comprising the following steps: (1) Preparation of reference solution: pentobarbital and alcohol antibacterial agent are diluted with a diluent to obtain a reference solution; the alcohol antibacterial agent is benzyl alcohol, and the diluent is a mobile phase; (2) Preparation of test solution: Take a sodium pentobarbital solution preparation and dilute it with a diluent to obtain a test solution; the sodium pentobarbital solution preparation contains an alcohol antibacterial agent; (3) The test solution and the reference solution are respectively aspirated and injected into a liquid chromatograph for detection to obtain a chromatogram, and then the contents of sodium pentobarbital and alcohol antibacterial agent in the sodium pentobarbital solution preparation are calculated; the chromatographic conditions for the detection are as follows: Chromatographic column: Gemini C18 column; Mobile phase: composed of phase A and phase B, wherein phase A is an aqueous solution containing potassium dihydrogen phosphate and tetrabutylammonium hydrogen sulfate, and the pH value of phase A is 2.8-3.2; phase B is acetonitrile; the volume ratio of phase A to phase B is (65-75): (35-25); Column temperature is 35-45°C; The detection wavelength is 214 nm.

2. The method according to claim 1, characterized in that In the phase A, the concentration of potassium dihydrogen phosphate is 0.005-0.02M, and the concentration of tetrabutylammonium hydrogen sulfate is 0.001-0.01M.

3. The method according to claim 2, characterized in that In the phase A, the concentration of potassium dihydrogen phosphate is 0.01 M, and the concentration of tetrabutylammonium hydrogen sulfate is 0.005 M.

4. The method according to claim 1, characterized in that: The pH value of phase A is 3.0, and the volume ratio of phase A to phase B is 70:

30.

5. The method according to claim 1, characterized in that: In the chromatographic conditions, the injection volume is 5-20 μl and the flow rate is 0.9-1.1 ml / min.

6. The method according to claim 5, characterized in that In the chromatographic conditions, the injection volume was 10 μl and the flow rate was 1.0 ml / min.

7. The method according to claim 6, characterized in that In the chromatographic conditions, the column temperature was 40°C.

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