Method for analyzing propylene glycol content by diazepam solution chromatograph and verification method thereof

Abstract

The invention discloses a method for analyzing propylene glycol content by a diazepam solution chromatograph and a verification method thereof, and relates to the field of medicine analysis, the key points of the technical scheme are as follows: a diazepam oral solution is analyzed by the chromatograph, and the conditions of the gas chromatograph are as follows: an SH-WAX polar capillary chromatographic column is used, the temperature of a sample inlet is 230 DEG C, and the temperature of a detector is 250 DEG C; the carrier gas is nitrogen, the flow velocity is 1.0 ml / min, and the split ratio is 10: 1; temperature programming is carried out, the initial temperature is kept at 130 DEG C for 1 minute, and the temperature is raised to 240 DEG C at the speed of 10 DEG C / min and kept for 3 minutes; determining the content of propylene glycol by using an internal standard method; the purposes of low analysis cost and high sensitivity are achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis, and more particularly to a method for analyzing propylene glycol content in a diazepam solution using a chromatograph and a verification method thereof. Background Art

[0002] Diazepam as a benzodiazepine For diazepam-type drugs, their preparations (such as injections and oral solutions) often contain propylene glycol as a cosolvent. However, the prior art generally believes that the propylene glycol content of diazepam solution is difficult to accurately analyze by chromatography. The reasons include: diazepam solution contains multiple excipients such as ethanol, benzyl alcohol, and propylene glycol, whose similar polarity leads to overlapping chromatographic peaks. Propylene glycol (boiling point 188°C) has too short a retention time on conventional chromatographic columns and is easily overlapped with solvent peaks, affecting quantitative accuracy. Diazepam is easily hydrolyzed under acidic or alkaline conditions to form 2-methylamino-5-chlorobenzophenone (impurity D), whose chromatographic behavior is similar to that of propylene glycol. Therefore, there is an urgent need to develop a highly specific and sensitive analytical method. Summary of the Invention

[0003] The present invention aims to provide a method for analyzing the propylene glycol content of a diazepam solution using a chromatograph and a verification method thereof, thereby achieving the goals of low analysis cost and high sensitivity.

[0004] The above technical purpose of the present invention is achieved through the following technical solutions: A method for analyzing the propylene glycol content of diazepam oral solution by chromatography, comprising the following steps: (1) solution preparation: taking an appropriate amount of diazepam oral solution, dissolving and diluting it with anhydrous ethanol to 2-3 mg / ml, to obtain a first test solution; taking an appropriate amount of a first internal standard, dissolving and diluting it with anhydrous ethanol to 15-20 mg / ml, to obtain a first internal standard solution; taking an appropriate amount of propylene glycol and an appropriate amount of the first internal standard solution, dissolving and diluting it with anhydrous ethanol to a propylene glycol concentration of 1-2 mg / ml, to obtain a first reference solution, and the first internal standard solution is used. The amount is 5% to 10% of the total volume of the first reference solution; (2) gas chromatography analysis: using an SH-WAX polar capillary column, the injection port temperature is 230°C, the detector temperature is 250°C; the carrier gas is nitrogen, the flow rate is 1.0 mL / min, and the split ratio is 10:1; program temperature rise, the initial temperature is 130°C and maintained for 1 minute, and then increased to 240°C at 10°C / min and maintained for 3 minutes; the first reference solution and the first test solution are injected respectively to obtain a chromatogram; (3) internal standard method to determine the propylene glycol content: the propylene glycol content is calculated by the peak area ratio of the internal standard to the propylene glycol, and the calculation formula is:

[0005]

[0006] Wherein, f is the correction factor value of the first reference solution, A wis the peak area of propylene glycol in the test sample, A s is the peak area of the first internal standard, C s is the concentration of the first internal standard, and m is the weight of the first test sample;

[0007]

[0008] Among them, A S is the peak area of the first internal standard, A R is the propylene glycol peak area of the first reference substance, C S is the concentration of the first internal standard, C R is the propylene glycol concentration in the first reference substance.

[0009] Furthermore, the first internal standard is 1,3-butanediol.

[0010] Furthermore, the first reference substance solution is configured in two portions, divided into a first reference substance solution group A and a first reference substance solution group B; the injection method is 5 injections of the first reference substance solution group A and 2 injections of the first reference substance solution group B; the correction factor value f of the first reference substance solution is the average value of f of a total of 7 injections of the first reference substance solution group A and group B.

[0011] In the second aspect, a verification method for analyzing the propylene glycol content of diazepam oral solution by chromatography is provided, which is used to verify the specificity, stability and other characteristics of the method in the first aspect. The verification method has low operating difficulty and ideal test results.

