Sodium nitroprusside pharmaceutical composition and method for determining cyanide therein

Abstract

The present invention relates to a sodium nitroprusside pharmaceutical composition and a method for determining cyanide therein. Specifically, on the one hand, it relates to a method for determining the cyanide content in a sodium nitroprusside pharmaceutical composition using gas chromatography, the method comprising the following steps: providing a gas chromatograph, a capillary chromatographic column and a derivatization reaction flask; preparing a derivatization reagent and a test sample solution, etc., placing them outside the outer bottle of the derivatization reaction flask, and adding the derivatization reagent into the inner bottle of the derivatization reaction flask, covering and sealing; using the headspace injection method, performing gas chromatography testing with high-purity nitrogen as the carrier gas, performing analysis using a programmed temperature rise, recording the chromatograms of the reference solution and the test sample solution, and calculating the cyanide content in the test sample by peak area according to the external standard method. It also relates to the derivatization reaction flask used in the above method, and a method for preparing the sodium nitroprusside pharmaceutical composition. The method of the present invention exhibits the effects of one or more aspects as described in the specification, such as high test efficiency and accurate test results.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and relates to a pharmaceutical composition for treating hypertension, in particular to a pharmaceutical composition of sodium nitroprusside, which is preferably provided in the form of a freeze-dried powder injection or in the form of a solution injection. Further, the present invention relates to a method for determining the cyanide content or limit in such sodium nitroprusside pharmaceutical compositions, and more specifically, to a method for determining the cyanide content or limit in sodium nitroprusside pharmaceutical compositions using gas chromatography. Background Art

[0002] Sodium nitroprusside, CAS No. 14402-89-2, molecular formula: C5FeN6Na2O or Na2[Fe(CN)5NO], molecular weight 261.92, chemical name: sodium nitroferricyanide, English chemical name: Pentakis(cyano-C)nitrosylferrate(2-)disodium. The sodium nitroprusside bulk drug included in the current edition of the Chinese Pharmacopoeia, as well as the United States Pharmacopoeia and the European Pharmacopoeia, is sodium nitroferricyanide dihydrate, with a molecular weight of 297.95. This bulk drug is a dark red crystalline or powder, soluble in water and slightly soluble in ethanol.

[0003] Sodium nitroprusside was first prepared in the 19th century (L. Playfair, Proc. Roy. Soc. London 5, 846 (1849)), and its clinical application can be traced back to the 1920s at the earliest. In the 1950s, its application in the treatment of hypertension had been effectively confirmed. There has always been concern about its application value and potential side effects. Although clinicians are more worried about its cyanide toxicity, it is still the most effective and sometimes the only drug for controlling acute hypertension at present.

[0004] Once sodium nitroprusside is administered into the body, it will rapidly decompose into cyanide and nitric oxide (NO). Nitric oxide is a very strong vasodilator. It can activate guanylate cyclase, then lead to an increase in the intracellular cyclic guanosine monophosphate (cGMP) concentration, and finally lead to a decrease in the calcium ions entering vascular smooth muscle cells, resulting in vasodilation.

[0005] Sodium nitroprusside has a direct dilating effect on arteries and veins, and at the same time can ensure the blood supply to important organs. It is also a very powerful pulmonary vasodilator. Sodium nitroprusside has a strong antagonistic effect on the pulmonary vasoconstriction caused by hypoxia. Its action time is very short, usually only 1-2 minutes. When left ventricular failure occurs, it can reduce peripheral vascular resistance, pulmonary vascular resistance and right atrial pressure, thereby improving left ventricular output. In normal people without cardiovascular diseases, if sodium nitroprusside is administered, the cardiac output will decrease because the cardiac return flow will decrease.

[0006] Due to the potential side effect of cyanide poisoning, sodium nitroprusside preparations are mainly used clinically for hypertensive emergencies, such as emergency antihypertensive treatment for hypertensive crisis, hypertensive encephalopathy, malignant hypertension, paroxysmal hypertension before and after surgery for pheochromocytoma, etc., and can also be used for controlled hypotension during surgical anesthesia; it is also used for acute heart failure, including acute pulmonary edema; it is also used for acute heart failure in acute myocardial infarction or valve (mitral or aortic valve) insufficiency.

[0007] Generally speaking, since 1960, sodium nitroprusside has been widely used in the treatment of hypertensive crisis. The characteristics of sodium nitroprusside, such as rapid onset of action, easy titration, rapid degradation, and high efficiency, make it a very popular drug for treating hypertensive crisis. Since 1972, there have been continuous research reports that sodium nitroprusside has a therapeutic effect on acute myocardial infarction by reducing the afterload of cardiac contraction, reducing the left ventricular filling pressure, and increasing the left ventricular output. Later studies have also confirmed that it has a good therapeutic effect on left ventricular failure, mitral valve, and tricuspid valve regurgitation accompanied by acute cardiac infarction. In recent years, clinicians tend to use nitroglycerin to replace sodium nitroprusside in the treatment of acute myocardial infarction. However, if acute myocardial infarction is accompanied by severe valvular insufficiency and regurgitation, sodium nitroprusside can play a role in rapidly improving cardiac function. Many studies have pointed out that sodium nitroprusside can improve cardiac function regardless of the cause of chronic heart failure. The combined use of sodium nitroprusside with dopamine or epinephrine can significantly improve cardiac function. Controlled hypotension is an effective technique often used by anesthesiologists to control intraoperative bleeding. The rapid hypotensive effect, rapid inactivation, and easy titration of sodium nitroprusside make it an ideal choice. Orthopedic surgeries, especially spinal surgeries, often result in a large amount of blood loss. Controlled hypotension can provide a relatively bloodless surgical field or reduce blood transfusion volume. Of course, considering the side effects of sodium nitroprusside, many anesthesiologists also use beta-blockers, calcium channel blockers, and nitroglycerin as alternative options. In the repair of aortic aneurysm and aortic stenosis, the combined application of sodium nitroprusside and beta-blockers can effectively reduce the aortic pressure and prevent the formation of dissecting aneurysm. In addition, pheochromocytoma resection is still a difficult surgery in medicine because of frequent blood pressure fluctuations during the operation, sometimes with a sharp increase in blood pressure. Sodium nitroprusside is very suitable for this situation, and its short half-life will not cause postoperative hypotension. In cardiac surgeries, especially coronary artery bypass grafting and aortic valve repair, intraoperative hypertension often occurs, and the mechanism causing hypertension is unknown. Regardless of the mechanism, sodium nitroprusside can effectively control it. 30% to 60% of cardiac surgery patients will develop postoperative hypertension, which can reduce left ventricular function, increase oxygen consumption, induce postoperative bleeding, and cause arrhythmia. Using sodium nitroprusside alone or in combination with other antihypertensive drugs is a suitable option for reducing postoperative hypertension. Sodium nitroprusside is also a venodilator and has an effective therapeutic effect on relieving pulmonary hypertension after cardiac valve replacement.

[0008] The current editions of the Chinese Pharmacopoeia and the United States Pharmacopoeia both include the raw material drug and freeze-dried powder injection of sodium nitroprusside. There are multiple approval numbers for the currently marketed freeze-dried powder injection of sodium nitroprusside, and the sodium nitroprusside injection was launched on the market by Hainan Poly Pharm Co., Ltd. in 2022. The specifications of the currently commercially available freeze-dried powder injection of sodium nitroprusside are 25 mg or 50 mg per vial, while the specification of the commercially available sodium nitroprusside injection is 50 mg:2 ml per vial.

[0009] Cyanide in sodium nitroprusside, especially in its injection preparation, is an important quality indicator of the product. Neither the raw materials nor the preparations in the European Pharmacopoeia and the United States Pharmacopoeia detect cyanide; the raw material medicine of sodium nitroprusside included in the Chinese Pharmacopoeia does not detect cyanide, while its preparation stipulates the use of the second method in General Chapter 0806, Part IV of the Chinese Pharmacopoeia (2020 edition) for detection, and the limit in the preparation is stipulated to be 8 ppm. However, this method has its inherent limitations. For example, it is suitable for limit inspection but not suitable for calculating the content of cyanide. In addition, the determination time of this pharmacopoeia method is too long and requires overnight treatment, which not only has low determination efficiency but also increases the cost of labor protection due to the handling of cyanide.

[0010] Therefore, those skilled in the art still expect to have a new method for determining the cyanide content in sodium nitroprusside pharmaceutical compositions. Summary of the Invention

[0011] The object of the present invention is to provide a method for determining the cyanide content in sodium nitroprusside pharmaceutical compositions, and it is expected that this method can present one or more beneficial effects, such as accurate and reliable determination method, easy qualitative or quantitative calculation of cyanide content, short determination time, low cost of labor protection, etc.

