A method for determining the dissolution curve of relugolix tablets

Abstract

The present invention belongs to the technical field of drug analysis, and provides a method for determining the dissolution curve of relugolix tablets with good durability, reproducibility and discrimination. The method uses a pH 5.6 phosphate buffer as a dissolution medium, and solves the problem that the dissolution method in the prior art cannot meet the requirement of the similarity factor (f2) method for comparing the similarity of dissolution curves in the "Guidelines for Determination and Comparison of Dissolution Curves of Ordinary Oral Solid Preparations", thereby affecting the process screening and in vitro consistency evaluation of relugolix tablets.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to a method for determining a dissolution curve of a relugolix tablet. Background Art

[0002] Relugolix is ​​the first oral gonadotropin-releasing hormone (GnRH) receptor antagonist used to treat endometriosis and advanced prostate cancer. It is a small molecule gonadotropin-releasing hormone (GnRH) receptor antagonist used in the treatment of uterine fibroids and symptom relief in 2019. Because it can quickly suppress the release of LH and FSH from the pituitary gland in a short period of time, and has been confirmed by multiple clinical phase I and phase II studies to reduce testosterone levels, it was later approved for the treatment of advanced prostate cancer.

[0003] According to the Biopharmaceutics Classification System (BCS), relugolix is ​​classified as BCS Class IV, which is a low solubility and low permeability drug. The dissolution and dissolution of the drug directly affect the absorption of the drug. Referring to the "Guidelines for Determination and Comparison of Dissolution Curves of Ordinary Oral Solid Preparations" and "Questions and Answers on Dissolution Curve Study Conditions in the "Technical Guidelines for Pharmaceutical Change Research of Marketed Chemical Drugs (Trial)" (Draft for Comments), it is recommended to select dissolution media with no less than 3 pH values ​​(at least pH 1.2, 4.5, and 6.8) for dissolution curve investigation. Some standard media (referring to the media used in the registration standard) may contain a small amount of surfactants. Except for the standard media, it is not recommended to add other media. For drugs whose solubility is greatly affected by pH, it may be necessary to investigate in dissolution media with more pH values. As a drug whose solubility is greatly affected by pH value (the higher the pH, the lower the solubility), relugolix dissolves too quickly under pH 1.2 and pH 4.5 medium conditions, with the dissolution exceeding 85% in 15 minutes; it dissolves too slowly under pH 6.8 medium conditions, and cannot reach 85% in 6 hours; the only medium available for reference for dissolution curve determination is pH 5.5 citrate buffer salt recommended in the FDA review report, but under this medium condition, the dissolution of the reference preparation of relugolix reaches 85% in 15 minutes, and it is impossible to refer to the similarity factor (f2) method provided in the "Guidelines for Determination and Comparison of Dissolution Curves of Ordinary Oral Solid Preparations" for evaluation, thus affecting the in vitro consistency evaluation of generic drugs. Therefore, the development of a discriminating dissolution curve is of great significance for guiding the research and development of relugolix generic preparations, evaluating the consistency of quality within and between batches of preparations, and evaluating the consistency of quality and efficacy before and after changes in drug prescription processes. Summary of the invention

[0004] The present invention provides a method for determining the dissolution curve of relugolix tablets with good durability, reproducibility and discrimination by examining the dissolution curves in different pH media, so as to solve the problem that the dissolution method in the prior art cannot meet the requirement of the similarity factor (f2) method for comparing the similarity of dissolution curves in the "Guidelines for Determination and Comparison of Dissolution Curves of Ordinary Oral Solid Preparations", thereby affecting the process screening and in vitro consistency evaluation of relugolix tablets.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] A method for determining the dissolution curve of relugolix tablets comprises the following steps:

[0007] Step (1): preparing pH 5.6 phosphate buffer as a dissolution medium;

[0008] Step (2): Measure the dissolution medium in step (1) and place it in a dissolution cup, raise the temperature, put the relugolix tablets into the dissolution cup under stirring conditions, start the time, take the dissolution liquid at different dissolution time points, detect the dissolution amount, and establish a dissolution curve based on the dissolution amount and dissolution time.