[0012] The above technical purpose of the present invention is achieved by the following technical solution: setting the gas chromatograph conditions as follows: using an SH-WAX polar capillary chromatographic column, an inlet temperature of 230°C, a detector temperature of 250°C; the carrier gas is nitrogen, the flow rate is 1.0 mL / min, and the split ratio is 10:1; program temperature increase, the initial temperature is 130°C and maintained for 1 minute, and then increased to 240°C at 10°C / min and maintained for 3 minutes; inputting the second reference solution into the gas chromatograph to obtain analysis results, the analysis results including multiple second reference solution solutions; if the relative standard deviation of the retention time of multiple second reference solution solutions is less than 1%, the relative standard deviation of the correction factor value of multiple second reference solution solutions is less than 2%, and the theoretical plate number of multiple second reference solution solutions is greater than 3000 0, then the system adaptability is determined to meet the requirements; the propylene glycol positioning solution, 1,3-butanediol positioning solution and the second test sample solution are input into the gas chromatograph to obtain analysis results; if the resolution between the component peak and the adjacent peak in the second test sample solution is ≥1.5, and the difference between the actual retention time of the component in the second test sample and the reference retention time of the propylene glycol positioning solution and the 1,3-butanediol positioning solution is ≤0.1min, then the specificity is determined to meet the requirements; 50%, 80%, 100%, 120% and 150% linear solutions are respectively input into the gas chromatograph to obtain analysis results, and a linear equation is established: propylene glycol peak area / 1,3-butanediol peak area and propylene glycol concentration / 1,3-butanediol concentration. If the correlation coefficient is ≥0.998, the ratio of the intercept to Y at 100% x is ≤5%. The linearity is determined to meet the requirements; 80%, 100%, and 120% spiked test solutions are respectively input into the gas chromatograph to obtain the analysis results, and the recovery rate and the relative standard deviation of the recovery rate are calculated. If the recovery rate is between 95% and 105%, and the relative standard deviation of the recovery rate is ≤5%, the accuracy is determined to meet the requirements; 6 parallel prepared third test solutions are input into the gas chromatograph to obtain the content of propylene glycol in each parallel prepared test solution. If the relative standard deviation of the content is ≤2%, the repeatability is determined to meet the requirements; 6 parallel prepared fourth test solutions are prepared, and the results are analyzed by single The operators (n=6) and double operators (n=12) operated the gas chromatograph to obtain the propylene glycol content. If the relative standard deviation of the propylene glycol content under the single operator condition is ≤2% and the relative standard deviation of the propylene glycol content under the double operator condition is ≤2%, the intermediate precision is judged to meet the requirements; the third reference solution and the fifth test solution are prepared and passed through the gas chromatograph at 0 hours, 4 hours, 8 hours, 12 hours, 24 hours, and 36 hours, respectively. If the relative standard deviations of the correction factors f of the third reference solution and the fifth test solution are both less than or equal to 2%, the stability is judged to meet the requirements.

[0013] Furthermore, the preparation method of the second reference solution is as follows: take an appropriate amount of 1,3-butanediol, dilute it with anhydrous ethanol to 20-25 mg / ml, and prepare it as a first internal standard stock solution; take an appropriate amount of propylene glycol and an appropriate amount of the first internal standard stock solution, dilute it with anhydrous ethanol to a propylene glycol concentration of 1-2 mg / ml, and the amount of the first internal standard stock solution is 5% to 10% of the total volume of the second reference solution.

[0014] Furthermore, the propylene glycol positioning solution uses anhydrous ethanol as a solvent, and the propylene glycol concentration is 1 to 1.5 mg / ml; the 1,3-butanediol positioning solution uses anhydrous ethanol as a solvent, and the 1,3-butanediol concentration is 1 to 2 mg / ml; the test sample in the second test solution is diazepam oral solution, the solvent is anhydrous ethanol, and the diazepam concentration is 2 to 3 mg / ml; the second test solution also contains a first internal standard stock solution, and the amount of the first internal standard stock solution is 5% to 10% of the total volume of the second test solution.

[0015] Furthermore, the linear solution preparation method is as follows: take an appropriate amount of propylene glycol, dissolve and dilute it with anhydrous ethanol to 10-15 mg / ml to obtain a first reference substance stock solution; take an appropriate amount of 1,3-butanediol, dissolve and dilute it with anhydrous ethanol to 10-15 mg / ml to obtain a second internal standard stock solution; prepare a linear solution by using the first reference substance stock solution, the second internal standard stock solution and anhydrous ethanol; every 10 ml of 50% linear solution contains 0.5 ml of the first reference substance stock solution and 1 ml of the second internal standard stock solution; every 10 ml of 80% linear solution contains 0.8 ml of the first reference substance stock solution and 1 ml of the second internal standard stock solution; every 10 ml of 100% linear solution contains 1 ml of the first reference substance stock solution and 1 ml of the second internal standard stock solution; every 10 ml of 120% linear solution contains 1.2 ml of the first reference substance stock solution and 1 ml of the second internal standard stock solution; every 10 ml of 150% linear solution contains 1.5 ml of the first reference substance stock solution and 1 ml of the second internal standard stock solution.