[0012] To this end, the first aspect of the present invention provides a method for determining the cyanide content in sodium nitroprusside pharmaceutical compositions using gas chromatography. This method includes the following steps:

[0013] (1) Provide a gas chromatograph, a capillary chromatographic column and a derivatization reaction bottle [the stationary liquid of the capillary chromatographic column is, for example, polyethylene glycol or other suitable stationary liquids such as methyl polysiloxane, phenylmethyl polysiloxane, etc., for example, 100% polyethylene glycol is used as the stationary liquid; the derivatization reaction bottle includes an open vial and a stoppered large bottle. The small bottle can be placed into the large bottle to form a structure of an outer bottle (large bottle) and an inner bottle (small bottle). After adding the derivatization reagent and the solution to be measured into the inner bottle and the outer bottle respectively, the outer bottle is tightly stoppered, and a gas atmosphere communicating the liquid levels of the outer bottle and the inner bottle is formed];

[0014] (2) Preparation of solutions

[0015] (2a) Diluent: Take an appropriate amount of inorganic acid and add water to make an acid aqueous solution (for example, make an acid aqueous solution with a concentration of 0.5% - 2%, for example, 1%, and the inorganic acid is, for example, phosphoric acid or sulfuric acid);

[0016] (2b) Derivatization reagent: Take chloramine T, dissolve it in water and dilute to make a chloramine T solution (for example, a chloramine T solution with a concentration of 1%, and this concentration is also its common concentration as a derivatization reagent), as the derivatization reagent (prepared freshly before use);

[0017] (2c) Blank solution: Take the diluent and place it in the outer bottle of the derivatization reaction bottle. Separately, add the derivatization reagent to the inner bottle of the derivatization reaction bottle, cap and seal it to obtain (for example, the volume ratio of the liquid in the outer bottle to that in the inner bottle is 1 - 5:1, especially 1 - 4:1, especially 2 - 3:1, for example 3:1 or 2:1, for example the volume of the liquid in the inner bottle is 0.5 ml);

[0018] (2d) Reference solution: Weigh an appropriate amount of potassium cyanide reference substance accurately, dissolve and dilute it with the diluent to prepare a reference solution (for example, its concentration is 0.2 - 10 μg / ml, for example 0.5 - 5 μg / ml, for example 0.5 - 2.5 μg / ml, for example 1 μg / ml);

[0019] Take the reference solution and place it in the outer bottle of the derivatization reaction bottle. Separately, add the derivatization reagent to the inner bottle of the derivatization reaction bottle, cap and seal it to obtain (for example, the volume ratio of the liquid in the outer bottle to that in the inner bottle is 1 - 5:1, especially 1 - 4:1, especially 2 - 3:1, for example 3:1 or 2:1, for example the volume of the liquid in the inner bottle is 0.5 ml);

[0020] (2f) Test solution: Accurately measure an aqueous solution of the sodium nitroprusside pharmaceutical composition (where the concentration of sodium nitroprusside is 20 - 50 mg / ml, for example 25 - 50 mg / ml, for example 25 mg / ml; for example, the sodium nitroprusside pharmaceutical composition is a solution-type injection or a freeze-dried powder injection. If it is an injection, directly sample it. If it is a powder injection, first dissolve and dilute it with water to prepare a solution containing 25 mg / ml of the main component) and place it in the outer bottle of the derivatization reaction bottle, and add an appropriate amount of inorganic acid (for example, its volume ratio to the aqueous solution of the sodium nitroprusside pharmaceutical composition is 0.5 - 5:100, for example 0.75 - 2:100, for example 1:100), shake well. Separately, add the derivatization reagent to the inner bottle of the derivatization reaction bottle, cap and seal it to obtain (for example, the volume ratio of the liquid in the outer bottle to that in the inner bottle is 1 - 5:1, especially 1 - 4:1, especially 2 - 3:1, for example 3:1 or 2:1, for example the volume of the liquid in the inner bottle is 0.5 ml);

[0021] (3) Chromatographic conditions:

[0022] When using headspace injection, the equilibrium temperature of the headspace vial is 45 - 60 °C, for example 50 °C, and the equilibrium time is 15 - 30 minutes, for example 20 minutes;

[0023] (Other gas chromatography conditions are conventional conditions. For example, the inlet temperature is 180 - 240 °C, for example 200 °C, the temperature of the quantitative loop is 80 - 120 °C, for example 90 °C, the transfer line temperature is 80 - 120 °C, for example 100 °C, the detector (ECD) temperature is 280 - 350 °C, for example 300 °C, the injection time is 0.5 - 5 min, for example 0.5 - 2 min, for example 1 min. These chromatographic conditions are conventional and appropriate adjustments within the above ranges do not affect the determination of the method of the present invention);

[0024] Using high-purity nitrogen as the carrier gas, with a flow rate of 0.5 - 2.0 mL / min, such as 1.0 mL / min, and a split ratio of 8 - 15:1, such as 10:1;

[0025] Programmed temperature rise: The initial column temperature is 38 - 45 °C, such as 40 °C, maintained for 4 - 6 minutes, such as 5 minutes, then heated at a rate of 28 - 35 °C per minute, such as 30 °C, to 180 - 240 °C, such as 200 °C, and then maintained for 3 - 5 minutes, such as 4 minutes;

[0026] (4) System suitability test requirements: Take the blank solution and the reference solution for headspace injection respectively, record the chromatogram, and determine the cyanide chromatographic peak; deduct the interference peak in the blank solvent. The resolution between the cyanide chromatographic peak and its adjacent chromatographic peak should be greater than 5, such as greater than 10. The RSD% of the peak area of the cyanide in the repeated injection of the reference solution (for example, at least 5 times) should not be greater than 10%, such as not greater than 8%;

[0027] (5) Determination: Take the blank solution, the reference solution, and the test solution for headspace injection respectively, record the chromatogram, and calculate the cyanide content in the test sample by the external standard method based on the peak area.

[0028] According to the method of the first aspect of the present invention, it further includes the following way to prepare the sensitivity solution:

[0029] (2e) Accurately pipette 1.0 ml of the reference solution obtained in step (2d) into a 100-ml volumetric flask, dilute to the mark with the diluent, shake well to obtain the sensitivity solution. Take the sensitivity solution and place it in the outer bottle of the derivatization reaction bottle. Then add the derivatization reagent to the inner bottle of the derivatization reaction bottle, cover and seal it (for example, the volume ratio of the liquid in the outer bottle to the inner bottle is 1 - 5:1, especially 1 - 4:1, especially 2 - 3:1, such as 3:1 or 2:1. For example, the volume of the liquid in the inner bottle is 0.5 ml).

[0030] According to the method of the first aspect of the present invention, the signal-to-noise ratio of the cyanide peak in the chromatogram obtained by using the sensitivity solution for headspace injection test is not less than 100.

[0031] According to the method of the first aspect of the present invention, the inner bottle and the outer bottle of the derivatization reaction bottle are of an integrated fusion design glass bottle structure or are composed of a combination of 2 independent glass bottles. For the integrally fused bottle, liquids can be added to the outer bottle and the inner bottle respectively, and then the outer bottle is tightly stoppered; for the bottle composed of a combination of 2 independent glass bottles, a liquid can be added to the outer bottle, and the inner bottle containing another liquid is placed into the outer bottle, and then the outer bottle is tightly stoppered.

[0032] The method according to the first aspect of the present invention, wherein the outer bottle of the derivatization reaction bottle is a brown glass bottle; and / or, the inner bottle of the derivatization reaction bottle is a brown glass bottle.

[0033] The method according to the first aspect of the present invention, wherein the stopper of the outer bottle of the derivatization reaction bottle is a rubber stopper. Such a rubber stopper configuration facilitates headspace injection for gas chromatography.

[0034] The method according to the first aspect of the present invention, wherein the inner bottle of the derivatization reaction bottle has a volume of 0.5 ml to 5 ml, preferably 1 ml to 4 ml. For example, the inner bottle volume is 1 ml or 2 ml or 3 ml or 4 ml. For example, the volume of the solution in the inner bottle of the derivatization reaction bottle accounts for 1 / 4 to 3 / 4 of the inner bottle volume, preferably 1 / 2.

[0035] The method according to the first aspect of the present invention, wherein the outer bottle of the derivatization reaction bottle has a volume of 5 ml to 50 ml, preferably 10 ml to 30 ml. For example, the outer volume is 10 ml or 15 ml or 20 ml or 25 ml.