[0009] Furthermore, in step (1), the dissolution medium comprises a mixed solution of potassium dihydrogen phosphate, a pH regulator and water, and the pH regulator is sodium hydroxide.

[0010] Furthermore, the specific preparation method of the dissolution medium in step (1) is to weigh 27.200±0.100 g of potassium dihydrogen phosphate and add 90 ml-97 ml of sodium hydroxide solution into 4 L of degassed purified water to dissolve.

[0011] Furthermore, the specific preparation method of the dissolution medium in step (1) is to weigh 27.200±0.100g of potassium dihydrogen phosphate, and weigh 90-97ml of 0.1mol / L sodium hydroxide solution (4.000±0.004g of sodium hydroxide is weighed in a 1L volumetric flask, dissolved in degassed purified water and diluted to the scale) and add it to 4L of degassed purified water to dissolve, and the volume of the added 0.1mol / L sodium hydroxide solution is preferably 94ml.

[0012] Furthermore, in step (2), the amount of the dissolution medium is 900 mL / cup, the stirring speed is 40-75 rpm / min, preferably 50 rpm / min, and the temperature of the dissolution medium is 37±0.5°C.

[0013] Furthermore, the different dissolution time points in step (2) are 5, 10, 15, 20, 30, 45, 60, 90, and 120 min.

[0014] Furthermore, the method for detecting the dissolution amount in step (2) is high performance liquid chromatography.

[0015] Furthermore, in the high performance liquid chromatography detection process, the chromatographic column used is a C8 chromatographic column, preferably an Agilent SB-C8 chromatographic column.

[0016] Furthermore, during the high performance liquid chromatography detection process, the mobile phase used includes a phosphate buffer solution and acetonitrile, the volume ratio of the phosphate buffer solution to the acetonitrile is 3:2; the pH value of the phosphate buffer is 2.2±0.1, preferably a phosphate buffer with a pH of 2.2; the volume ratio of the phosphate buffer solution to acetonitrile is 3:2; preferably, the pH value of the phosphate buffer is adjusted by phosphoric acid.

[0017] The phosphate buffer solution is a potassium dihydrogen phosphate solution, which includes potassium dihydrogen phosphate and water. The molar volume ratio of the potassium dihydrogen phosphate to water is 0.01-0.03:1, preferably 0.02:1.

[0018] Furthermore, during the high performance liquid chromatography detection process, the mobile phase used is acetonitrile and 0.02 mol / L potassium dihydrogen phosphate solution, and the pH value of the 0.02 mol / L potassium dihydrogen phosphate solution is adjusted to 2.2±0.1 with phosphoric acid, wherein the volume ratio of acetonitrile to 0.02 mol / L potassium dihydrogen phosphate solution is 3:2.

[0019] Furthermore, during the HPLC detection process, the column temperature of the chromatographic column was 40° C., the detection wavelength was 291 nm, the injection volume was 5 μl, and the flow rate was 1.0 ml / min.

[0020] Furthermore, the chromatographic conditions of the high performance liquid chromatography are:

[0021] Chromatographic column: Agilent SB-C8 column; detection wavelength: 291nm; flow rate 1.0ml / min; injection volume: 5μl; column temperature: 40℃; mobile phase: a mixed solution of 0.02mol / L potassium dihydrogen phosphate solution and acetonitrile, the pH value of 0.02mol / L potassium dihydrogen phosphate solution was adjusted to 2.2±0.1 with phosphoric acid, and the volume ratio of 0.02mol / L potassium dihydrogen phosphate solution to acetonitrile was 3:2.

[0022] Furthermore, the mobile phase is prepared by dissolving 2.72 g of potassium dihydrogen phosphate in 1 L of purified water, adjusting the pH to 2.2±0.1 with phosphoric acid, filtering to obtain a phosphate buffer, and mixing it with acetonitrile so that the volume ratio of the two is 3:2.

[0023] Furthermore, during the HPLC detection process, the test solution is prepared by taking 10 ml of the dissolution solution at different time points in step (2), filtering with a 0.45 μm filter membrane, discarding 5-7 ml of the initial filtrate, and taking the subsequent filtrate as the test solution.