[0016] Furthermore, the preparation method of the spiked test solution is as follows: take an appropriate amount of 1,3-butanediol, dissolve and dilute it with anhydrous ethanol to 18-25 mg / ml to obtain a third internal standard stock solution; take an appropriate amount of propylene glycol, dissolve and dilute it with anhydrous ethanol to 20-25 mg / ml to obtain a second reference substance stock solution; prepare the spiked test solution by using the second reference substance stock solution, the third internal standard stock solution, diazepam oral solution and anhydrous ethanol; prepare 80% spiked test solution for every 20 ml Each 20 ml of 100% spiked test solution contains 0.5 ml of the second reference stock solution, 1 ml of the third internal standard stock solution, and 20-30 mg of diazepam oral solution; each 20 ml of 120% spiked test solution contains 0.7 ml of the second reference stock solution, 1 ml of the third internal standard stock solution, and 20-30 mg of diazepam oral solution.

[0017] Furthermore, the third test solution is prepared as follows: take an appropriate amount of 1,3-butanediol, dissolve it in anhydrous ethanol and dilute it to 20-22 mg / ml to obtain a fourth internal standard stock solution; the third test solution is prepared by diazepam oral solution, the fourth internal standard stock solution and anhydrous ethanol; each 20 ml of the third test solution contains 1 ml of the fourth internal standard stock solution and 45-60 mg of diazepam oral solution.

[0018] Furthermore, the preparation method of the third reference solution and the fifth test solution is as follows: take an appropriate amount of 1,3-butanediol, dissolve and dilute it with anhydrous ethanol to 19-22 mg / ml to obtain a sixth internal standard stock solution; the third reference solution is prepared by propylene glycol, the sixth internal standard stock solution and anhydrous ethanol; the fifth test solution is prepared by diazepam oral solution, the sixth internal standard stock solution and anhydrous ethanol; every 20 ml of the third reference solution contains 1 ml of the sixth internal standard stock solution and 20-25 mg of propylene glycol solution; every 20 ml of the fifth test solution contains 1 ml of the sixth internal standard stock solution and 50-55 mg of propylene glycol solution.

[0019] In summary, the present invention has the following beneficial effects: by optimizing the chromatographic conditions, the problem of chromatographic peak interference between propylene glycol and excipients and impurities in diazepam solution is effectively solved, and highly specific and sensitive quantitative analysis is achieved. The method is simple to operate and has comprehensive verification, meeting the strict requirements of drug quality control. BRIEF DESCRIPTION OF THE DRAWINGS DETAILED DESCRIPTION

[0020] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments.

[0021] The terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features specified as "first" or "second" may explicitly or implicitly include one or more of such features. In the description of the present invention, "plurality" means two or more, unless otherwise specifically defined.

[0022] Example 1:

[0023] A method for analyzing propylene glycol content in a diazepam solution using a chromatograph and a verification method thereof

[0024] Solution preparation:

[0025] Diluent: anhydrous ethanol

[0026] Blank solution: diluent

[0027] First internal standard solution: Accurately weigh about 400 mg of 1,3-butanediol, place it in a 20 ml volumetric flask, dissolve it in diluent and dilute to the scale, and shake well.

[0028] First reference solution: Accurately weigh 20 mg of propylene glycol reference solution and 1 ml of internal standard solution, place them in the same 20 ml volumetric flask, dilute to the mark with diluent, and shake well. Prepare two portions in parallel, each as first reference solution A and first reference solution B.

[0029] First test solution: Accurately weigh 50 mg of diazepam oral solution, place it in a 20 ml volumetric flask, dissolve it with diluent and dilute to the scale, and shake well.

[0030] Chromatographic conditions:

[0031] An SH-WAX polar capillary column (30 m × 0.25 mm, 0.25 μm) was used with an inlet temperature of 230°C and a detector temperature of 250°C. The carrier gas was nitrogen at a flow rate of 1.0 mL / min and a split ratio of 10:1. The temperature was programmed, starting at 130°C for 1 minute, then increasing at 10°C / min to 240°C and holding for 3 minutes.

[0032] Injection procedure:

[0033] Blank solution, 1 injection; first reference solution A, 5 injections; first reference solution B, 2 injections, first test solution 2 injections.

[0034] System recovery calculation:

[0035] F=(C R / A R ) / (C i / A i ) (Formula 1)

[0036] Where: C R is the propylene glycol concentration of the first reference solution A; A R is the peak area of propylene glycol in the first control solution A;

[0037] C i is the concentration of 1.3-butanediol in the first reference solution A; A i is the peak area of 1.3-butanediol in the first reference solution A.