[0036] The method according to the first aspect of the present invention, wherein the inner bottle of the derivatization reaction bottle has a volume of 2 ml and the outer bottle has a volume of 20 ml.

[0037] The method according to the first aspect of the present invention, wherein the inner bottle of the derivatization reaction bottle has a volume of 2 ml and the outer bottle has a volume of 20 ml. During testing, 0.5 ml of derivatization reagent is added to the inner bottle, and 1.0 ml of test solution (such as reference solution, test sample solution, sensitivity solution, blank solution, etc.) is added to the outer bottle.

[0038] Essentially, the outer bottle of the derivatization reaction bottle of the present invention can be a conventional headspace bottle for gas chromatography testing, and the inner bottle is a conventional injection vial for gas chromatography testing. The two conventional glass bottles can be combined and used in a split manner, or the latter can form an integrated fused bottle as described herein. At this time, the sizes of the inner bottle and the outer bottle are equivalent to those of the above-mentioned conventional headspace bottle and conventional injection vial.

[0039] The method according to the first aspect of the present invention, wherein the capillary chromatographic column is a quartz capillary chromatographic column.

[0040] The method according to the first aspect of the present invention, wherein the specification of the capillary chromatographic column is 30 m * 0.25 mm, 0.5 μm.

[0041] The method according to the first aspect of the present invention, wherein the capillary chromatographic column is a DB-WAXetr quartz capillary chromatographic column, with 100% polyethylene glycol as the stationary liquid, and the specification is 30 m * 0.25 mm, 0.5 μm.

[0042] The method according to the first aspect of the present invention, wherein when adding the blank solution, reference solution, test solution and optionally the sensitivity solution to the derivatization reaction flask, the addition is carried out as follows: Take 1.0 ml of these solutions and place them in the outer flask of the derivatization reaction flask, and add 0.5 ml of the derivatization reagent to the inner flask of the derivatization reaction flask, cover and seal it to obtain the required solution.

[0043] The method according to the first aspect of the present invention, wherein in the chromatogram obtained by measuring with the reference solution, the theoretical plate number of the cyanide peak is greater than 20000, for example, greater than 25000.

[0044] The method according to the first aspect of the present invention, wherein after sealing the reference solution or the test solution and the derivatization reagent in the derivatization reaction flask and placing it at room temperature for 5 hours, headspace gas chromatography determination is carried out at 0 h, 1 h, 2 h, 3 h, 4 h, and 5 h respectively, and the RSD of the 6 results is less than 15%, for example, less than 12%, for example, less than 10%.

[0045] Furthermore, the second aspect of the present invention provides a derivatization reaction flask for gas chromatography determination of the cyanide content in a sodium nitroprusside pharmaceutical composition, which comprises an open vial and a stoppered large flask. The vial can be placed into the large flask to form a structure of an outer flask (large flask) and an inner flask (vial). After adding the derivatization reagent and the solution to be measured to the inner flask and the outer flask respectively, the outer flask is tightly stoppered, and the liquid levels of the outer flask and the inner flask form a communicating gas atmosphere.

[0046] For the derivatization reaction flask according to the second aspect of the present invention, the inner flask and the outer flask of the derivatization reaction flask are of an integrally fused design glass bottle structure or are composed of a combination of 2 independent glass bottles. For the integrally fused bottle, liquids can be added to the outer flask and the inner flask respectively, and then the outer flask is tightly stoppered; for the bottle composed of a combination of 2 independent glass bottles, a liquid can be added to the outer flask, and the inner flask added with another liquid is placed into the outer flask, and then the outer flask is tightly stoppered.

[0047] For the derivatization reaction flask according to the second aspect of the present invention, the outer flask is a brown glass bottle; and / or, the inner flask of the derivatization reaction flask is a brown glass bottle.

[0048] For the derivatization reaction flask according to the second aspect of the present invention, the stopper of the outer flask is a rubber stopper. This kind of rubber stopper configuration is convenient for headspace injection in gas chromatography.

[0049] For the derivatization reaction flask according to the second aspect of the present invention, the volume of the inner flask is 0.5 ml to 5 ml, preferably 1 ml to 4 ml. For example, the volume of the inner flask is 1 ml or 2 ml or 3 ml or 4 ml. For example, the volume of the solution in the inner flask of the derivatization reaction flask accounts for 1 / 4 to 3 / 4 of the volume of the inner flask, preferably 1 / 2.

[0050] The derivatization reaction flask according to the second aspect of the present invention, wherein the outer flask has a volume of 5 ml to 50 ml, preferably 10 ml to 30 ml, for example, the outer volume is 10 ml or 15 ml or 20 ml or 25 ml.

[0051] The derivatization reaction flask according to the second aspect of the present invention, wherein the inner flask has a volume of 2 ml and the outer flask has a volume of 20 ml.

[0052] The derivatization reaction flask according to the second aspect of the present invention, wherein the inner flask has a volume of 2 ml and the outer flask has a volume of 20 ml. During the test, 0.5 ml of the derivatization reagent is added to the inner flask, and 1.0 ml of the test solution (such as a reference solution, a test sample solution, a sensitivity solution, a blank solution, etc.) is added to the outer flask.

[0053] The derivatization reaction flask according to the second aspect of the present invention, which is used for the method for determining the cyanide content in a sodium nitroprusside pharmaceutical composition by gas chromatography, includes the following steps:

[0054] (1) Provide a gas chromatograph, a capillary chromatographic column, and a derivatization reaction flask [the stationary liquid of the capillary chromatographic column is, for example, polyethylene glycol or other suitable stationary liquids such as methyl polysiloxane, phenylmethyl polysiloxane, etc., for example, using 100% polyethylene glycol as the stationary liquid];

[0055] (2) Preparation of solutions

[0056] (2a) Diluent: Take an appropriate amount of inorganic acid and add water to make an acidic aqueous solution (for example, make an acidic aqueous solution with a concentration of 0.5% to 2%, for example, 1%, and the inorganic acid is, for example, phosphoric acid or sulfuric acid);

[0057] (2b) Derivatization reagent: Take chloramine T, dissolve it in water and dilute it to make a chloramine T solution (for example, a chloramine T solution with a concentration of 1%, and this concentration is also its common concentration as a derivatization reagent), as the derivatization reagent (freshly prepared before use);

[0058] (2c) Blank solution: Take the diluent and place it in the outer flask of the derivatization reaction flask. Additionally, add the derivatization reagent to the inner flask of the derivatization reaction flask, cover it and seal it, and that's it (for example, the volume ratio of the liquid in the outer flask to the inner flask is 1 to 5:1, especially 1 to 4:1, especially 2 to 3:1, for example, 3:1 or 2:1, for example, the volume of the liquid in the inner flask is 0.5 ml);

[0059] (2d) Reference solution: Weigh an appropriate amount of potassium cyanide reference substance precisely, dissolve it with the diluent and dilute it to make a reference solution (for example, its concentration is 0.2 to 10 μg / ml, for example, 0.5 to 5 μg / ml, for example, 0.5 to 2.5 μg / ml, for example, 1 μg / ml);

[0060] Place the reference solution in the outer bottle of the derivatization reaction flask. Separately, add the derivatization reagent to the inner bottle of the derivatization reaction flask, cover and seal it to obtain (for example, the volume ratio of the liquid in the outer bottle to that in the inner bottle is 1-5:1, especially 1-4:1, especially 2-3:1, for example 3:1 or 2:1. For example, the volume of the liquid in the inner bottle is 0.5 ml);

[0061] (2f) Test solution: Accurately measure an aqueous solution of the sodium nitroprusside pharmaceutical composition (where the concentration of sodium nitroprusside is 20-50 mg / ml, for example 25-50 mg / ml, for example 25 mg / ml; for example, the sodium nitroprusside pharmaceutical composition is a solution-type injection or a freeze-dried powder injection. If it is an injection, directly sample it. If it is a powder injection, first dissolve it with water and dilute it to a solution containing 25 mg / ml of the main component) and place it in the outer bottle of the derivatization reaction flask, and add an appropriate amount of inorganic acid (for example, the volume ratio of it to the aqueous solution of the sodium nitroprusside pharmaceutical composition is 0.5-5:100, for example 0.75-2:100, for example 1:100), shake well. Separately, add the derivatization reagent to the inner bottle of the derivatization reaction flask, cover and seal it to obtain (for example, the volume ratio of the liquid in the outer bottle to that in the inner bottle is 1-5:1, especially 1-4:1, especially 2-3:1, for example 3:1 or 2:1. For example, the volume of the liquid in the inner bottle is 0.5 ml);