[0024] Furthermore, the preparation method of the test solution is: take 10 ml of the dissolution solution at different time points in step (2) respectively, and replenish 10 ml of the dissolution medium in time, filter the 10 ml of the dissolution solution using a PES (0.45 μm, 25 mm) filter head, discard 6 ml of the initial filtrate, and take the subsequent filtrate as the test solution.

[0025] Furthermore, the filter head PES (0.45μm, 25mm) used for the dissolution solution is a commonly used filter head for water systems. However, it was found during the application process that after the same filter head was used continuously for a long time, the dissolution amount obtained increased abnormally, indicating that the filter head was damaged or the relugoli adsorbed on the filter membrane was re-dissolved. The preferred method is to replace a new filter head after each time point.

[0026] Furthermore, the dissolution operation process of step (2) can refer to the conventional dissolution process, such as using the paddle method to perform the dissolution operation, and the dissolution apparatus can refer to the second method of General Rules 0931 of Part 4 of the 2020 edition of the Chinese Pharmacopoeia.

[0027] Furthermore, the specific implementation process of the high performance liquid chromatography method can refer to the conventional process in the prior art, and preferably the external standard method is used for quantitative analysis.

[0028] Furthermore, the method for determining the dissolution curve of the relugolix tablets of the present invention comprises the following steps:

[0029] Step (1): Weigh 27.200±0.100 g of potassium dihydrogen phosphate and add 90 ml-97 ml of sodium hydroxide solution to 4 L of degassed purified water to dissolve;

[0030] Step (2): using the paddle method, 900 ml of the dissolution medium in step (1) was measured and placed in a dissolution cup, the temperature of the dissolution medium was set to 37±0.5°C, the stirring speed was 50 rpm / min, and the relugolix tablets were placed in the dissolution cup. The time was counted, and 10 ml of the dissolution solution was taken out after 5, 10, 15, 20, 30, 45, 60, 90, and 120 minutes, respectively, and 10 ml of the dissolution medium was replenished in time. The 10 ml of the dissolution solution taken out was filtered with a 0.45 μm filter membrane, 5-7 ml of the initial filtrate was discarded, and the subsequent filtrate was taken as the test solution. The dissolution amount was detected by high performance liquid chromatography, and a dissolution curve was established according to the dissolution amount and dissolution time;

[0031] Furthermore, a method for determining the dissolution curve of relugolix tablets of the present invention comprises the following steps:

[0032] Step (1): Weigh 27.200±0.100 g of potassium dihydrogen phosphate and add 90 ml-97 ml of sodium hydroxide solution to 4 L of degassed purified water to dissolve;

[0033] Step (2): using the paddle method, 900 ml of the dissolution medium in step (1) was measured and placed in a dissolution cup, the temperature of the dissolution medium was set to 37±0.5°C, the stirring speed was 50 rpm / min, relugolix tablets were placed in the dissolution cup, and timing was performed. 10 ml of the dissolution solution was taken out after 5, 10, 15, 20, 30, 45, 60, 90, and 120 min, respectively, and 10 ml of the dissolution medium was replenished in time. The 10 ml of the dissolution solution was filtered through a 0.45 μm filter membrane, 5-7 ml of the initial filtrate was discarded, and the subsequent filtrate was taken as the test solution. The dissolution amount was detected by high performance liquid chromatography, and a dissolution curve was established according to the dissolution amount and dissolution time;

[0034] The chromatographic conditions of the high performance liquid chromatography method are as follows: chromatographic column: Agilent SB-C8 chromatographic column; detection wavelength: 291 nm; flow rate: 1.0 ml / min; injection volume: 5 μl; column temperature: 40° C.; mobile phase: a mixed solution of 0.02 mol / L potassium dihydrogen phosphate solution and acetonitrile, the pH value of the 0.02 mol / L potassium dihydrogen phosphate solution is adjusted to 2.2 with phosphoric acid, wherein the volume ratio of 0.02 mol / L potassium dihydrogen phosphate solution to acetonitrile is 3:2.