[0038] Recovery rate % = f × (C i / A i )×A R / C R ×100(Formula 2)

[0039] Where: f is the average of the f values in 5 reference solutions A; C i A is the concentration of 1.3-butanediol in the first reference solution B; i is the average peak area of 1.3-butanediol in reference solution B; A R is the average peak area of propylene glycol in the first reference solution B; C R is the concentration of propylene glycol in the first reference solution B.

[0040] Propylene glycol content calculation

[0041] The propylene glycol content was calculated by the peak area ratio of the internal standard to the propylene glycol. The calculation formula is:

[0042]

[0043] Wherein, f is the correction factor value of the first reference solution, A w is the peak area of propylene glycol in the test sample, A s is the peak area of the first internal standard, C s is the concentration of the first internal standard, and m is the weight of the first test sample;

[0044]

[0045] Among them, A S is the peak area of the first internal standard, A R is the propylene glycol peak area of the first reference substance, C S is the concentration of the first internal standard, C R is the propylene glycol concentration in the first reference substance.

[0046] Example 2:

[0047] A validated method for chromatographic analysis of propylene glycol content in diazepam oral solution.

[0048] Verification items:

[0049] System suitability, specificity, linearity, accuracy, repeatability, precision, and stability.

[0050] Chromatographic conditions:

[0051] An SH-WAX polar capillary column (30 m × 0.25 mm, 0.25 um) was used, with an inlet temperature of 230°C and a detector temperature of 250°C. The carrier gas was nitrogen with a flow rate of 1.0 mL / min and a split ratio of 10:1. The temperature was programmed, with an initial temperature of 130°C maintained for 1 minute, then increased to 240°C at a rate of 10°C / min and maintained for 3 minutes.

[0052] System suitability verification

[0053] Diluent: anhydrous ethanol

[0054] Blank solution: diluent

[0055] First internal standard stock solution: Accurately weigh 1,3-butanediol, place it in a 20ml volumetric flask, dissolve it in diluent and dilute to the scale, and shake well.

[0056] Second Reference Solution: Accurately weigh propylene glycol reference solution and pipette 1 mL of the first internal standard stock solution into the same 20 mL volumetric flask. Dissolve and dilute to volume with diluent and shake well. Prepare two parallel aliquots to create Second Reference Solution A and Second Reference Solution B.

[0057] Three tests of system suitability:

[0058] Dosage of 1,3-butanediol: first time, 403.20 mg; second time, 400.56 mg; third time: 399.94 mg.

[0059] Propylene glycol dosage: 20.11mg, 20.12mg for the first time; 20.79mg, 20.48mg for the second time; 21.98mg, 22.70mg for the third time.

[0060] Needle insertion procedure:

[0061] Second reference solution A, 5 injections; second reference solution B, 2 injections.

[0062] The first system suitability results are shown in the following table

[0063]

[0064] The second system suitability results are shown in the following table:

[0065]

[0066] The results of the third system suitability are shown in the following table:

[0067]

[0068]

[0069] The recovery rate (%) is calculated using Formula 1 and Formula 2. Those skilled in the art should know that when applying the formula, the relevant code of the first reference solution in the formula should be replaced by the second reference solution. For example: C in Formula 2 i is the concentration of 1.3-butanediol in the first reference solution B, where C i is the concentration of 1,3-butanediol in the second reference solution B. In this patent, unless otherwise specified, those skilled in the art should be aware of the code replacement method when applying the formula.

[0070] Conclusion: The relative standard deviation (RSD) of retention time (RT) was 0.02% to 0.10%, less than 1.0%; the RSD of f value was 0.15% to 0.26%, less than 2.0%; and the number of theoretical plates was greater than 30,000. The method has good system applicability and meets the applicability requirements.

[0071] Example 3:

[0072] Specificity Verification

[0073] Diluent: anhydrous ethanol

[0074] Blank solution: diluent

[0075] Propylene glycol reference solution: Accurately weigh 22.65 mg of propylene glycol reference substance into a 20 ml volumetric flask, dissolve it in diluent and dilute to the mark, shake well;

[0076] 1,3-Butanediol positioning stock solution: Accurately weigh 403.20 mg of 1,3-butanediol, place it in a 20 ml volumetric flask, dissolve it with diluent and dilute to the scale, and shake well.

[0077] 1,3-Butanediol positioning solution: Accurately pipette 1 ml of 1,3-butanediol positioning stock solution into a 20 ml volumetric flask, dilute to the scale with diluent, and shake well.

[0078] Negative blank solution: Accurately weigh sorbitol (18.7 mg), polyethylene glycol 1000 (13.6 mg), citric acid (10.9 mg), and diazepam (101.3 mg) in the same 100 ml volumetric flask, dissolve them with diluent and dilute to the mark.

[0079] Glycerol positioning solution: accurately weigh 27.38 mg of glycerol, place it in a 50 ml volumetric flask, dissolve it with diluent and dilute to the scale, shake well;

[0080] Second test solution: Accurately weigh 50.57 mg of diazepam oral solution, accurately pipette 1 ml of internal standard stock solution, place it in the same 20 ml volumetric flask, dissolve it and dilute it to the scale with diluent, and shake well.