[0062] (3) Chromatographic conditions:

[0063] When using headspace injection, the equilibrium temperature of the headspace vial is 45-60 °C, for example 50 °C, and the equilibrium time is 15-30 minutes, for example 20 minutes;

[0064] (Other gas chromatography conditions are conventional conditions. For example, the inlet temperature is 180-240 °C, for example 200 °C, the temperature of the quantitative loop is 80-120 °C, for example 90 °C, the transfer line temperature is 80-120 °C, for example 100 °C, the detector (ECD) temperature is 280-350 °C, for example 300 °C, the injection time is 0.5-5 min, for example 0.5-2 min, for example 1 min. These chromatographic conditions are conventional and appropriate adjustments within the above ranges do not affect the determination of the method of the present invention);

[0065] High-purity nitrogen is used as the carrier gas, with a flow rate of 0.5-2.0 mL / min, for example 1.0 mL / min, and a split ratio of 8-15:1, for example 10:1;

[0066] Programmed temperature rise: The initial column temperature is 38-45 °C, for example 40 °C, maintained for 4-6 minutes, for example 5 minutes, then heated at a rate of 28-35 °C per minute, for example 30 °C, to 180-240 °C, for example 200 °C, and then maintained for 3-5 minutes, for example 4 minutes;

[0067] (4) System suitability test requirements: Separately take the blank solution and the reference solution for headspace injection, record the chromatogram, and determine the cyanide chromatographic peak; deduct the interference peaks in the blank solvent. The resolution between the cyanide chromatographic peak and its adjacent chromatographic peak should be greater than 5, for example, greater than 10. The RSD% of the peak area of cyanide in the repeated injection of the reference solution (for example, at least 5 times) should not be greater than 10%, for example, not greater than 8%.

[0068] (5) Determination: Separately take the blank solution, the reference solution, and the test solution for headspace injection, record the chromatogram, and calculate the cyanide content in the test sample by the external standard method based on the peak area.

[0069] According to the derivatization reaction bottle of the second aspect of the present invention, it further includes the preparation of a sensitivity solution in the following manner:

[0070] (2e) Accurately pipette 1.0 ml of the reference solution obtained in step (2d) into a 100-ml volumetric flask, dilute to the mark with the diluent, and shake well to obtain the sensitivity solution. Take the sensitivity solution and place it in the outer bottle of the derivatization reaction bottle. Additionally, add the derivatization reagent to the inner bottle of the derivatization reaction bottle, cover and seal it to obtain (for example, the volume ratio of the liquid in the outer bottle to the inner bottle is 1-5:1, especially 1-4:1, especially 2-3:1, for example, 3:1 or 2:1, for example, the volume of the liquid in the inner bottle is 0.5 ml).

[0071] According to the derivatization reaction bottle of the second aspect of the present invention, the signal-to-noise ratio of the cyanide peak in the chromatogram obtained by headspace injection testing using the sensitivity solution is not less than 100.

[0072] According to the derivatization reaction bottle of the second aspect of the present invention, the capillary chromatographic column is a fused-silica capillary chromatographic column.

[0073] According to the derivatization reaction bottle of the second aspect of the present invention, the specifications of the capillary chromatographic column are 30 m * 0.25 mm, 0.5 μm.

[0074] According to the derivatization reaction bottle of the second aspect of the present invention, the capillary chromatographic column is a DB-WAXetr fused-silica capillary chromatographic column with 100% polyethylene glycol as the stationary liquid, and the specifications are 30 m * 0.25 mm, 0.5 μm.

[0075] According to the derivatization reaction bottle of the second aspect of the present invention, when adding the blank solution, the reference solution, the test solution, and optionally the sensitivity solution to the derivatization reaction bottle, they are added in the following manner: Take 1.0 ml of these solutions and place them in the outer bottle of the derivatization reaction bottle. Additionally, add 0.5 ml of the derivatization reagent to the inner bottle of the derivatization reaction bottle, cover and seal it to obtain.

[0076] The derivatization reaction flask according to the second aspect of the present invention, wherein in the chromatogram obtained by measuring with the reference solution, the number of theoretical plates of the cyanide peak is greater than 20,000, for example, greater than 25,000.

[0077] The derivatization reaction flask according to the second aspect of the present invention, wherein after sealing the reference solution or the test sample solution and the derivatization reagent in the derivatization reaction flask, it is left at room temperature for 5 hours, and headspace gas chromatography determination is carried out at 0 h, 1 h, 2 h, 3 h, 4 h, and 5 h respectively, and the RSD of the 6 results is less than 15%, for example, less than 12%, for example, less than 10%.

[0078] Furthermore, the third aspect of the present invention provides a sodium nitroprusside pharmaceutical composition, which is in the form of a pharmaceutical preparation of an injection or a freeze-dried powder injection sealed in a glass bottle. Calculated as sodium nitroferricyanide dihydrate, each bottle of the injection or the freeze-dried powder injection contains 25 mg or 50 mg of sodium nitroprusside.

[0079] Furthermore, the fourth aspect of the present invention provides a method for preparing a sodium nitroprusside pharmaceutical composition in a commercial form, including the following steps:

[0080] i) Prepare a sodium nitroprusside pharmaceutical composition in the form of a pharmaceutical preparation of an injection or a freeze-dried powder injection sealed in a glass bottle according to the injection preparation method or the freeze-dried powder injection preparation method. Calculated as sodium nitroferricyanide dihydrate, each bottle of the injection or the freeze-dried powder injection contains 25 mg or 50 mg of sodium nitroprusside;

[0081] ii) Measure the cyanide content in the sodium nitroprusside pharmaceutical composition obtained in step i) by using the method described in any one of the first aspects of the present invention;

[0082] iii) If the cyanide content measured in step ii) is less than 20 ppm, for example, 15 ppm, for example, 10 ppm, package the injection or the freeze-dried powder injection sealed in a glass bottle with a paper packaging box to obtain a sodium nitroprusside pharmaceutical composition in a commercial form.

[0083] The sodium nitroprusside pharmaceutical composition of the present invention, its preparation method, and their method for measuring the cyanide content present the effects of at least one aspect as described herein, such as simple preparation of the test solution, short GC determination time, etc.

[0084] Sodium nitroprusside is a nitrohydrocyanate and a potent vasodilator that acts directly on the arteriovenous vascular bed. This drug has a direct dilating effect on both resistance and capacitance vessels, and its effect on afterload is greater than that of nitroglycerin. Therefore, it can reduce the left ventricular filling pressure of patients and increase cardiac output. For the acute decompensation of patients with chronic left ventricular failure, sodium nitroprusside is more effective and faster-acting than furosemide. Since sodium nitroprusside mainly acts on the resistance vessels in the coronary circulation, it can cause coronary ischemia. Sodium nitroprusside can increase the arteriovenous shunt in the myocardium and lungs. Therefore, the increase in total blood flow does not necessarily manifest as an increase in the blood flow that shows improved perfusion. The increase in stroke volume can counteract the decrease in peripheral vascular resistance, so arterial blood pressure will not drop significantly. The heart rate generally does not increase and may even decrease due to the improvement of hemodynamics. Its mechanism of action is the same as that of nitrates, which can cause vascular endothelial cells to release NO and activate guanylate cyclase, increasing the intracellular cGMP level and dilating blood vessels. Clinically, the specific types of hemodynamic changes and the underlying pathological basis may help with the selection of drugs. For patients with obvious pump dysfunction, increased left ventricular filling pressure and significantly increased peripheral vascular resistance, with reduced cardiac output and normal or increased arterial pressure, it is advisable to use sodium nitroprusside for short-term intravenous infusion. Sodium nitroprusside needs to be freshly prepared for intravenous administration. The half-life of sodium nitroprusside is extremely short. Its duration of action is only 5 - 15 minutes, so intravenous infusion should be used to maintain the efficacy. It is first converted to cyanide by red blood cells and then to the end metabolite thiocyanate by rhodanase in the liver. Thiocyanate is excreted by the kidneys, and the half-life in patients with normal renal function is 4 - 7 days. There is accumulation in patients with renal failure. If the dose is too large, the metabolite thiocyanate in the blood is too high and poisoning is likely to occur.