[0035] Compared with the prior art, the beneficial effects achieved by the present invention are:

[0036] (1) The present invention solves the defect that the conventional dissolution curve in the prior art has no discriminating power and cannot accurately measure the difference in in vitro dissolution behavior between the generic preparation of relugolix tablets and the original preparation. By applying the technical scheme of the present invention, the dissolution rate of relugolix tablets is significantly improved, the dissolution amount in 15 minutes is less than 85%, and the dissolution amount in 2 hours is greater than 85%, which meets the requirements of the "Guidelines for Determination and Comparison of Dissolution Curves of Ordinary Oral Solid Preparations" on the use of the similarity factor (f2) method to compare the similarity of dissolution curves. The obtained dissolution curve can be used for process screening and in vitro consistency evaluation, can better reflect the intrinsic quality of relugolix tablets, and has good in vitro and in vivo correlation, which is of great significance for improving the efficiency of bioequivalence tests, accelerating the development of generic drugs, enriching domestic drug supply, and reducing medical costs.

[0037] (2) This method is scientific, durable, and reproducible. It can effectively distinguish the effects of different formulation and process variables on the in vitro release of relugolix tablets. It can not only be used for the quality consistency evaluation of generic drugs and original drugs, but also provide a guarantee for the consistency of drug quality between batches, thereby ensuring the quality of drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 These are the dissolution curves of three batches of relugolix tablets reference preparations and two batches of homemade preparations provided in Example 1 of the present invention. DETAILED DESCRIPTION

[0039] The method of the present invention is described below by specific examples to make the technical solution of the present invention easier to understand and grasp, but the present invention is not limited thereto. The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified.

[0040] In the following examples, with reference to the "Guidelines for Determination and Comparison of Dissolution Curves of Ordinary Oral Solid Preparations", the similarity factor method was adopted to calculate the similarity factor (f2) using the dissolution data of the reference preparation and the homemade preparation measured at different time points. The similarity factor (f2) was used to evaluate the similarity between the reference preparation and the homemade preparation.

[0041] Similarity factor (f2) calculation formula:

[0042] Among them, Rt is the average dissolution amount measured by the reference preparation at time t; Tt is the average dissolution amount measured by the homemade sample at time t; n is the number of sampling time points.

[0043] Example 1

[0044] (1) Relugolix Tablets Sample

[0045] Reference preparation (Batch number: S11399A, T04328A, T05195A)

[0046] Homemade preparation (Batch No.: 22070402, 220705)

[0047] (2) Dissolution curve determination method:

[0048] 1. Dissolution conditions

[0049] Paddle method, dissolution medium volume 900 ml / cup, rotation speed 50 rpm / min, dissolution medium temperature 37±0.5℃.

[0050] 2. Dissolution medium preparation

[0051] Weigh 27.200±0.100g of potassium dihydrogen phosphate, and 90-97ml of 0.1mol / L sodium hydroxide solution (weigh 3.996-4.004g of sodium hydroxide in a 1L volumetric flask, dissolve it in degassed purified water and dilute to the scale) and add it to 4L of degassed purified water to dissolve.

[0052] 3. Detection method and solution preparation

[0053] The dissolution amount was detected by high performance liquid chromatography, and the chromatographic conditions were: Agilent SB-C8 column; detection wavelength: 291nm; flow rate: 1.0ml / min; injection volume: 5μl; column temperature: 40°C. Mobile phase: a mixed solution of 0.02mol / L potassium dihydrogen phosphate solution and acetonitrile (pH adjusted to 2.2±0.1 with phosphoric acid) (3:2).

[0054] Preparation of mobile phase: dissolve 2.72 g of potassium dihydrogen phosphate in 1 L of purified water, adjust the pH to 2.2±0.1 with phosphoric acid, filter to obtain phosphate buffer, and mix it with acetonitrile so that the volume ratio of the two is 3:2.

[0055] Preparation of reference solution: Accurately weigh 13.33 mg of relugolix reference solution into a 100 ml volumetric flask, add 5 ml of acetonitrile and ultrasonically dissolve it, then add dissolution medium and dilute to the scale.

[0056] Test solution: Using the paddle method, measure 900 ml / cup of the prepared dissolution medium and place it in the dissolution cup. Set the dissolution medium temperature at 37±0.5℃ and the stirring speed at 50 rpm / min. Put relugolix tablets in the dissolution cup, take out 10 ml of the dissolution solution at 5, 10, 15, 20, 30, 45, 60, 90, and 120 min, respectively, then add 10 ml of the dissolution medium, filter with a PES (0.45 μm, 25 mm) filter head, discard 6 ml of the initial filtrate, and take the subsequent filtrate directly as the test solution. Replace the filter head after sampling at each time point.