[0081] result:

[0082]

[0083] RT in the table refers to retention time.

[0084] Conclusion: Blank, impurities and other excipients had no interference with the determination. The separation between each component peak and adjacent peaks was ≥1.5; the RT difference was ≤0.1min. The specificity of the verification method met the requirements.

[0085] Example 4:

[0086] Linear validation

[0087] First reference substance stock solution: Accurately weigh 102.44 mg of propylene glycol reference substance, place it in a 10 ml volumetric flask, dilute to the scale with diluent, and shake well.

[0088] Second internal standard stock solution: Accurately weigh 203.36 mg of 1,3-butanediol, place it in a 20 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0089] 50% linear solution: Accurately pipette 0.5 ml of the first reference stock solution and 1 ml of the second internal standard stock solution into the same 10 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0090] 80% linear solution: Accurately pipette 0.8 ml of the first reference stock solution and 1 ml of the second internal standard stock solution into the same 10 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0091] 100% linear solution: Accurately pipette 1.0 ml of the first reference stock solution and 1 ml of the second internal standard stock solution into the same 10 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0092] 120% linear solution: Accurately pipette 1.2 ml of the first reference stock solution and 1 ml of the second internal standard stock solution into the same 10 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0093] 150% linear solution: Accurately pipette 1.5 ml of the first reference stock solution and 1 ml of the second internal standard stock solution into the same 10 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0094] result:

[0095]

[0096] The regression equation established through the experiment is: y = 0.8711x - 0.0431; the correlation coefficient R is 0.9998. The ratio of the intercept b to y when 100% x is 5%.

[0097] Conclusion: The correlation coefficient r = 0.9998 ≥ 0.998; the ratio of the intercept b to Y at 100% X is ≤ 5%, and the method meets the linear requirements.

[0098] Example 5:

[0099] Accuracy verification

[0100] Third internal standard stock solution: Accurately weigh approximately 403.1 mg of 1,3-butanediol, place it in a 20 ml volumetric flask, dissolve it in diluent and dilute to the mark, and shake well.

[0101] Second reference substance stock solution: Accurately weigh 202.05 mg of propylene glycol reference substance, place it in a 10 ml volumetric flask, dissolve it with diluent and dilute to the scale, and shake well.

[0102] 80% spiked test solution: Accurately weigh 25 mg of diazepam oral solution, 0.3 ml of the second reference stock solution, and 1 ml of the third internal standard stock solution into a 20 ml volumetric flask. Dilute to the mark with diluent and shake well. (Prepare three parallel aliquots: 25.37 mg, 25.52 mg, and 29.08 mg)

[0103] 100% spiked test solution: Accurately weigh 25 mg of diazepam oral solution, 0.5 ml of the second reference stock solution, and 1 ml of the third internal standard stock solution into a 20 ml volumetric flask. Dilute to the mark with diluent and shake well. (Prepare three parallel aliquots: 26.19 mg, 26.91 mg, and 27.40 mg)

[0104] 120% spiked test solution: Accurately weigh 25 mg of diazepam oral solution, 0.7 ml of the second reference stock solution, and 1 ml of the third internal standard stock solution, place them in the same 20 ml volumetric flask, dilute to the mark with diluent, and shake well. (Prepare three parallel aliquots: 26.45 mg, 26.87 mg, and 28.74 mg)

[0105] Calculation formula:

[0106]

[0107] Measured amount: The content of propylene glycol measured in the accuracy solution;

[0108] Existing amount: the content of propylene glycol in the test solution;

[0109] Added amount: the actual amount of propylene glycol added to the accuracy solution;

[0110] Existing amount (mg) = m w ×W;

[0111] Where: m w is the sample weight of the test sample (mg); W is the mean value of the content measured under the repeatability experiment (%);

[0112] Addition amount = m s / 10×V s ;

[0113] Where: m s is the sample weight of the reference substance (mg); 10 is the dilution volume of the reference substance stock solution (ml); V s is the pipetting volume of the reference stock solution (ml);

[0114] Measured quantity = f × A w ×(C s / A s )×20

[0115] Where: f is the average value of f in the reference solution of 7 samples in the system suitability verification; A w is the peak area of propylene glycol in the test solution; A s is the peak area of the internal standard; 20 is the dilution multiple of the test sample; C s is the internal standard concentration.

[0116] result:

[0117]

[0118]

[0119] Conclusion: The recovery rate was 98.65% to 103.85%, between 95.0% and 105.0%, and the recovery rate RSD was 1.6% ≤ 5.0%. The accuracy of the verification method met the requirements.

[0120] Example 6:

[0121] Repeatability verification

[0122] Fourth internal standard stock solution: Accurately weigh about 403.20 mg of 1,3-butanediol, place it in a 20 ml volumetric flask, dissolve it in diluent and dilute it to the scale, and shake well.