[0085] Indications of sodium nitroprusside: For various hypertensive emergencies such as malignant hypertension, hypertensive crisis, hypertensive encephalopathy or hypertension complicated with aortic dissection (dissecting hematoma formation), sodium nitroprusside should be the first choice; for reactive hypertension after cardiac surgery, sodium nitroprusside can also be used; for hypertension complicated with acute left heart failure and pulmonary edema, sodium nitroprusside should be used early; for acute myocardial infarction complicated with left heart failure, if the onset has exceeded 8 hours and general treatment is ineffective, sodium nitroprusside can be considered; for refractory heart failure caused by various etiologies, sodium nitroprusside can be combined with dobutamine for "pulse therapy" for several days, which can often relieve the condition, and sometimes the efficacy can be maintained for several weeks; acute mitral and aortic valve insufficiency, ventricular septal perforation: After using sodium nitroprusside, the forward resistance of blood flow decreases, and the regurgitation volume and left-to-right shunt volume can both be reduced, thus alleviating the condition, and it can be used as a preparation before surgical operation.

[0086] Dosage and administration of sodium nitroprusside: The usual adult dosage is by intravenous infusion. Start at 0.5 μg / kg / min based on body weight and increase by 0.5 μg / kg / min according to the therapeutic response, gradually adjusting the dose. The usual dose is 3 μg / kg / min based on body weight. The maximum dose is 10 μg / kg / min based on body weight; the total dose is 3.5 mg / kg based on body weight; for short-term controlled hypotension during anesthesia, the maximum infusion rate is 0.5 mg / kg / min based on body weight; the usual pediatric dosage is by intravenous infusion at 1.4 μg / kg / min, and the dosage is gradually adjusted according to the effect. Description of the drawings

[0087] Figure 1 : Schematic diagram of the structure of the cyanide derivatization reaction bottle

[0088] Figure 2 : Chromatogram of the reference solution

[0089] Figure 3 : Chromatogram of the sensitivity solution

[0090] Figure 4 : Chromatogram of the test solution Detailed implementation mode

[0091] The following provides some specific examples to further illustrate the present invention. All kinds of reagents, instruments, etc. used in these examples are easily available in the market if not otherwise specified. In the present invention, if not otherwise specified, the headspace vial (derivatization reaction bottle) used is a brown glass bottle.

[0092] Example 1: Method for Determining Cyanide Content in Sodium Nitroprusside Injection

[0093] The basic principle of the determination method in this example is that in a closed container and at a certain temperature, cyanide in the sample is derivatized to cyanogen chloride with chloramine T under acidic conditions. However, due to the strong activity of sodium nitroprusside, the derivatization reagent cannot be directly added to the test solution. The method of the present invention uses Figure 1 the headspace vial device shown as the cyanide derivatization reaction bottle. This headspace vial is composed of two glass bottles, an inner bottle and an outer bottle, with an integrated design. The inner bottle is open, and the outer bottle can be sealed with a rubber stopper. The space inside the inner bottle is connected to the space inside the outer bottle. The chloramine T solution of the derivatization reagent is placed in the inner bottle of the headspace vial, and the test solution (or reference solution) is added to the outer bottle of the headspace vial (or the derivatization reagent can be placed in the outer bottle and the test solution or reference solution can be placed in the inner bottle). The headspace vial is sealed with a rubber stopper. In the closed space, the hydrogen cyanide gas volatilized from the test solution combines with the chlorine gas volatilized from the chloramine T solution to form the gas derivative cyanogen chloride. After the cyanogen chloride reaches equilibrium in the gas phase and liquid phase, the gas phase part is introduced into the gas chromatograph for separation, detected with an electron capture detector, and calculated by the external standard method to quantitatively determine the cyanide in the test sample.

[0094] The inner bottle volume of the above derivatization reaction bottle is 0.5 ml to 5 ml, preferably 1 ml to 4 ml. For example, the inner bottle volume is 1 ml or 2 ml or 3 ml or 4 ml. For example, the volume of the solution in the inner bottle of the derivatization reaction bottle accounts for 1 / 4 to 3 / 4 of the inner bottle volume, preferably 1 / 2.

[0095] The outer bottle volume of the above derivatization reaction bottle is 5 ml to 50 ml, preferably 10 ml to 30 ml. For example, the outer volume is 10 ml or 15 ml or 20 ml or 25 ml. Of course, the present invention can also use independent glass bottles of small and large sizes. During the test, the small bottle is placed inside the large bottle to form a spatial structure similar to the inner and outer bottles of the above integrated design.

[0096] The specific determination method includes the following steps:

[0097] (1) Provide a gas chromatograph, a capillary chromatographic column with polyethylene glycol as the stationary liquid, and a derivatization reaction bottle [in this example, an Agilent 7890B GC 7697A HS gas chromatograph, a DB-WAXetr quartz capillary chromatographic column (100% polyethylene glycol as the stationary liquid, 30 m * 0.25 mm, 0.5 μm), and the derivatization reaction bottle is Figure 1 a glass bottle structure with an integrated fusion design with an inner bottle volume of 2 ml and an outer bottle volume of 20 ml as shown; other similar models of gas chromatographs, capillary chromatographic columns, and derivatization reaction bottles will also be applicable];

[0098] (2) Preparation of solutions

[0099] (2a) Diluent: Take an appropriate amount of phosphoric acid and add water to make a 1% phosphoric acid solution;

[0100] (2b) Derivatization reagent: Take 0.5 g of chloramine T, place it in a 50 ml volumetric flask, dissolve it with water and dilute to the mark, shake well, and prepare a 1% chloramine T solution as the derivatization reagent (freshly prepared before use);

[0101] (2c) Blank solution: Take 1.0 ml of the diluent and place it in the outer bottle of the derivatization reaction bottle. Additionally, add 0.5 ml of the derivatization reagent to the inner bottle of the derivatization reaction bottle, cover and seal it to obtain;

[0102] (2d) Reference substance solution:

[0103] Precisely weigh about 125 mg of potassium cyanide reference substance, place it in a 100 ml volumetric flask, dissolve it with water (or the diluent) and dilute to the mark, shake well, and use it as reference substance stock solution ①;

[0104] Precisely measure 2.0 ml of reference substance stock solution ① and place it in a 100 ml volumetric flask, dilute it to the mark with the diluent, shake well, and use it as reference substance stock solution ②;

[0105] Accurately measure 1.0 ml of the reference stock solution ② and transfer it to a 25-ml volumetric flask. Dilute it to the mark with the diluent and mix well to obtain the reference solution (equivalent to 1 μg / ml).

[0106] Take 1.0 ml of the reference solution and place it in the outer flask of the derivatization reaction flask. Separately, add 0.5 ml of the derivatization reagent to the inner flask of the derivatization reaction flask, cover it with a lid and seal it to obtain the solution.

[0107] (2e) Sensitivity solution: Accurately pipette 1.0 ml of the reference solution into a 100-ml volumetric flask, dilute it to the mark with the diluent and mix well to obtain the sensitivity solution (equivalent to 0.01 μg / ml). Take 1.0 ml of the sensitivity solution and place it in the outer flask of the derivatization reaction flask. Separately, add 0.5 ml of the derivatization reagent to the inner flask of the derivatization reaction flask, cover it with a lid and seal it to obtain the solution.

[0108] (2f) Test solution: Accurately measure 1.0 ml of sodium nitroprusside injection (produced by Hainan Puli Pharmaceutical Co., Ltd., specification 50 mg / 2 ml) and place it in the outer flask of the derivatization reaction flask, then add 10 μl of phosphoric acid and mix well. Separately, add 0.5 ml of the derivatization reagent to the inner flask of the derivatization reaction flask, cover it with a lid and seal it to obtain the solution.

[0109] (3) Chromatographic conditions:

[0110] When using headspace injection, the equilibrium temperature of the headspace vial is 50 °C and the equilibrium time is 20 minutes.

[0111] The injection port temperature is 200 °C, the temperature of the quantitative loop is 90 °C, the temperature of the transfer line is 100 °C, the temperature of the detector (ECD) is 300 °C, and the injection time is 1 min. High-purity nitrogen is used as the carrier gas, the flow rate is 1.0 mL / min, and the split ratio is 10:1.

[0112] Programmed temperature rise: The initial column temperature is maintained at 40 °C for 5 minutes, then it is heated at a rate of 30 °C per minute to 200 °C, and then maintained for 4 minutes.

[0113] (4) Requirements for system suitability test: Separately take the blank solution, the reference solution, and the sensitivity solution for headspace injection, record the chromatogram, and determine the cyanide chromatographic peak (under the conditions of this example, the retention time of the cyanide chromatographic peak is approximately 2.7 min). After deducting the interference peaks in the blank solvent, the resolution between the cyanide chromatographic peak and its adjacent chromatographic peak should be greater than 5. The RSD% of the peak area of the cyanide peak in the repeated injection of the reference solution (at least 5 times, 6 times in this example) should not be greater than 10%; the signal-to-noise ratio of the cyanide peak in the chromatogram of the sensitivity solution should not be less than 100.