[0057] Determination method: Take 5μl of the reference solution and the test solution and inject them into the high performance liquid chromatograph respectively, and calculate the dissolution amount.

[0058] (3) Dissolution curve measurement results

[0059] According to the above determination method, the dissolution curves of three batches of reference preparations (batch numbers: S11399A, T04328A, T05195A) and two batches of homemade preparations (batch numbers: 22070402, 220705) were determined. The specific dissolution curve determination results (dissolution amount and relative standard deviation) are shown in Table 1. The dissolution curves are shown in Appendix Figure 1The similarity factor (f2) was calculated with reference to the “Guidelines for Determination and Comparison of Dissolution Curves of Ordinary Oral Solid Preparations” to evaluate the similarity between the reference preparation and the homemade preparation. The results are shown in Table 2.

[0060] Table 1 Dissolution curve data of each batch of preparations

[0061]

[0062] Table 2 Similarity factors (f2) of each batch of preparations

[0063] batch T04328A T05195A S11399A 22070402 30 34 31 220705 42 50 45

[0064] It can be seen from Table 1 that the dissolution curve data of the three batches of reference preparations, whether in terms of dissolution amount or relative standard deviation (RSD%), meet the data requirements for similarity comparison using the similarity factor (f2) in the "Guidelines for Determination and Comparison of Dissolution Curves of Ordinary Oral Solid Preparations".

[0065] It can be seen from Table 2 that the similarity factor (f2) of the homemade preparation batch 22070402 is significantly smaller than that of batch 220705, indicating that the dissolution curve method has a strong ability to distinguish and can distinguish the differences between different prescriptions. The uniformity and repeatability meet the requirements, indicating that this method is suitable for evaluating the differences between different batches of generic relugolix tablets. At the same time, it can also evaluate the differences in in vitro dissolution behavior between it and the original preparation, make predictions for in vivo bioequivalence tests, and improve the success rate of bioequivalence tests.

[0066] Test Example 1 Dissolution Medium pH Investigation Test

[0067] Relugoli tablets sample: homemade preparation (batch 220117)

[0068] According to the determination method described in Example 1, the homemade preparation (220117 batches) was used as the sample, the added volume of 0.1 mol / L sodium hydroxide solution in the dissolution medium preparation method of Example 1 was changed, and 87 ml, 90 ml, 92 ml, 95 ml and 97 ml were investigated respectively, and the pH of the dissolution medium was tested, and the dissolution data under different pH dissolution medium conditions were measured, and the results are shown in Table 3. And taking the added volume of 92 ml (first measurement) as the standard, the similarity factor (f2) of the dissolution curve obtained by other added volumes was calculated, and the results are shown in Table 4.

[0069] Table 3 Dissolution data under different pH dissolution media conditions

[0070]

[0071] Table 4 Similarity factors under different pH dissolution media conditions

[0072] Add volume 87ml 90ml 92ml(second time) 95ml 97ml Similarity factor (f2) 63 80 89 80 88 Dissolution medium pH 5.54 5.56 5.60 5.62 5.62

[0073] It can be seen from Table 4 that when the added volume is 90, 92, 95, and 97 ml, the pH of the dissolution medium is between 5.56 and 5.62, and the similarity factor (f2) is above 80; when the added volume is 87 ml, the pH of the dissolution medium is 5.54, and the similarity factor (f2) is significantly reduced to 63, indicating that the dissolution curve of relugolix tablets is extremely sensitive to slight changes in the pH of the medium under the condition of pH 5.6 medium, and the pH of the dissolution medium needs to be strictly controlled. When the added volume of 0.1 mol / L sodium hydroxide is between 90 and 97 ml, a stable and reproducible dissolution curve result can be obtained; when the added volume of 0.1 mol / L sodium hydroxide is not within this range (such as the 87 ml mentioned above), the result consistent with the added volume of 90-97 ml cannot be obtained.