[0123] Third test solution: Accurately weigh 50 mg of diazepam oral solution and place it in a 20 ml volumetric flask. Accurately add 1 ml of the fourth internal standard stock solution, dilute to the mark with diluent, and shake well. (Prepare 6 parallel portions: 51.90 mg, 51.23 mg, 50.94 mg, 49.33 mg, 53.04 mg, and 51.18 mg)

[0124] Calculation formula:

[0125] The content of propylene glycol in the test solution was calculated by the internal standard method.

[0126] f=(C R / A R ) / (C s / A s );

[0127] Where: A s is the peak area or peak height of the internal standard substance; A R is the peak area or peak height of the reference substance;

[0128] C s is the concentration of the internal standard substance; C R is the concentration of the reference substance.

[0129] Content (%) = f × A w ×(C s / A s )×20 / m×100

[0130] Where: f is the average value of f in the reference solution during the system suitability verification; Aw is the peak area of propylene glycol in the test solution; A s . is the peak area of the internal standard; 20 is the dilution factor of the test sample; C s is the concentration of the internal standard; m is the sample weight of the test sample.

[0131] result:

[0132]

[0133] Conclusion: RSD of content = 0.33% ≤ 2.0%, the repeatability of this method meets the requirements.

[0134] Example 7:

[0135] Precision verification

[0136] Fifth internal standard stock solution: Accurately weigh approximately 399.94 mg of 1,3-butanediol, place it in a 20 ml volumetric flask, dissolve it in diluent and dilute it to the mark, and shake well.

[0137] Fourth test solution: Accurately weigh 50 mg of diazepam oral solution and place it in a 20 ml volumetric flask. Accurately add 1 ml of the fifth internal standard stock solution, dilute to the mark with diluent, and shake well. (Prepare 6 parallel portions: 53.74 mg, 50.93 mg, 55.10 mg, 54.64 mg, 54.05 mg, and 52.50 mg)

[0138] Calculation formula

[0139] Same as repeatability test.

[0140]

[0141] Conclusion: Content RSD% (n=6) = 0.67≤2.0%; Content RSD% (n=12) = 0.56≤2.0%; The intermediate precision of this method meets the requirements.

[0142] Example 8:

[0143] Stability verification

[0144] Sixth internal standard stock solution: Accurately weigh about 400.74 mg of 1,3-butanediol, place it in a 20 ml volumetric flask, dissolve it in diluent and dilute it to the scale, and shake well.

[0145] Third reference solution: Accurately weigh 21.33 mg of propylene glycol reference solution and accurately pipette 1 ml of the sixth internal standard stock solution into the same 20 ml volumetric flask. Dissolve and dilute to the mark with diluent and shake well. (1 mg / ml)

[0146] Fifth test solution: Accurately weigh 52.93 mg of diazepam oral solution, accurately pipette 1 ml of the sixth internal standard stock solution, place it in a 20 ml volumetric flask, dissolve it with diluent and dilute to the scale, and shake well.

[0147] The third reference solution and the fifth test solution were measured at 0 hours, 4 hours, 8 hours, 12 hours, 24 hours and 36 hours, respectively, to investigate the stability of the solutions under room temperature conditions.

[0148] result

[0149] Fifth test solution

[0150] 0 hours 4 hours 8 hours 12 hours 24 hours 36 hours RSD% Propylene glycol peak area 677925 673941 687204 702475 683902 695983 N / A 1,3-Butanediol peak area 675779 674294 689053 697639 683035 699034 N / A f-number 1.2331 1.2377 1.2403 1.2285 1.2354 1.2424 0.41

[0151] The third reference solution

[0152] 0 hours 4 hours 8 hours 12 hours 24 hours 36 hours RSD% Propylene glycol peak area 581819 600435 601421 585640 582756 593036 N / A 1,3-Butanediol peak area 651088 684414 689896 666359 660468 671026 N / A f-number 1.2033 1.2257 1.2335 1.2235 1.2187 1.2167 0.83

[0153] Conclusion: The RSD of f value is 0.41%, 0.83%, both ≤ 2.0%. The stability of the solution within 36 hours meets the requirements.