[0114] (5) Determination: Separately take the blank solution, the reference solution, the sensitivity solution, and the test solution for headspace injection, record the chromatogram, and calculate the cyanide content in the test sample by the external standard method based on the peak area.

[0115] Example 2: Methodological Performance and Sample Determination Using the Method of Example 1

[0116] The determination was carried out using the method of Example 1, and some typical results are provided as follows.

[0117] 1. Retention time and resolution

[0118] The results of cyanide retention time, resolution, peak area and related RSD% in the chromatograms obtained from a typical six - time determination of the reference solution are shown in the following table.

[0119] Item 1 2 3 4 5 6 Average Value RSD% Retention Time (min) 2.732 2.732 2.732 2.732 2.732 2.732 2.732 0.00 Peak Area 15581.9 17508.6 17295.0 17715.4 17881.1 18573.8 17426.0 5.76 Resolution 16.62 16.62 16.62 16.43 16.52 16.43 16.5 0.57

[0120] The RSD of the cyanide retention time in the reference solution was less than 0.1%, the RSD of the peak area was less than 6.0%, and the resolution between cyanide and adjacent chromatographic peaks was greater than 16, meeting the general requirements of verification.

[0121] A typical chromatogram of the reference solution is as Figure 2 shown, showing a cyanide retention time of 2.732 min, a resolution greater than 5.0 from its adjacent peak, and the theoretical plate number of the chromatographic column calculated for the cyanide peak in this chromatogram was 33740. It was observed that the theoretical plate number could remain above 28000 for more than 200 determinations of the test solution or reference solution using the method of Example 1, and no phenomenon of decreasing theoretical plate number due to the increase in the number of determinations was seen.

[0122] 2. Signal - to - noise ratio

[0123] A typical chromatogram of the sensitivity solution is as Figure 3 shown, and the recorded signal - to - noise ratio S / N was 182.

[0124] 3. Sample determination results and stability

[0125] According to the method of Example 1, three batches of sodium nitroprusside injection (provided by Hainan Puli Pharmaceutical) were determined, and the results are shown in the following table.

[0126] Test Sample Measured Cyanide Concentration (n = 6) RSD RSDx Sodium Nitroprusside Injection LOT1 9.46ppm 4.84％ 8.53％ Sodium Nitroprusside Injection LOT2 10.37ppm 5.16％ 9.86％ Sodium Nitroprusside Injection LOT3 9.18ppm 4.17％ 8.79％

[0127] In the above table, the determination results in the RSDx column are the RSD statistical values of six results obtained from headspace gas chromatography determinations at 0 h, 1 h, 2 h, 3 h, 4 h, and 5 h after the test solution prepared from the same sample and the derivatization reagent were sealed in a derivatization reaction bottle and placed at room temperature for 5 hours. The RSDs were all less than 15%. These results indicate that using the method of this example, the test solution and the derivatization reagent are stable within 5 hours after being sealed in a derivatization reaction bottle and placed at room temperature.

[0128] A typical chromatogram of the test solution of Puli sodium nitroprusside injection (LOT1) is shown inFigure 4 。

[0129] In addition, the inventor of the present invention sealed a reference solution and a derivatization reagent in a derivatization reaction bottle, prepared 6 portions in parallel, placed them at room temperature, and performed headspace gas chromatography determination at 0 h, 1 h, 2 h, 3 h, 4 h, and 5 h respectively. The RSD of the 6 results was 9.26%, which also indicates that the method of this example is stable for the reference solution placed at room temperature within 5 hours.

[0130] Example 3: Using a Split Headspace Vial to Determine Cyanide Content in Sodium Nitroprusside Injection

[0131] This example refers to the methods of Example 1 and Example 2. The only difference is that the integrated headspace bottle is changed to a split type, that is, the outer bottle is a 20 ml stoppered glass bottle (which can be a conventional 20 ml headspace bottle for gas chromatography injection), and the inner bottle is a 2 ml open glass bottle (which can be a conventional 2 ml small vial for gas injection). The inner bottle can be placed into the outer bottle from the bottle mouth, forming a structure similar to but with the inner and outer bottles separable; during the test, 1.0 ml of a test solution such as a reference solution or a test sample solution is added to the outer bottle, and then the inner bottle containing 0.5 ml of the derivatization reagent is carefully placed into the outer bottle, and the outer bottle is sealed with a cap. For example, the blank solution, reference solution, sensitivity solution, and test sample solution are prepared as follows: Figure 1 (2c) Blank solution: Take 1.0 ml of diluent and place it in a 20 ml headspace bottle. In the same headspace bottle, carefully place a 2 ml small vial for gas injection containing 0.5 ml of the derivatization reagent, and seal it with a cap to obtain;

[0132] (2d) Reference solution: Prepare the reference stock solution ①, reference stock solution ②, and reference solution respectively as described in Example 1. Then, take 1.0 ml of the reference solution and place it in a 20 ml headspace bottle. In the same headspace bottle, carefully place a 2 ml small vial for gas injection containing 0.5 ml of the derivatization reagent, and seal it with a cap to obtain;

[0133] (2e) Sensitivity solution: Precision transfer 1.0 ml of the reference solution into a 100 ml volumetric flask, dilute it to the mark with diluent, and shake well to obtain the sensitivity solution (equivalent to 0.01 μg / ml). Take 1.0 ml of the sensitivity solution and place it in a 20 ml headspace bottle. In the same headspace bottle, carefully place a 2 ml small vial for gas injection containing 0.5 ml of the derivatization reagent, and seal it with a cap to obtain;

[0134] (2f) Test sample solution: Take the test sample, weigh it accurately, add an appropriate amount of diluent to dissolve it, and transfer it to a 100 ml volumetric flask. Dilute it to the mark with diluent, shake well, and then transfer an appropriate amount of the solution to a 20 ml headspace bottle. In the same headspace bottle, carefully place a 2 ml small vial for gas injection containing 0.5 ml of the derivatization reagent, and seal it with a cap to obtain;

[0135] (2f)Test solution: Accurately measure 1.0 ml of sodium nitroprusside injection (produced by Hainan Pulitzer Pharmaceutical Co., Ltd., specification 50 mg / 2 ml) and place it in a 20-ml headspace vial. Add 10 μl of phosphoric acid, shake well. In the same headspace vial, carefully place a 2-ml gas chromatograph injection vial containing 0.5 ml of derivatization reagent, cover and seal tightly to obtain the test solution.

[0136] The remaining operations and test conditions are the same as those in Example 1 and Example 2, and the results of each determination are basically the same as those recorded in Example 2:

[0137] The average retention time of the reference solution measured 6 times was 2.733 (RSD < 0.1%), the average peak area was 18142.4 (RSD = 6.13%), and the average resolution was 16.8 (RSD = 0.64%); the signal-to-noise ratio S / N of the sensitivity solution was 173; the test results of 3 batches of sodium nitroprusside injection (measured 6 times within 5 hours, n = 6) were as follows: Pulitzer sodium nitroprusside injection LOT1 = 9.34 ppm (RSD = 6.57%), Pulitzer sodium nitroprusside injection LOT2 = 10.92 ppm (RSD = 5.84%), Pulitzer sodium nitroprusside injection LOT3 = 9.48 ppm (RSD = 6.23%).

[0138] Example 4: Changing the Diluent to Determine Cyanide Content in Sodium Nitroprusside Injection

[0139] This example refers to the methods of Example 1 and Example 2. The only difference is that the phosphoric acid used therein is replaced with sulfuric acid, hydrochloric acid or nitric acid. For example, the diluent is replaced with 1% sulfuric acid solution, hydrochloric acid solution or nitric acid solution, and 10 μl of phosphoric acid is replaced with an equal volume of sulfuric acid, hydrochloric acid or nitric acid when preparing the test solution. The remaining operations and test conditions are the same as those in Example 1 and Example 2. It was found that when using sulfuric acid, the results of each determination were basically the same as those recorded in Example 2: the average retention time of the reference solution measured 6 times was 2.731 (RSD < 0.1%), the average peak area was 17884.7 (RSD = 5.74%), and the average resolution was 15.7 (RSD = 0.56%); the signal-to-noise ratio S / N of the sensitivity solution was 194; the test results of 3 batches of sodium nitroprusside injection (measured 6 times within 5 hours, n = 6) were as follows: Pulitzer sodium nitroprusside injection LOT1 = 9.31 ppm (RSD = 6.03%), Pulitzer sodium nitroprusside injection LOT2 = 10.73 ppm (RSD = 6.31%), Pulitzer sodium nitroprusside injection LOT3 = 9.74 ppm (RSD = 5.78%), and there was no decrease in the theoretical plate number of the cyanide chromatographic peak during the test. However, in the case of using hydrochloric acid and nitric acid, it was found that after more than 20 determinations, the theoretical plate number calculated from the cyanide chromatographic peak would rapidly decrease from about 33000 to below 15000, which is completely unacceptable. Therefore, according to the method of the present invention, the acid used can be phosphoric acid or sulfuric acid.