[0074] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for determining the dissolution curve of relugolix tablets, characterized in that: Step (1): preparing pH 5.6 phosphate buffer as a dissolution medium; Step (2): taking a quantity of the dissolution medium in step (1) and placing it in a dissolution cup, adding the relugolix tablets into the dissolution cup under stirring conditions, timing, taking the dissolution liquid at different dissolution time points, testing the dissolution amount, and establishing a dissolution curve based on the dissolution amount and dissolution time; Wherein, the dissolution medium in step (1) consists of potassium dihydrogen phosphate, sodium hydroxide and water.

2. The dissolution curve determination method according to claim 1, characterized in that: The dissolution medium is specifically prepared by weighing 27.200±0.100 g of potassium dihydrogen phosphate and adding 90-97 ml of sodium hydroxide solution into 4 L of degassed purified water to dissolve.

3. The dissolution curve determination method according to claim 1, characterized in that: The method for detecting the dissolution amount in step (2) is high performance liquid chromatography.

4. The dissolution curve determination method according to claim 1, characterized in that: The amount of the dissolution medium is 900 mL / cup, the stirring speed is 40-75 rpm / min, and the temperature of the dissolution medium is 37±0.5°C.

5. The dissolution curve determination method according to claim 4, characterized in that: The stirring speed is 50 rpm / min.

6. The dissolution curve determination method according to claim 1, characterized in that: The different dissolution time points are 5, 10, 15, 20, 30, 45, 60, 90, and 120 min.

7. The dissolution curve determination method according to claim 3, characterized in that: During the high performance liquid chromatography detection process, the chromatographic conditions are as follows: the chromatographic column used is a C8 chromatographic column; the column temperature of the chromatographic column is 40°C; the detection wavelength is 291nm, the injection volume is 5μl, and the flow rate is 1.0ml / min; the mobile phase is a mixed solution of phosphate buffer solution and acetonitrile, the pH value of the phosphate buffer solution is 2.2±0.1; and the volume ratio of the phosphate buffer solution to acetonitrile is 3:

2.

8. The dissolution curve determination method according to claim 7, characterized in that: The C8 chromatographic column is an Agilent SB-C8 chromatographic column.

9. The dissolution curve determination method according to claim 7, characterized in that: The phosphate buffer solution is a potassium dihydrogen phosphate solution, which consists of potassium dihydrogen phosphate and water, and the molar volume ratio of the potassium dihydrogen phosphate to water is 0.01-0.03:

1.

10. The dissolution curve determination method according to claim 3, characterized in that: During the HPLC detection process, the test solution is prepared by taking 10 ml of the dissolution solution at different time points in step (2), filtering with a 0.45 μm filter membrane, discarding 5-7 ml of the initial filtrate, and taking the subsequent filtrate as the test solution.

11. The dissolution curve determination method according to claim 1, characterized in that: The steps include: Step (1): Weigh 27.200±0.100 g of potassium dihydrogen phosphate and add 90-97 ml of sodium hydroxide solution to 4 L of degassed purified water to dissolve; Step (2): using the paddle method, 900 ml of the dissolution medium in step (1) was measured and placed in a dissolution cup, the temperature of the dissolution medium was set at 37±0.5°C, the stirring speed was 50 rpm / min, and the relugolix tablets were placed in the dissolution cup. The time was counted, and 10 ml of the dissolution solution was taken after 5, 10, 15, 20, 30, 45, 60, 90, and 120 min, respectively. The solution was filtered through a 0.45 μm filter membrane, and 5-7 ml of the initial filtrate was discarded. The subsequent filtrate was taken as the test solution, and the dissolution amount was detected by high performance liquid chromatography. A dissolution curve was established based on the dissolution amount and dissolution time. The chromatographic conditions of the high performance liquid chromatography method are as follows: chromatographic column: Agilent SB-C8 chromatographic column; detection wavelength: 291 nm; Flow rate: 1.0 ml / min; injection volume: 5 μl; Column temperature: 40°C; Mobile phase: a mixed solution of 0.02 mol / L potassium dihydrogen phosphate solution and acetonitrile. The pH value of the 0.02 mol / L potassium dihydrogen phosphate solution was adjusted to 2.2±0.1 with phosphoric acid. The volume ratio of 0.02 mol / L potassium dihydrogen phosphate solution to acetonitrile was 3:2.

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