[0154] This specific embodiment is merely an explanation of the present invention and is not intended to limit the present invention. After reading this specification, those skilled in the art may make non-creative modifications to this embodiment as needed. However, as long as such modifications are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. A method for analyzing the propylene glycol content of diazepam oral solution by chromatography, characterized in that: The following steps are included: (1) Solution configuration: Take an appropriate amount of diazepam oral solution and dissolve it in anhydrous ethanol to dilute it to 2-3 mg / ml to obtain the first test solution; Take an appropriate amount of the first internal standard substance and dissolve it in anhydrous ethanol to dilute it to 15-20 mg / ml to obtain a first internal standard solution; Take an appropriate amount of propylene glycol and an appropriate amount of the first internal standard solution, dissolve and dilute with anhydrous ethanol to a propylene glycol concentration of 1 to 2 mg / ml to obtain a first reference solution, wherein the amount of the first internal standard solution is 5% to 10% of the total volume of the first reference solution; (2) Gas chromatography analysis: An SH-WAX polar capillary column was used with an inlet temperature of 230°C and a detector temperature of 250°C. The carrier gas was nitrogen at a flow rate of 1.0 mL / min and a split ratio of 10:

1. The temperature was programmed to maintain the initial temperature at 130 °C for 1 min, then to 240 °C at a rate of 10 °C / min and maintain for 3 min; Injecting the first reference solution and the first test solution respectively to obtain chromatograms; (3) Determination of propylene glycol content by internal standard method: The propylene glycol content was calculated by the peak area ratio of the internal standard to the propylene glycol. The calculation formula is: Wherein, f is the correction factor value of the first reference solution, A w is the peak area of propylene glycol in the test sample, A s is the peak area of the first internal standard, C s is the concentration of the first internal standard, and m is the weight of the first test sample; Among them, A S is the peak area of the first internal standard, A R is the propylene glycol peak area of the first reference substance, C S is the concentration of the first internal standard, C R is the propylene glycol concentration in the first reference substance.

2. The method for analyzing the propylene glycol content of a diazepam oral solution by chromatography according to claim 1, wherein: The first internal standard is 1,3-butanediol.

3. The method for analyzing the propylene glycol content of a diazepam oral solution by chromatography according to claim 1, wherein: The first reference solution is prepared in two portions, namely, a first reference solution group A and a first reference solution group B; The injection method is as follows: 5 injections of the first reference solution group A and 2 injections of the first reference solution group B; The correction factor value f of the first reference solution is the average value of f of a total of 7 injections of the first reference solution group A and group B.

4. A method for verifying the content of propylene glycol in diazepam oral solution by chromatographic analysis, characterized in that: Set the gas chromatograph conditions as follows: An SH-WAX polar capillary column was used with an inlet temperature of 230°C and a detector temperature of 250°C. The carrier gas was nitrogen at a flow rate of 1.0 mL / min and a split ratio of 10:

1. The temperature was programmed to maintain the initial temperature at 130 °C for 1 min, then to 240 °C at a rate of 10 °C / min and maintain for 3 min; Inputting a second reference solution into a gas chromatograph to obtain an analysis result, the analysis result including a plurality of second reference solutions; if a relative standard deviation of retention times of the plurality of second reference solutions is less than 1%, a relative standard deviation of correction factor values of the plurality of second reference solutions is less than 2%, and the number of theoretical plates of the plurality of second reference solutions is greater than 30,000, then determining that the system adaptability meets the requirements; Input the propylene glycol positioning solution, 1,3-butanediol positioning solution, and the second test solution into the gas chromatograph to obtain analysis results; if the resolution between the component peak and the adjacent peak in the second test solution is ≥1.5, and the difference between the actual retention time of the component in the second test solution and the reference retention time of the propylene glycol positioning solution and 1,3-butanediol positioning solution is ≤0.1 min, then the specificity is determined to meet the requirements; Input 50%, 80%, 100%, 120% and 150% linear solutions into the gas chromatograph respectively, obtain analysis results, and establish a linear equation of propylene glycol peak area / 1,3-butanediol peak area and propylene glycol concentration / 1,3-butanediol concentration. If the correlation coefficient is ≥0.998 and the ratio of the intercept to Y at 100% x is ≤5%, it is determined that the linearity meets the requirements; Input 80%, 100%, and 120% spiked test solution into the gas chromatograph respectively, obtain the analysis results, calculate the recovery rate and the relative standard deviation of the recovery rate. If the recovery rate is between 95% and 105% and the relative standard deviation of the recovery rate is ≤5%, it is determined that the accuracy meets the requirements; Input the six parallel prepared third test solutions into the gas chromatograph to obtain the content of propylene glycol in each parallel prepared test solution. If the relative standard deviation of the content is ≤2%, it is determined that the repeatability meets the requirements; Prepare six parallel preparations of the fourth test solution, and obtain the propylene glycol content by gas chromatograph using a single operator (n=6) and a dual operator (n=12). If the relative standard deviation of the propylene glycol content is ≤2% under the single operator condition and ≤2% under the dual operator condition, the intermediate precision is determined to meet the requirements; Prepare a third reference solution and a fifth test solution, and pass them through a gas chromatograph at 0 hours, 4 hours, 8 hours, 12 hours, 24 hours, and 36 hours, respectively. If the relative standard deviations of the correction factors f of the third reference solution and the fifth test solution are both less than or equal to 2%, it is determined that the stability meets the requirements.