[0140] This example refers to the methods of Example 1 and Example 2. The only difference is that the diluent used therein is changed to 0.5% phosphoric acid solution or 2% phosphoric acid solution, and the remaining operations and test conditions are the same as those in Example 1 and Example 2. It was found that when using phosphoric acid at two concentrations, the results of each determination were basically the same as those recorded in Example 2. For example, the average peak area of the reference solution was 18063.2 (RSD = 5.12%) for 6 determinations, and the cyanide determination results of the corresponding 3 batches of sodium nitroprusside injection were all within the range of 9 - 11 ppm, and the RSDs of 6 determinations were all within the range of 4.8% - 6.1%. During the test process, no decrease in the theoretical plate number of the cyanide chromatographic peak was observed.

[0141] This example refers to the methods of Example 1 and Example 2. The only difference is that 7.5 μl or 15 μl of phosphoric acid is added when preparing the test solution, and the remaining operations and test conditions are the same as those in Example 1 and Example 2. It was found that when using phosphoric acid at two concentrations, the results of each determination were basically the same as those recorded in Example 2. For example, the average peak area of the reference solution was 17897.4 (RSD = 5.47%) for 6 determinations, and the cyanide determination results of the corresponding 3 batches of sodium nitroprusside injection were all within the range of 9 - 11 ppm, and the RSDs of 6 determinations were all within the range of 4.6% - 5.7%. During the test process, no decrease in the theoretical plate number of the cyanide chromatographic peak was observed.

[0142] Example 5: Changing the Equilibrium Temperature or Equilibrium Time to Determine Cyanide Content in Sodium Nitroprusside Injection

[0143] As described in Example 1 of the present invention, when headspace sampling, the equilibration temperature of the headspace vial is 50 °C and the equilibration time is 20 minutes.

[0144] This example examines the influence of changing the equilibration temperature and / or equilibration time on the method. Referring to the methods of Example 1 and Example 2, the difference is that the equilibration temperature of the headspace vial is set to 45 °C or 60 °C during headspace sampling, and other conditions remain unchanged. It was found that when using two equilibration temperatures, the results of each determination were basically the same as those recorded in Example 2. For example, the average peak areas of the reference solution were 17584.2 (RSD = 5.35%) and 17937.4 (RSD = 5.82%) for 6 determinations.

[0145] Referring to the methods of Example 1 and Example 2, the difference is that the equilibration time of the headspace vial is set to 15 minutes or 30 minutes during headspace sampling, and other conditions remain unchanged. It was found that when using two equilibration times, the results of each determination were basically the same as those recorded in Example 2. For example, the average peak areas of the reference solution were 17821.6 (RSD = 6.07%) and 18224.8 (RSD = 5.44%) for 6 determinations. According to the above results, it is feasible that the equilibration temperature of the headspace vial is 45 - 60 °C and the equilibration time is 15 - 30 minutes during headspace sampling of the present invention.

[0146] However, in further investigations, when the equilibrium temperature was further decreased to 35 °C or increased to 75 °C, it was found that the main peak area of the reference solution decreased significantly at low equilibrium temperatures. The average peak area of the reference solution measured 6 times was 11473.4 (RSD = 6.43%); while at high equilibrium temperatures, although the main peak area of the reference solution did not change significantly, the deviation became larger. The average peak area of the reference solution measured 6 times was 18024.3 (RSD = 14.62%). Therefore, it is not advisable to have too high or too low an equilibrium temperature.

[0147] In addition, in further investigations referring to the methods of Example 1 and Example 2, when the equilibrium time was further decreased to 10 minutes or increased to 60 minutes, it was found that the main peak area of the reference solution decreased significantly at low equilibrium times. The average peak area of the reference solution measured 6 times was 12424.6 (RSD = 5.75%); while at high equilibrium times, although the main peak area of the reference solution did not change significantly, the deviation became larger. The average peak area of the reference solution measured 6 times was 17863.1 (RSD = 12.84%). Therefore, it is not advisable to have too high or too low an equilibrium time.

[0148] Example 6: Determining Cyanide Content in Sodium Nitroprusside Freeze-Dried Powder for Injection

[0149] Referring to the methods of Example 1 and Example 2, 3 batches of freeze-dried sodium nitroprusside for injection were determined. The test method only changed the preparation of the test solution: First, accurately add 2 ml (for the specification of 50 mg per bottle) of 5% glucose injection to each bottle of freeze-dried powder to dissolve the powder, and then precisely measure 1.0 ml of the obtained solution and place it in a derivatization reaction bottle for subsequent treatment.

[0150] The parameters such as retention time, resolution, signal-to-noise ratio, and theoretical plate number obtained using the reference solution were the same as those in Example 2. The determination results of 3 batches of freeze-dried sodium nitroprusside for injection were as follows: Cyanide concentration of powder injection 1 (n = 6) = 11.24 ppm (RSD = 5.78%), cyanide concentration of powder injection 2 (n = 6) = 9.32 ppm (RSD = 4.46%), cyanide concentration of powder injection 3 (n = 6) = 10.67 ppm (RSD = 6.13%). The preparation method of the above powder injection 1 is as follows: Prepared according to the method of Example 2 in the specification of CN107412259B

[0062] -

[0069] , with 50 mg of the active ingredient per bottle; the preparation method of powder injection 2 is as follows: Prepared according to the method of Example 3 in the specification of CN 115350140 A

[0059] -

[0066] , with 50 mg of the active ingredient per bottle; the preparation method of powder injection 3 is as follows: Prepared according to the method of Example 1 in the specification of CN 103768096 A

[0028] -

[0036] , with 50 mg of the active ingredient per bottle.

[0151] In some other examples of the present invention, tests were also carried out using methylpolysiloxane or phenylmethylpolysiloxane as the stationary liquid of the capillary chromatographic column, and the results all showed excellent test results. For example, the theoretical plate number of the main component peak reached more than 30,000, the resolution between the main component peak and the adjacent impurity peak was greater than 13, the RSD of the control solution tested 6 times was less than 8%, and the signal-to-noise ratio of the cyanide peak in the chromatogram obtained by headspace injection of the sensitivity solution was greater than 150.

[0152] Example 7: Preparing a Sodium Nitroprusside Pharmaceutical Composition in a Commercial Form

[0153] This embodiment provides a method for preparing a commercially available form of a sodium nitroprusside pharmaceutical composition, which is a pharmaceutical preparation form of an injection sealed in a glass bottle or a freeze-dried powder injection. Calculated as sodium nitroferricyanide dihydrate, each bottle of the injection or freeze-dried powder injection contains 25 mg or 50 mg of sodium nitroprusside.

[0154] The method for preparing a commercially available form of a sodium nitroprusside pharmaceutical composition in this embodiment includes the following steps:

[0155] i) According to the injection preparation method or the freeze-dried powder injection preparation method, prepare a sodium nitroprusside pharmaceutical composition in the form of a pharmaceutical preparation of an injection sealed in a glass bottle or a freeze-dried powder injection. Calculated as sodium nitroferricyanide dihydrate, each bottle of the injection or freeze-dried powder injection contains 25 mg or 50 mg of sodium nitroprusside;

[0156] ii) Use the method of Examples 1-2 of the present invention to determine the cyanide content in the sodium nitroprusside pharmaceutical composition obtained in step i);

[0157] iii) If the cyanide content measured in step ii) is less than 20 ppm, such as 15 ppm, such as 10 ppm, pack the injection sealed in a glass bottle or the freeze-dried powder injection with a paper packaging box to obtain a commercially available form of the sodium nitroprusside pharmaceutical composition.

[0158] It is well known to those of ordinary skill in the pharmaceutical manufacturing technology field that when preparing a commercially available form of a pharmaceutical preparation, the pharmaceutical preparation is usually embodied in the form of a commercially available package. For example, each bottle of an injection or a freeze-dried powder for injection is packed in a (small) paper box, and if necessary, multiple small paper boxes are packed into a large paper box for sale in this packaging form. Before loading the medicine bottle into the paper box, it is necessary to first inspect the medicine, and it can be further packed into the paper box only when the inspection is qualified or determined to be qualified after the inspection. Therefore, the process of determining the cyanide content in the medicine inside the medicine bottle is also an essential step in preparing a commercially available form of the pharmaceutical composition.