5. The method for verifying the content of propylene glycol in diazepam oral solution by chromatography according to claim 4, wherein: The second reference substance solution is prepared as follows: Take an appropriate amount of 1,3-butanediol, dilute it with anhydrous ethanol to 20-25 mg / ml, and prepare a first internal standard stock solution; take an appropriate amount of propylene glycol, and dilute it with anhydrous ethanol to a propylene glycol concentration of 1-2 mg / ml. The amount of the first internal standard stock solution is 5%-10% of the total volume of the second reference solution.

6. The method for verifying the content of propylene glycol in diazepam oral solution by chromatography according to claim 5, wherein: The propylene glycol positioning solution uses anhydrous ethanol as the solvent, and the propylene glycol concentration is 1-1.5 mg / ml; the 1,3-butanediol positioning solution uses anhydrous ethanol as the solvent, and the 1,3-butanediol concentration is 1-2 mg / ml; the test sample in the second test sample solution is diazepam oral solution, the solvent is anhydrous ethanol, and the diazepam concentration is 2-3 mg / ml; the second test sample solution also contains a first internal standard stock solution, and the amount of the first internal standard stock solution is 5% to 10% of the total volume of the second test sample solution.

7. The method for verifying the content of propylene glycol in diazepam oral solution by chromatography according to claim 4, wherein: The linear solution was prepared as follows: Take an appropriate amount of propylene glycol and dissolve it in anhydrous ethanol and dilute it to 10-15 mg / ml to obtain a first reference stock solution; take an appropriate amount of 1,3-butanediol and dissolve it in anhydrous ethanol and dilute it to 10-15 mg / ml to obtain a second internal standard stock solution; prepare a linear solution with the first reference stock solution and the second internal standard stock solution and anhydrous ethanol; Each 10 ml of 50% linear solution contains 0.5 ml of the first reference stock solution and 1 ml of the second internal standard stock solution; Each 10 ml of 80% linear solution contains 0.8 ml of the first reference substance stock solution and 1 ml of the second internal standard stock solution; Each 10 ml of 100% linear solution contains 1 ml of the first reference stock solution and 1 ml of the second internal standard stock solution; Each 10 ml of 120% linear solution contains 1.2 ml of the first reference stock solution and 1 ml of the second internal standard stock solution; Each 10 ml of 150% linear solution contains 1.5 ml of the first reference stock solution and 1 ml of the second internal standard stock solution.

8. The method for verifying the content of propylene glycol in diazepam oral solution by chromatography according to claim 4, wherein: The preparation method of the spiked test solution is as follows: Take an appropriate amount of 1,3-butanediol and dissolve it in anhydrous ethanol to dilute it to 18-25 mg / ml to obtain a third internal standard stock solution; take an appropriate amount of propylene glycol and dissolve it in anhydrous ethanol to dilute it to 20-25 mg / ml to obtain a second reference substance stock solution; prepare a spiked test solution using the second reference substance stock solution, the third internal standard stock solution, diazepam oral solution, and anhydrous ethanol; Each 20 ml of 80% spiked test solution contains 0.3 ml of the second reference stock solution, 1 ml of the third internal standard stock solution, and 20-30 mg of diazepam oral solution; Each 20 ml of 100% spiked test solution contains 0.5 ml of the second reference stock solution, 1 ml of the third internal standard stock solution, and 20-30 mg of diazepam oral solution; Each 20 ml of 120% spiked test solution contains 0.7 ml of the second reference stock solution, 1 ml of the third internal standard stock solution and 20-30 mg of diazepam oral solution.

9. The method for verifying the content of propylene glycol in diazepam oral solution by chromatography according to claim 4, wherein: The third test solution was prepared as follows: Take an appropriate amount of 1,3-butanediol and dissolve it in anhydrous ethanol to dilute it to 20-22 mg / ml to obtain the fourth internal standard stock solution; the third test solution is prepared by using diazepam oral solution, the fourth internal standard stock solution and anhydrous ethanol; Each 20 ml of the third test solution contains 1 ml of the fourth internal standard stock solution and 45-60 mg of diazepam oral solution.

10. The method for verifying the content of propylene glycol in diazepam oral solution by chromatography according to claim 4, wherein: The preparation methods of the third reference solution and the fifth test solution are as follows: Take an appropriate amount of 1,3-butanediol and dissolve it in anhydrous ethanol to dilute it to 19-22 mg / ml to obtain the sixth internal standard stock solution; prepare the third reference solution by using propylene glycol, the sixth internal standard stock solution and anhydrous ethanol; The fifth test solution was prepared by diazepam oral solution, the sixth internal standard stock solution, and anhydrous ethanol; Each 20 ml of the third reference solution contains 1 ml of the sixth internal standard stock solution and 20-25 mg of propylene glycol solution; Each 20 ml of the fifth test solution contains 1 ml of the sixth internal standard stock solution and 50-55 mg of propylene glycol solution.

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