[0159] The above descriptions are given for various aspects of the present invention. It should be understood that these examples are only illustrative, and the protection scope of the present invention is not limited to such examples.

Claims

1. A method for determining the cyanide content in a sodium nitroprusside pharmaceutical composition using gas chromatography, the method comprising the following steps: (1) Provide a gas chromatograph, a capillary chromatographic column, and a derivatization reaction flask; (2) Preparation of solutions: (2a) Diluent: Take an appropriate amount of inorganic acid and add water to make an acid aqueous solution with a concentration of 0.5% - 2%; (2b) Derivatization reagent: Take chloramine T, dissolve it in water and dilute to make a chloramine T solution as the derivatization reagent; (2c) Blank solution: Place the diluent in the outer flask of the derivatization reaction flask, and add the derivatization reagent to the inner flask of the derivatization reaction flask, cover and seal to obtain; (2d) Reference solution: Weigh accurately an appropriate amount of potassium cyanide reference substance, dissolve and dilute it with the diluent to make a reference solution with a concentration of 0.2 - 10 mg / ml; Place the reference solution in the outer flask of the derivatization reaction flask, and add the derivatization reagent to the inner flask of the derivatization reaction flask, cover and seal to obtain; (2f) Test solution: Accurately measure an aqueous solution of the sodium nitroprusside pharmaceutical composition with a concentration of 20 - 50 mg / ml and place it in the outer flask of the derivatization reaction flask, and add an appropriate amount of inorganic acid. The volume ratio of the inorganic acid to the aqueous solution of the sodium nitroprusside pharmaceutical composition is 0.75 - 2:100, shake well, and add the derivatization reagent to the inner flask of the derivatization reaction flask, cover and seal to obtain; (3) Chromatographic conditions: When using headspace injection, the equilibrium temperature of the headspace vial is 45 - 60 °C, and the equilibrium time is 15 - 30 minutes; The injection port temperature is 180 - 240 °C, the quantitative loop temperature is 80 - 120 °C, the transfer line temperature is 80 - 120 °C, the ECD detector temperature is 280 - 350 °C, and the injection time is 0.5 - 5 min; High-purity nitrogen is used as the carrier gas, with a flow rate of 0.5 - 2.0 mL / min and a split ratio of 8 - 15:1; Programmed temperature rise: The initial column temperature is maintained at 38 - 45 °C for 4 - 6 minutes, then heated at a rate of 28 - 35 °C per minute to 180 - 240 °C, and then maintained for 3 - 5 minutes; (4) Requirements for system suitability test: Take the blank solution and the reference solution for headspace injection respectively, record the chromatogram, and determine the cyanide chromatographic peak; deduct the interference peak in the blank solvent. The resolution between the cyanide chromatographic peak and its adjacent chromatographic peak is greater than 5, and the RSD% of the cyanide peak area of the repeated injection of the reference solution shall not be greater than 10%; (5) Determination: Take the blank solution, the reference solution, and the test solution for headspace injection respectively, record the chromatogram, and calculate the cyanide content in the test sample by the external standard method based on the peak area; Wherein: The stationary liquid of the capillary chromatographic column is selected from polyethylene glycol, methyl polysiloxane, and phenyl methyl polysiloxane; The derivatization reaction flask includes an open vial and a stoppered large bottle. The small bottle is placed in the large bottle, forming a structure of an outer bottle (the large bottle) and an inner bottle (the small bottle). After adding the derivatization reagent and the test solution to the inner bottle and the outer bottle respectively, the outer bottle is tightly stoppered, and the liquid levels of the outer bottle and the inner bottle form a communicating gas atmosphere; The volume ratio of the blank solution, the reference solution or the test solution and the derivatization reagent added to the liquids in the outer bottle and the inner bottle is 1 - 4:1; The inorganic acid is selected from phosphoric acid or sulfuric acid.

2. The method according to claim 1, wherein in the test sample solution in step (2f), the volume ratio of the inorganic acid to the aqueous solution of the sodium nitroprusside pharmaceutical composition is 1:

100.

3. The method according to claim 1, wherein in the chromatographic conditions of step (3), when headspace sampling is performed, the equilibrium temperature of the headspace vial is 50°C and the equilibrium time is 20 minutes.

4. The method according to claim 1, wherein the volume ratio of the liquids added to the outer vial and the inner vial for the blank solution, the reference solution or the test sample solution and the derivatization reagent is 2 - 3:

1.

5. The method according to claim 1, which further comprises the following step (2e) of preparing a sensitivity solution: (2e)Precisely pipette 1.0 ml of the reference solution obtained in step (2d) into a 100-ml volumetric flask, dilute to the mark with the diluent, shake well to obtain the sensitivity solution. Transfer the sensitivity solution to the outer flask of the derivatization reaction flask. Separately, add the derivatization reagent to the inner flask of the derivatization reaction flask, cover and seal it to obtain the product; among which, The volume ratio of the liquids in the outer vial and the inner vial is 1 - 4:

1.

6. The method according to claim 5, wherein: The signal-to-noise ratio of the cyanide peak in the chromatogram obtained by headspace sampling and testing with the sensitivity solution is not less than 100.

7. The method according to claim 1, wherein the inner vial and the outer vial of the derivatization reaction vial are of an integrally fused glass bottle structure or are composed of a combination of 2 independent glass bottles.

8. The method according to claim 1, wherein the outer vial and the inner vial of the derivatization reaction vial are each independently a brown glass bottle.

9. The method according to claim 5, wherein the stopper of the outer vial of the derivatization reaction vial is a rubber stopper.

10. The method according to claim 1, wherein the inner vial of the derivatization reaction vial has a volume of 1 ml - 4 ml, and the volume of the solution in the inner vial of the derivatization reaction vial accounts for 1 / 4 - 3 / 4 of the volume of the inner vial.

11. The method according to claim 1, wherein the outer vial of the derivatization reaction vial has a volume of 10 ml - 30 ml.

12. The method according to claim 1, wherein the inner vial of the derivatization reaction vial has a volume of 2 ml and the outer vial has a volume of 20 ml. During testing, 0.5 ml of the derivatization reagent is added to the inner vial and 1.0 ml of the test solution is added to the outer vial.

13. The method according to claim 1, wherein the capillary chromatographic column is a fused silica capillary chromatographic column with a specification of 30 m * 0.25 mm, 0.5 μm.

14. The method according to claim 1, wherein the capillary chromatographic column is a DB-WAXetr fused silica capillary chromatographic column with 100% polyethylene glycol as the stationary liquid and a specification of 30 m * 0.25 mm, 0.5 μm.

15. The method according to claim 5, when adding the blank solution, the reference solution, the test sample solution and optionally the sensitivity solution to the derivatization reaction vial, it is added in the following manner: Take 1.0 ml of these solutions and place them in the outer vial of the derivatization reaction vial, and add 0.5 ml of the derivatization reagent to the inner vial of the derivatization reaction vial, then seal it with a cap to obtain the sample.

16. The method according to claim 15, in the chromatogram obtained by measuring with the reference solution, the number of theoretical plates of the cyanide peak is greater than 20000.

17. The method according to claim 15, after sealing the reference solution or the test sample solution and the derivatization reagent in the derivatization reaction vial and placing it at room temperature for 5 hours, headspace gas chromatography determination is performed at 0 h, 1 h, 2 h, 3 h, 4 h, and 5 h respectively, and the RSD of the 6 results is less than 15%.

18. A method for preparing a commercial form of a sodium nitroprusside pharmaceutical composition, comprising the following steps: i) Prepare a sodium nitroprusside pharmaceutical composition in the form of an injection or a freeze-dried powder injection sealed in a glass bottle according to the injection preparation method or the freeze-dried powder injection preparation method. Calculated as sodium nitroferricyanide dihydrate, each bottle of the injection or freeze-dried powder injection contains 25 mg or 50 mg of sodium nitroprusside; ii) Determine the cyanide content in the sodium nitroprusside pharmaceutical composition obtained in step i) by using the method described in any one of claims 1 to 17; iii) If the cyanide content measured in step ii) is less than 15 ppm, pack the injection or freeze-dried powder injection sealed in a glass bottle with a paper packaging box to obtain a commercially available form of the sodium nitroprusside pharmaceutical composition.

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